Olympus FV1000 User Manual

User’s Manual

A

FLUOVIEW

FV1000

CONFOCAL LASER SCANNING

BIOLOGICAL MICROSCOPE

[OPERATION] FV10-SW Ver 5.0c

Petition

This user’s manual is for the software to be run on Olympus FLUOVIEW FV1000 Confocal Laser
Scanning Biological Microscope. To ensure safety, obtain optimum performance and familiarize
yourself fully with this product, we recommend that you study this manual thoroughly before operation.
This user’s manual is composed of two volumes including “OPERATION INSTRUCTIONS” and
“MAINTENANCE”. Together with this manual, please also read the “SAFETY GUIDE”, “HARDWARE
GUIDE” of User’s manual FLUOVIEW FV1000 and the instruction manual of the microscope in order to
understand overall operation methods. To ensure the safety operation of laser system, we recommend
you to study the manual of each laser and the light source equipment besides this manual.
Retain this manual in an easily accessible place near a system for future reference.

X7274

CAUTION

CAUTION

1. Reproduction, copying or duplication of a part or all of this software and manual is prohibited.

2. The information described in this manual may be subject to change without notice.

Registered Trademarks

Microsoft, Microsoft Windows, Excel for Windows are registered trademarks of Microsoft Corporation.

Other brand names and product names are trademarks or registered trademarks of their respective owners.

Page

1

FLUOVIEW MANUAL CONFIGURATION

FLUOVIEW MANUAL CONFIGURATION

The FLUOVIEW system uses two manuals including this “User’s Manual” and the on-screen

manual built into the software (“Online Help”).

The User’s Manual is composed of the five following volumes and subject matter:

OPERATION INSTRUCTIONS

Describes the operation procedures of the FLUOVIEW system, for example, methods

for image acquisition and various image processing.

1 Getting Started FLUOVIEW................................................................................ 1-1

2 APPLIED OPERATIONS.................................................................................... 2-1

Appendix A List of Hot Keys.................................................................................. A-1

Appendix B Glossary............................................................................................. B-1

Appendix C USER REGISTRATION OF FV1000 ................................................. C-1

Appendix D Change of Default Folder for [File I/O] Panel..................................... D-1

Appendix E List of Functions in the [Active Overlays] Dialog Box......................... E-1

Appendix F Hand Switch and Microscope Frame Function Allocation .................. F-1

MAINTENANCE

Describes maintenance of the FLUOVIEW system.

1 Software Setup................................................................................................... 1-1

2 Maintenance of Major System Units................................................................... 2-1

3 Setting the Confocal Aperture ............................................................................3-1

TROUBLESHOOTING

Describes countermeasures in case trouble occurs.

1 TROUBLESHOOTING GUIDE ........................................................................... 1-1

For Online Help, please see “1-3 Online Help” in “OPERATION INSTRUCTIONS” of this

manual.

2

Page

NOTATIONS IN THIS MANUAL

This manual complies with the following notations.

 Notation of Caution, Notes and Tips

Notation Description

NOTATIONS IN THIS MANUAL

Caution to prevent injuries to the user or damage to the

product (including surrounding objects).

NOTE

TIP

Note for the user.

Hint or one-point advice for user reference.

 Notation of panel, Command Buttons and Dialog Boxes

Notation Description

[Acquire] panel The name of a panel, dialog box, list box or check box is

enclosed inside square brackets ([ ]).

<OK> button

<Open File> button

The name of a button in a panel or dialog box is enclosed

inside angled brackets (< >).

 Notation of Mouse Operations

Notation Description

clicking Action of pressing, then immediately releasing the mouse

button.

double-clicking Action of clicking the mouse button twice in quick succession.

dragging Action of moving the mouse while holding down the mouse

button, then releasing the mouse button at the desired

destination.

(Note) In this manual, clicking, double-clicking and dragging involves pressing the left button

of the mouse, unless otherwise specified.

Page

3

NOTATIONS IN THIS MANUAL

 Notation of key operations

Notation Description

Enter

The name of a key is enclosed inside .

Alt

+

Direction keys

F1

The positive sign (+) expresses the combination of more than
one key operation.

For example,

key while holding the

F1

Generic names given to the

keys.

↓

Alt

+

refers to pressing the

F1

key down.

Alt

→

,

←

,

and

↑

 Notation of system-specific terms

Notation Description

XY observation

(Other observations)

Note that some of the panels and dialog boxes shown in this manual are not the precise

reproductions of the originals. Some windows are resized to facilitate the reading and some

grayed-out characters are printed in readable characters.

Refers to observation with XY scanning.

(The same principle also applies to other observations such

as XZ, Xt, XYZ, XYt and XYZt.)

4

Page

Software Functional Configuration

This software uses panel-type windows.

Usually, it is required to “select a menu then select the command to be executed” in order to

execute a function provided by software. With the panel system, a software function can be

executed easily by “selecting the panel page tab of the function to be executed”, just like

when using a system notebook or file folder.

Function Window and Image Window

The FLUOVIEW software is organized by two kinds of windows, the function windows and

the image .window.

The function windows include the [Acquire], [File I/O], [Tile], [Process], [Analyze] and

[Visualize] panels.

The image .window shows either the [Live] panel or the panel image loaded from a file

([(filename)] window).

The function window

Software Functional Configuration

NOTE

The image window

In this manual, the function windows are referred to simply using their

page tabs.

Namely, the [File I/O] panel of function windows is referred to simply as the

[File I/O] panel.

Page

5

Software Functional Configuration

A

A

Panel Structure of the Software

This software cannot show the all function panels at a glance.Please use the following list of

the panels for reference in scrolling.

Acquire

Settings

Z Stage

Time Series

Dyes

Lasers Sets the intensity of laser and other option for image acquisition.

File I/O

Tile

Process

Math

Filters

Histogram

Experiment Editor

Analyze

Single

Sets up the image acquisition and executes actual acquisition.

Sets the zooming ratio and observation mode for image acquisition.
Sets the microscope light path for image acquisition.

Sets the Z-direction scanning range for image acquisition.

Sets the interval period for image acquisition.

Sets the fluorescence dye method for image acquisition and reagent for
each channel.

Saves, loads and deletes images.

Changes the image display method.

Processes the acquired images.

Performs mathematical and logical operations between images.

Filters images.

Changes the image contrast.

ppends two images, adds and extracts the channel.

nalyzes image data.

Obtains the intensity values, intensity distribution, length, area and
average intensity in images.

Series

Visualize

Orientation

Other Options

6

Page

Obtains the change in the sum of intensity values in images.

Constructs an image from a different viewpoint or displays a 3D image.

Sets the image rotation angle, direction and number.

Sets the various 3D Rendering.

Icons Executed by Dragging & Dropping

This software selects image files and observation methods (dye name) by means of

dragging & dropping. This allows simple selection based on an intuitive operation of

“selecting an icon (image file or observation method), dragging it to the desired position and

dropping it there”.

Software Functional Configuration

Page

7

Software Functional Configuration

Identification of Images Depending on the Observation Methods

Image Icon Significance

XZ observation

XZ observation, 2-channel mode

Xt observation

Xt observation, 2-channel mode

XZT observation

XZT observation, 2-channel

mode

XY observation

XY observation, 2-channel mode

XYt observation

XYt observation, 2-channel
mode

On many occasions, FLUOVIEW displays image

icons to allow identification of the observation

method used when each image is acquired. (See

table on the left.)

When the [File I/O], [Tile], [Process], [Analyze] or

[Visualize] panel is selected, the icon of the image

selected in the image window is displayed in a

frame at the top of the function panel. The image

icons are also displayed in the [Icon] field in the

[Files] list box in the [File I/O] panel or during

dragging of an image file.

Use these icons to identify the observation methods

used in image acquisition.

TIP

In all observation modes, the icons for 3

XYZ observation

XYZ observation, 2-channel
mode

XYZt observation

XYZt observation, 2-channel

mode

Point Scan

Animation image

Stereo 3D image: Image to be
viewed with color eyeglasses.

3 or more channels

or more channels are identical.

8

Page

O

O

P

P

E

E

N

IIN

R

R

S

A

A

S

TII

T

T

T

R

R

O

O

U

U

N

N

C

C

TII

T

O

O

N

N

S

S

On This Volume

This volume describes the operating procedures of the

FLUOVIEW FV1000 system.

“Getting Started FLUOVIEW” contains information on the basic

operation flow until acquisition of XY images.

“APPLIED OPERATIONS” provides detailed operating

procedures of the system.

Please read this volume so that you can understand the system

before use.

CONTENTS

1 Getting Started FLUOVIEW 1-1

1-1 Basic Operations .............................................................................1-1

1-1-1 Microscope .................................................................................................... 1-1

1-1-2 General Mouse Operation Procedures.......................................................... 1-7

1-1-3 Names of Major Panel and Window Controls and Their Functions............... 1-8

1-2 Outline of LSM Observation Procedures ......................................1-9

1-2-1 Turning Power On ....................................................................................... 1-11

1-2-2 Focusing on the Specimen.......................................................................... 1-12

1-2-2-1 Combination with BX .......................................................................................1-12

1-2-2-2 Combination with IX81 FVF .............................................................................1-14

1-2-3 Setting the LSM Light Path.......................................................................... 1-16

1-2-3-1 Combination with Upright Microscope (BX).....................................................1-16

1-2-3-2 Combination with Inverted Microscope (IX81 FVF) .........................................1-18

1-2-4 Selecting the Dyeing Method ...................................................................... 1-20

1-2-5 Selecting the Filters..................................................................................... 1-23

1-2-6 Setting the ND Filters ..................................................................................1-26

1-2-7 Setting the Observation Condition............................................................... 1-27

1-2-7-1 Setting the Objective Magnification .................................................................1-27

1-2-7-2 Setting the Zoom Ratio to 1X...........................................................................1-28

1-2-7-3 Setting the Channels .......................................................................................1-28

1-2-7-4 Setting the Highest Scan Speed......................................................................1-29

1-2-7-5 Setting the XY Observation Mode ...................................................................1-30

1-2-7-6 Repeated Scanning Operation ........................................................................1-30

1-2-7-7 Setting the Cross-section to be Observed.......................................................1-31

1-2-7-8 Setting the Area to be Observed .....................................................................1-32

1-2-7-9 Setting a Lower Scan Speed ...........................................................................1-32

1-2-7-10 Stopping Repeated Scanning........................................................................1-33

1-2-8 Acquiring Image ..........................................................................................1-33

1-2-9 Saving Image .............................................................................................. 1-34

1-2-10 Exiting from the Software, Turning Power Off ........................................... 1-35

CONTENTS

1-3 Online Help..................................................................................... 1-36

2 APPLIED OPERATIONS 2-1

2-1 General Operation Procedure ........................................................2-1

1-3-1 Function Help ..............................................................................................1-36

1-3-2 Microscope Help..........................................................................................1-37

1-3-2-1 Configuring the Microscope ............................................................................ 1-40

1-3-2-2 Parfocality Correction and Jog Sensitivity Adjustment ................................... 1-46

1-3-2-3 Configuring the Filters (When using a filter system) ....................................... 1-52

1-3-2-4 Configuring the filters (When using a spectral detecting system)................... 1-56

1-3-2-5 Setting the C.A. Diameters ............................................................................. 1-59

2-1-1 Image Acquisition Procedure (Section (A))....................................................2-3

2-1-2 Image Acquisition Procedure in an Observation Mode (Section (B)) ............2-4

2-1-3 Examples of Operation Procedures...............................................................2-5

2-2 Image Acquisition............................................................................ 2-7

2-2-1 Image Acquisition in XY Observation Mode ..................................................2-8

2-2-1-1 Configuring the Microscope .............................................................................. 2-9

2-2-1-2 Setting the Filters ............................................................................................ 2-15

2-2-1-3 Setting the ND Filters...................................................................................... 2-17

2-2-1-4 Setting the Observation Condition .................................................................. 2-19

2-2-1-5 Acquiring Image .............................................................................................. 2-28

2-2-1-6 Acquiring Image in Accumulation Mode.......................................................... 2-29

2-2-1-7 Saving the Acquired Image in File .................................................................. 2-33

2-2-2 Image Acquisition in Other Observation Modes ..........................................2-34

2-2-2-1 XZ Observation Mode ..................................................................................... 2-34

2-2-2-2 XT Observation Mode ..................................................................................... 2-38

2-2-2-3 XZT Observation Mode................................................................................... 2-40

2-2-2-4 XYZ Observation Mode................................................................................... 2-46

2-2-2-5 XYT Observation Mode................................................................................... 2-51

2-2-2-6 XYZT Observation Mode ................................................................................ 2-55

2-2-3 Differences in Image Acquisition Method Between Fluorescent and Transmitted

CONTENTS

Images......................................................................................................... 2-61

2-2-3-1 Monochrome Image.........................................................................................2-61

2-2-3-2 Dual-Fluorochrome Image ...............................................................................2-64

2-2-3-3 Transmitted Image...........................................................................................2-67

2-2-4 Image Acquisition by Rotating It (Rotation Scan)........................................ 2-71

2-2-5 Image Acquisition of Only the Rectangular Position (Clip Scan)................. 2-72

2-2-6 Image Acquisition by Magnifying the Rectangular Position (Zoom-In Scan)2-75

2-2-7 High-Speed Image Acquisition .................................................................... 2-78

2-2-8 Image acquisition to prevent crosstalk between fluorescence (Sequential Scan)

.................................................................................................................... 2-79

2-2-8-1 Virtual Channel Function .................................................................................2-82

2-2-9 Image Acquisition of a Line at Desired Angle.............................................. 2-88

2-2-10 Display the change of image intensity (Point Scan) .................................. 2-89

2-2-11 Image Acquisition on Desired Line (XZ, XT or XZT Observation)............. 2-94

2-2-12 Image Acquisition in the Laser Excitation Mode........................................ 2-97

2-2-12-1 Making REX Mask File ..................................................................................2-98

2-2-12-2 Example of FRAP experiment .....................................................................2-103

2-2-13 Notes for image acquisition ..................................................................... 2-112

2-2-13-1 Memory of setting information for scanning region......................................2-112

2-3 Saving, Opening and Shredding Images................................... 2-113

2-3-1 Saving Images........................................................................................... 2-116

2-3-1-1 Saving Images As a Series............................................................................2-116

2-3-1-2 Saving a Display ............................................................................................2-118

2-3-1-3 Saving Specified Area of Image ....................................................................2-120

2-3-1-4 Saving Animation Images ..............................................................................2-123

2-3-1-5 File Types Available for Save ........................................................................2-125

2-3-2 Opening Previously Saved Images ........................................................... 2-132

2-3-3 Shredding Images .....................................................................................2-133

2-3-4 Saving Comment Together with Image ..................................................... 2-135

2-3-5 Checking the Image Information/Acquisition Parameters.......................... 2-138

2-3-6 Saving the Image Information/Observation Condition............................... 2-142

2-3-7 Saving/Reading the Region File................................................................ 2-145

CONTENTS

2-4 Protocol processor...................................................................... 2-150

2-3-7-1 Saving the Region File.................................................................................. 2-145

2-3-7-2 Reading the Region File ............................................................................... 2-147

2-4-1 Starting the Protocol Processor.................................................................2-151

2-4-2 Editing the Protocol ...................................................................................2-152

2-4-2-1 Description of Setting Items .......................................................................... 2-153

2-4-2-2 Command List ............................................................................................... 2-160

2-4-2-3 Protocol Repetition Processing..................................................................... 2-165

2-4-2-4 Input supporting function............................................................................... 2-167

2-4-2-5 Protocol procedure supporting function ........................................................ 2-169

2-4-3 Saving the Protocol....................................................................................2-170

2-4-4 Executing the Protocol...............................................................................2-172

2-4-5 Loading a Protocol.....................................................................................2-174

2-4-6 Loading a protocol of previous format .......................................................2-175

2-4-7 COM Communication function................................................................... 2-177

2-4-8 Example of assembling a protocol.............................................................2-178

2-4-8-1 Example of Setting Procedure of FRAP Observation ................................... 2-178

2-4-8-2 Example of Setting Procedure of XYT Observation for hours (Protocol using

repetition processing) ........................................................................................... 2-187

2-4-9 Pop-up Menu .............................................................................................2-195

2-4-10 Restrictions of [PAPP] setting.................................................................. 2-196

2-4-10-1 Restrictions with [Mode] and [Sub Mode] ................................................... 2-196

2-5 Changing the Image Display Method......................................... 2-198

2-5-1 Displaying an Image in Simulated Colors .................................................. 2-198

2-5-2 Editing the LUT (Look Up Table) ...............................................................2-200

2-5-2-1 LUT Graph Editing According to Colors........................................................ 2-200

2-5-2-2 LUT Graph Editing by Gamma Correction.................................................... 2-202

2-5-3 Switching the Displayed Channels (Ch1 – Ch5)........................................2-203

2-5-4 Displaying Images of Multiple Channels Simultaneously (Side By Side Views,

Over And Under Views, Single View) ........................................................2-204

2-5-4-1 Displaying Images Separately Per Channel (Side By Side Views, Over And Under

Views) ................................................................................................................... 2-205

CONTENTS

2-5-4-2 Displaying Merged Image of Multiple Channels (Single View)......................2-206

2-5-5 Changing the Number of Divided Images ................................................. 2-208

2-5-5-1 Increasing the Number of Divided Images ....................................................2-208

2-5-5-2 Decreasing the Number of Divided Images...................................................2-210

2-5-6 Switching the Display Method of Multiple Images ..................................... 2-211

2-5-7 Displaying Multiple Image Slices Together ............................................... 2-213

2-5-7-1 Displaying Multiple Images Per Channel.......................................................2-214

2-5-7-2 Displaying Images of Two Channels Together..............................................2-216

2-5-7-3 Displaying Time-Lapse Images .....................................................................2-217

2-5-7-4 Displaying Multiple Multiple sections Images ................................................2-218

2-5-7-5 Displaying Same Images in Different Display Methods.................................2-218

2-5-7-6 Re-arranging Images Using the Same Display Method ................................2-221

2-5-7-7 Displaying Different Images Together ...........................................................2-221

2-5-8 Magnifying/Reducing an Image................................................................. 2-224

2-6 Image Processing ........................................................................ 2-225

2-6-1 Filtering...................................................................................................... 2-225

2-6-1-1 Contour Enhancement...................................................................................2-226

2-6-1-2 Noise Reduction ............................................................................................2-229

2-6-1-3 Image Sharpening .........................................................................................2-230

2-6-1-4 DIC Correcting DIC Level Irregularities .........................................................2-231

2-6-2 Contrast Conversion.................................................................................. 2-233

2-6-3 Mathematical Operations Between Images............................................... 2-236

2-6-3-1 Image Addition...............................................................................................2-236

2-6-3-2 Image Subtraction..........................................................................................2-239

2-6-3-3 Image Multiplication .......................................................................................2-239

2-6-3-4 Image Division ...............................................................................................2-240

2-6-3-5 NOT Image ....................................................................................................2-241

2-6-3-6 Image AND Image .........................................................................................2-243

2-6-3-7 Image OR Image ...........................................................................................2-244

2-6-3-8 Image XOR Image .........................................................................................2-244

2-6-4 Brightness overlap level between 2 channels (Colocalization).................. 2-246

2-6-4-1 Annotation Mode............................................................................................2-247

CONTENTS

2-7 Image Analysis............................................................................. 2-274

2-6-4-2 Colocalization for series image data set ....................................................... 2-254

2-6-4-3 Image measurement ..................................................................................... 2-255

2-6-5 Appending image (Append).......................................................................2-258

2-6-5-1 Appending two images.................................................................................. 2-258

2-6-5-2 Appending image from several image data set ............................................ 2-262

2-6-6 Extract image (Crop)..................................................................................2-267

2-7-1 Checking the Intensity of a Specific Part ...................................................2-275

2-7-1-1 Intensity Values on a Line (Line Profile) ....................................................... 2-275

2-7-1-2 Intensity Values on a Planar Region (Bird’s Eye View) ................................ 2-278

2-7-2 Checking the Intensity Distribution of a Specific Part ................................2-283

2-7-2-1 Intensity Distribution on a Line (Histogram) .................................................. 2-283

2-7-2-2 Intensity Distribution on a Planar Region (Histogram).................................. 2-284

2-7-3 Image Measurement..................................................................................2-286

2-7-3-1 Length Measurement .................................................................................... 2-286

2-7-3-2 Area Measurement........................................................................................ 2-286

2-7-3-3 Measuring the Change in Mean Value of Intensity ....................................... 2-287

2-7-3-4 Measuring the Change in Integrated Intensity .............................................. 2-293

2-8 Building an Image from a Different Viewpoint.......................... 2-295

2-8-1 Building Extended Focus Image from XYZ Image.....................................2-295

2-8-1-1 Display Switching to Built Image................................................................... 2-295

2-8-1-2 Turning Built Image into Single Image .......................................................... 2-298

2-8-1-3 Turning Built Image into time series image................................................... 2-300

2-8-2 Building line images to be viewed in Z direction........................................2-301

2-9 Viewing 3D Image ........................................................................2-305

2-9-1 Successive Display of Images...................................................................2-307

2-9-1-1 Changing the Successive Display Speed ..................................................... 2-308

2-9-1-2 Changing the successive image display position ......................................... 2-309

2-9-2 Animation...................................................................................................2-310

2-9-3 Building Stereo 3D Images........................................................................2-312

2-9-4 Building a 3D Image to be Viewed Through Color (Red/Green) Eyeglasses2-314

2-10 Viewing Images Following the Progress of Time................... 2-316

CONTENTS

2-10-1 Displaying Images Together.................................................................... 2-316

2-10-2 Displaying Images Successively .............................................................2-316

2-11 Transferring Data to Another Application...............................2-319

2-11-1 Transferring Analysis Data to Another Application.................................. 2-319

2-11-2 Transferring the Plot Image of Analysis Data to Another Application...... 2-322

2-11-3 Transferring Image Data to Another Application (Paint, etc.).................. 2-323

2-12 Entering Comment in Image .....................................................2-324

2-12-1 Writing Characters in Image.................................................................... 2-324

2-12-2 Displaying the Image Intensity ................................................................ 2-326

2-12-3 Displaying the X-coordinate/Y-coordinate of the Image .......................... 2-327

2-12-4 Drawing a Figure in Image ...................................................................... 2-328

2-12-5 Drawing a Scale in Image ....................................................................... 2-329

2-12-6 Drawing an Arrow in Image ..................................................................... 2-330

2-12-7 Drawing Color Bars in Image .................................................................. 2-331

2-12-8 Deleting Comment................................................................................... 2-332

2-12-9 Moving Comment ....................................................................................2-333

2-12-10 Changing the Comment Size ................................................................ 2-334

2-12-11 Changing the Comment Color............................................................... 2-335

2-12-12 Changing the Comment Font ................................................................ 2-336

2-13 Image Output at Printer.............................................................2-337

2-14 Merger/Extraction of Image Channels ..................................... 2-338

2-14-1 Setting the Range of Multiple Image Slices............................................. 2-338

2-14-2 Merging Image Channels ........................................................................ 2-339

2-14-3 Extracting Channels from Image ............................................................. 2-344

2-15 Changing the Chart Display Method........................................ 2-348

2-15-1 [Chart] Panel ...........................................................................................2-348

2-15-2 [Series] Panel .......................................................................................... 2-356

2-16 Pop-up Menus ............................................................................ 2-359

Appendix A List of Hot Keys A-1

CONTENTS

Appendix B Glossary B-1

Appendix C USER REGISTRATION OF FV1000 C-1

Appendix C-1 User Registration.......................................................... C-1

Appendix C-2 Logging into the FV1000.............................................. C-4

Appendix C-3 Deleting a User ............................................................. C-5

Appendix D Change of Default Folder for [File I/O] Panel

D-1

Appendix E List of Functions in the [Active Overlays]

Dialog Box E-1

Appendix E-1 Coordinate Position Data..............................................E-1

Appendix E-1-1 X-Coordinate ................................................................................ E-1

Appendix E-1-2 Y-Coordinate ................................................................................ E-2

Appendix E-1-3 Other ............................................................................................ E-3

1 Z Position....................................................................................................................E-3

2 T Position....................................................................................................................E-3

3 Animation.................................................................................................................... E-3

Appendix E-2 Intensity Data .................................................................E-4

Appendix E-3 Other ...............................................................................E-5

Appendix E-3-1 Channel Number .......................................................................... E-5

Appendix E-3-2 Objective Power ........................................................................... E-5

Appendix E-3-3 Date of Image Capturing .............................................................. E-5

Appendix E-3-4 Time of Image Capturing.............................................................. E-5

Appendix E-3-5 Image File Name .......................................................................... E-5

Appendix F Hand Switch and Microscope Frame Function

CONTENTS

Allocation F-1

Appendix F-1 Hand Switch Functions .................................................F-1

Appendix F-1-1 BX/BXWI .......................................................................................F-1

Appendix F-1-2 IX...................................................................................................F-2

Appendix F-2 Microscope Frame Functions .......................................F-3

Appendix F-2-1 BX .................................................................................................F-3

Appendix F-2-2 IX...................................................................................................F-4

Appendix F-2-3 Focus Adjustment Knob ................................................................F-5

1 BX............................................................................................................................... F-5

2 IX ................................................................................................................................ F-5

Getting Started FLUOVIEW/ Basic Operations

r

r

)

1 Getting Started FLUOVIEW

1-1 Basic Operations

1-1-1 Microscope

The following figure shows the major controls of a microscope. The actual configuration of

the modules including the specimen stage, revolving nosepiece and lighting equipment may

differ from those shown below.

For detailed microscope operation procedures, refer to the instruction manuals of your

microscope.

Combination with BX61

(1) Light path

selector knob

(2) Cube turret /Cube display window

(This figure shows Cube display

window)

(4) Transmitted light DIC

FV5-DICTS/ WI-DICTH (with
BX61WI) (optional)

(3) Analyze

IX2-MDICT

(6) Universal

condense

(This figure shows the case of BX)

(5) Filters

LBD
ND6

ND25

When using BX61WI,

LBD

FFR

(7)Hand switch

U-HSTR2

(U-FH is optionally
available with BX61

OPERATION INSTRUCTIONS

Page

1-1

Getting Started FLUOVIEW/Basic Operations

2

(1) Light path selector knob

Select the light path between the visual observation and photography observation.

• See the following table and set the knob to the position corresponding to the

required light path.

Light path selector knob Symbol Intensity Ratio

Pushed in 100% for the visual observation

Middle position

Pulled out 100% for photography observation

(2) Cube turret

Select the fluorescence observation tube by rotating the turret.

20% for the visual observation, 80%
for photography observation

• Engage the desired cube in the light path for visual fluorescence observation or

visual transmitted light observation.

• For laser microscopy, rotate the turret to page tab

state that mirror cube is entered.)

(3) Analyzer IX2-MDICT

Polarizing plate for use in differential interference observation and polarized light

observation.

• Rotate the turret to engage the IX2-MDICT in the light path for visual transmitted

light differential interference observation or transmitted polarized light observation.

(4) Transmitted light DIC slider U-DICTS/WI-DICTHRA (built-in FV10-SRE or FV10-

SNPXLU of BX61WI)

This is the prism for use in differential interference observation.

• Engage the transmitted light DIC in the light path for laser differential interference

observation or visual transmitted light differential interference observation.

Leaving the transmitted light DIC engaged during laser fluorescence observation will

1

. (Set cube turret in the

degrade the resolution somewhat. We recommend disengaging the transmitted light

DIC from the light path when simple laser fluorescence observation is required.

OPERATION INSTRUCTIONS

1-

Page

Getting Started FLUOVIEW/Basic Operations

NOTE

(5) Filters

These filters are used to adjust the transmitted light.

• Be sure to disengage any filter from the light path for transmitted observation using

lasers. Leaving a filter engaged in the light path will degrade the image quality.

When you perform transmitted observation using laser with BX61WI, use the filter

knob to disengage the LBD from the light path and engage the FR (Frost) into the

light path. Disengaging the FR (Frost) from the light path may generate interference

fringes on an image.

(6) Universal condenser

With transmitted light differential interference observation using an

immersion objective, set the microscope’s field diaphragm so that

it circumscribes the field of view. Otherwise the contrast may

degrade. (This applies to both visual observation and laser

differential interference observation.)

Condenser for transmitted lighting. In addition, the rotary turret for the transmitted light

DIC prism and the polarizing plate for differential interference observation (polarizer)

are also provided.

• To perform differential interference observation, engage the transmitted light DIC

prism matching the objective in use in the light path (For both visual observation and

laser differential interference observation).

• To perform visual differential interference observation or laser differential

interference observation, engage the polarizing plate in the light path.

(7) Hand switch U-HSTR2 (U-FH is optionally available with BX61/BX61WI)

This is the hand switch to operate the BX motorized system.

NOTE

Connect the filter wheel of BX to connector of the following in the

back of UCB.

FW0: FW1

•

FWR: FW2

•

FWT: FW3

•

OPERATION INSTRUCTIONS

Page

1-3

Getting Started FLUOVIEW/Basic Operations

y

)

r

Combination with IX81 FVF

(4) Condense

(5) Filters

(6) Light path selector button

(1)Fluorescence mirror
unit

((2) Analyzer IX-MDICT

Optionall

installed

(4) Transmitted light

DIC slider

U-DICTS (optional)

(6)Hand switch

U-HSTR2

(U-FH is optionally
available.)

OPERATION INSTRUCTIONS

1-4

Page

Getting Started FLUOVIEW/Basic Operations

5

(1) Fluorescence mirror unit

Select the fluorescence observation tube by rotating the turret.

• Engage the desired cube in the light path for visual fluorescence observation.

• For laser microscopy, rotate the turret to page tab

mirror cube is entered.)

(2) Analyzer IX2-MDICT

Polarizing plate for use in differential interference observation and polarized light

observation.

• Rotate the cube turret to engage the IX2-MDICT analyzer into the light path for visual

transmitted light differential interference observation or transmitted polarized light

1

. (Set turret in the state that

observation.

(3) Transmitted light DIC slider U-DICTS

This is a prism for use in differential interference observation.

• Engage U-DICTS in the light path for laser differential interference observation or

visual transmitted light differential interference observation.

Leaving U-DICTS engaged during laser fluorescence observation will degrade image

quality somewhat. We recommend disengaging the U-DICTS from the light path

when simple laser fluorescence observation is required.

NOTE

With transmitted light differential interference observation using an

immersion objective, set the microscope’s field diaphragm so that it

inscribes the field of view. Otherwise the contrast may degrade.

(This applies to both visual observation and laser differential

interference observation.)

OPERATION INSTRUCTIONS

Page

1-

Getting Started FLUOVIEW/Basic Operations

6

(4) Condenser, polarizing plate

Condenser for transmitted lighting.

In addition, the rotary turret for the transmitted light DIC and the polarizing plate for

differential interference observation (analyzer) are also provided.

• To perform differential interference observation, engage the transmitted light DIC

(optional) matching the objective in use in the light path (For both visual observation

and laser differential interference observation).

• To perform visual differential interference observation or laser differential

interference observation, engage the polarizing plate in the light path.

(5) Filters

These filters are used to adjust transmitted light.

• For transmitted observation using laser, disengage the LBD filter from the light path

and engage the FR (frost) filter in the light path by operating the filter levers. If the FR

filter is disengaged from the light path, the image may suffer from stripe interference.

(6) Light path selector button

Select the light path between the visual observation and photography observation.

• When <

• When <

(7) Hand switch (U-FH is optionally available.)

This is the hand switch to operate the IX motorized system.

> LED is lighted, visual observation can be done.

> LED is lighted, TV or photography observation can be done.

OPERATION INSTRUCTIONS

1-

Page

7

1-1-2 General Mouse Operation Procedures

Use the mouse to select a command, character string or button. Use the left

button of the mouse unless otherwise specified.

To select or execute something: Clicking

To click the mouse, place the mouse pointer on the desired function and

press the mouse button once.

(Pressing the right button of the mouse is referred to as right-clicking.)

To select something and execute its function: Double clicking

To double-click, place the mouse pointer on the desired function and press the

Getting Started FLUOVIEW/Basic Operations

mouse button successively twice.

To move something: Dragging

To drag, place the mouse pointer on the desired function, and while pressing

and holding the mouse button, move the mouse to the desired destination. At

the desired destination, release the mouse button.

(Dragging by pressing the right button of the mouse is referred to as right-

dragging.)

One Point!

When the mouse is moved, the picture of arrow on the screen moves

accordingly. The picture which moves on the screen as the mouse is

moved is referred to as the mouse pointer.

OPERATION INSTRUCTIONS

Page

1-

Getting Started FLUOVIEW/Basic Operations

f

r

g

r

f

1-1-3 Names of Major Panel and Window Controls and Their Functions

The window as shown below is displayed when FLUOVIEW starts up. FLUOVIEW uses

panel-type windows.

This section describes the names of the major controls displayed in panels and windows.

Click to turn the window into an icon.

Original size button <

Click each button to

execute the processing

Drop-down list

Click
the list of available

items for selection.

To select an item in

the list, click the item.

Buttons

indicated on it.

to display

Control menu box

Clicking this box displays a control
menu, which contains the commands
for use in controllin

the window.

Click to return the maxim-size window to its original size.

Title bar

Shows the title of the window. The title ba
of a window that is active is displayed in a

different color from that of other windows.

Information

Shows information on the operations and
meanings of functions.

Scale

The scale is used to set a value which is
continuously variable in a certain range.
Clicking a point in the scale area allows the
value to change on a large scale.
Clicking the top or bottom arrow button allows
fine adjustment of the value.
Dragging the square knob allows the value to
vary directly.

Option buttons

This is a group of multiple items among
which only one can be selected.
Clicking one of the round buttons selects
the corresponding item.
The option button of the selected item is
displayed with a black dot in the center o

Minimize button < >

>

Sub-panel

A sub-panel is

provided for use in

detailed setting o

information display o

a function.

Fig. 1-1 Window and Major Functions

Check box

Clicking this box enables or disables the indicated item. The
item is enabled when the check box is checked (X).

Scroll bar

The scroll bar is displayed when there are too many data items to be displayed in a field at
once, and is used to display the data items outside the field. Clicking a point in the scroll area
allows data items to be scrolled in large steps.
Clicking the top or bottom arrow button allows fine scrolling of the data items.
Dragging the square knob allows direct scrolling.

List box

Shows the list of available items for selection. All items in the list can be displayed by
scrolling. To select an item in the list, double-click or drag the item.

Fig. 1-2 Sub-panel and Major Functions

OPERATION INSTRUCTIONS

1-8

Page

Group box

The group box groups functions with
specific meanings and encloses
frame.

them in a

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2 Outline of LSM Observation Procedures

• Fluorescence observation procedure

Start the system.

•

Turn the system power ON.

•

Start the FLUOVIEW software.

z

Let an appropriate cube for

(Section 1-2-1)

(Section 1-2-1)

dyeing method into the light
path.

z

Select the light path for 100
binocular tube and focus on
the specimen.

(Section 1-2-2)

z

Select the LSM light path.

z

Let the mirror cube into the
light path.

(Section 1-2-3)

Set the dyeing method.

(Section 1-2-41-2-5)

When the combination using the laser

combiner is used, set the optimum ND

filters for the laser.

(Section 1-2-6)

Change the ND filter with a filter

with higher transmittance

(Section 1-2-6)

Set the observation condition.

Set the objective magnification.

(Section 1-2-7-1)

•

Set the zoom ratio to 1X.

•

Set the channels.

•

Set the highest scan speed.

•

Select the XY observation mode.

•

Perform repeated scanning.

(Section 1-2-7-6)

(Section 1-2-7-3)

(Section 1-2-7-4)

(Section 1-2-7-5)

(Section 1-2-7-2)

If no image is
displayed

Image is

displayed in the

Adjust PMT Voltage.

(Section 2-1-1-4-9)

[Live] panel of

the software

•

Set the multiple sections to be

observed.

•

Set the area to be observed.

(Section 1-2-7-8)

If an image
is displayed

(Section 1-2-7-7)

• Set a lower scan speed.

(Section 1-2-7-9)

•

Stop repeated scanning.

(Section 1-2-7-10)

If the image is still not displayed

Acquire an image.

(Sec 1-2-8)

Save the image.

(Sec 1-2-9)

Exit from the FLUOVIEW software.

(Sec 1-2-10)

Turn the system power OFF.

(Sec 1-2-10)

OPERATION INSTRUCTIONS

Page

1-9

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

• Transmitted light observation procedure

Start the system

Turn the system power ON.

•

(Section 1-2-1)

Start the FLUOVIEW software.

•

(Section 1-2-1)

Change the ND filter with a higher

transmittance filter.

(Section 1-2-6)

z

Let the transmitted light DIC

slider into the light path.

z

Select the light path for 100%

binocular tube and focus on the
specimen.

(Section 1-2-2)

Select the LSM light path.

(Section 1-2-3)

When the combination using the laser

combiner is used, set the optimum ND

filters for the laser.

(Section 1-2-6)

Set the observation condition.

Set the objective magnification.

•

(Section 1-2-7-1)

Set the zoom ratio to 1X.

•

(Section 1-2-7-2)

Set the channels.

•

Set the highest scan speed.

•

(Section 1-2-7-4)

Select the XY observation mode.

•

(Section 1-2-7-5)

Perform repeated scanning.

•

(Sec tion 1-2-7-6)

(Section 1-2-7-3)

If no image is
displayed.

Image is
displayed in
the [Live]

Adjust PMT Voltage.

(Section 2-2-1-4-9)

panel of the
software.

If an image is
displayed

• Set the multiple sections to be

observed. (Section 1-2-7-7)

Set the area to be observed.

•

(Section 1-2-7-8)

Set a lower scan speed.

•

(Section 1-2-7-9)

Stop repeated scanning.

•

(Section 1-2-7-10)

If the image is still not displayed

OPERATION INSTRUCTIONS

1-10

Page

Acquire an image.

(Section 1-2-8)

Save the image.

(Section 1-2-9)

Exit from the FLUOVIEW software.

(Section 1-2-10)

Turn the system power OFF.

(Section 1-2-10)

1-2-1 Turning Power On

Set the power switch of each unit to ON, then start the software.

For details, see sections 1-1 “Turning the Power On” and section 1-2 “Starting the Software”

in Volume II [PREPARATION For OPERATION] of [HARDWARE GUIDE] of the FV1000

User’s Manual.

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

OPERATION INSTRUCTIONS

Page

1-11

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-2 Focusing on the Specimen

1-2-2-1 Combination with BX

1. Select the light path for 100% eyepiece by pushing in the light path selector

knob (1) fully to the stop position.

2. From the page tabs on the bottom right of the [Acquire] panel, select the

[Settings] sub-panel.

(2)

OPERATION INSTRUCTIONS

1-12

Page

(1)

(3)

(4)

Fig. 1-3 [Settings] Sub-panel

3. Select the <BI> button in the [Light Path] group box.

The <BI> button looks pushed in to indicate that it is selected.

4. Push the cube button (5) of the hand switch (4) to engage the optimum

cube for specimen dye. In the cube display window (2), the selected cube

is displayed.

5. When you do the transmitted light observation, push the transmitted light

DIC slider (3) and let it into the light path.

6. Focus on the specimen by looking into the eyepiece. Be sure to adjust

(5)

the diopter of the eyepiece in advance. (Refer to the instruction manual of

the BX microscope.)

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

When focus is aligned with use of focus handle of microscope

or U-FH, uncheck checkbox of [Locked] on [Z Stage] sub

panel inside [Acquire] panel. (See 2-2-1-4-7 of this manual.)

When [Locked] checkbox is checked, the handle cannot be

operated.

Clip for immersion
objective

NOTE

If you want to use a differential interference unit in transmitted light observation,

refer to the instruction manual of your microscope.

NOTE

The specimen may float during oil-immersed observation. In

this case, prepare an optional clip for immersion objective and

attach it as shown on the left.

With transmitted light differential interference observation

using an immersion objective, set the microscope’s field

diaphragm so that it circumscribes the field of view. Otherwise

the contrast may become degrade.

OPERATION INSTRUCTIONS

Page

1-13

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-2-2 Combination with IX81 FVF

1. Push the light path selector button (1) on the front of the microscope to

(when using Manual microscope).

From the page tabs on the bottom right of the [Acquire] panel, select the [Scan]

sub-panel, and Select the <BI> button in the [Light Path] group box (when using

Motorized microscope).

The <BI> button looks pushed in to indicate that it is selected.

(1)

(3)

OPERATION INSTRUCTIONS

Fig. 1-4 [Settings] Sub-panel

2. Engage the optimum cube for specimen dye by pressing the cube turret (2).

(when using Manual microscope)

Engage the optimum cube for specimen dye by operating the cube button (4) on

the hand switch (5).(when using Motorized microscope)

3. When you do the transmitted light observation, push the transmitted light DIC

slider (3) and let it into the light path.

4. Focus on the specimen by looking into the eyepiece. Be sure to adjust the diopter

of the eyepiece in advance. (Refer to the instruction manual of the IX71/81

(4)

microscope.)

(5)

1-14

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

When focus is aligned with use of focus handle of microscope or

U-FH, uncheck checkbox of [Locked] on [Z Stage] sub panel

inside [Acquire] panel. (See 2-2-1-4-7 of this manual.) When

[Locked] checkbox is checked, the handle cannot be operated.

NOTE

The specimen may float during oil-immersed observation. In this

case, prepare a stage clip (U-SCL) and attach it to the microscope

as shown on the left.

OPERATION INSTRUCTIONS

Page

1-15

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-3 Setting the LSM Light Path

1-2-3-1 Combination with Upright Microscope (BX)

1. From the page tabs on the bottom right of the [Acquire] panel, select the [Settings]

sub-panel.

OPERATION INSTRUCTIONS

1-16

Page

Fig. 1-5 [Settings] Sub-panel

2. Select the <LSM> button in the [Light Path] group box.

The <LSM> button looks pushed in to indicate that it is selected.

(When scanning is started while the <BI> button is selected, the LSM light path is

selected automatically. It is switched back to the visual observation automatically when

scanning completes.)

3. Push the hand switch button to (8) set

window (1) on the reflected light fluorescence vertical illuminator

to be displayed in the cube display

1

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

r

4. When only fluorescence observation is required, disengage the transmitted DIC slider

(3) by setting the switch to the pulled-out position. When transmitted light differential

interference observation or simultaneous fluorescence + transmitted light differential

interference observation is required, engage the transmitted DIC slider and the optimum

transmitted light DIC for the objective in the light path by operating the universal

condenser (5).

5. For transmitted light observation, disengage any filter (4) from the light path.

When you perform transmitted observation using laser with BX61WI, use the filter knob

to disengage the LBD from the light path and engage the FR (Frost) into the light path.

Disengaging the FR (Frost) from the light path may generate interference fringes on an

image.

(1) Cube display window

(2) Analyze

IX2-MDICT

(3) Transmitted light DIC slider

(5) Universal condenser

(4) Filters

LBD
ND6

ND25

When using

BX61WI,

LBD
FFR

(This figure shows

the case of BX)

(6) Hand switch

U-HSTR2(U-FH)

OPERATION INSTRUCTIONS

Page

1-17

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-3-2 Combination with Inverted Microscope (IX81 FVF)

1. From the page tabs on the bottom right of the [Acquire] panel, select the [Settings]

sub-panel, and .select the <LSM> button in the [Light Path] group box.

The <LSM> button looks pushed in to indicate that it is selected.

(When scanning is started while the <BI> button is selected, the LSM light path is

selected automatically. It is switched back to the visual observation automatically when

scanning completes.)

OPERATION INSTRUCTIONS

1-18

Page

2. Push the hand switch button (7) to set reflected light fluorescence mirror unit to

3. When a fluorescence observation alone is required, disengage the U-DICTS

transmitted DIC slider (3) by setting the switch to the pulled-out position. When a

transmitted light DIC observation or a simultaneous fluorescence & transmitted light

DIC observation is required, engage the transmitted DIC slider (3) and the optimum

transmitted DIC slider for the objective into the light path by operating the universal

condenser (4).

In a simultaneous fluorescence & transmitted light differential interference observation,

leaving the transmitted DIC slider (3) within the light path degrades the fluorescence

image resolution to some extent.

1

.

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

(5)

)

4. During the transmitted light observation, be sure to disengage the filter (5) from the light

path.

Filters

(3) Transmitted DIC

U-DICTS

(1) Fluorescence mirror unit

(4) Condenser

((2) Analyzer IX-MDICT

Optionally installed.)

(6) Hand switch

U-HSTR

(U-FH is optionally

available.

OPERATION INSTRUCTIONS

Page

1-19

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

A

1-2-4 Selecting the Dyeing Method

1. From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-

panel.

2. Select the specimen dyeing method by dragging [FITC] and [TRITC] in the [Available

Dyes] list box in the [Selected Dyes] group box to the field immediately above the list

box.

Place the pointer on the icon displayed
in the [Selected Dyes], and the dyeing
method is shown in the pop-up display.

[Available Dyes] list box

Lists the available dyes. Select the
desired items from this list and drag
them to the field above it to select the
dyeing method.

<Clear> button

Clear the set dyeing method.

<Prev.> button

Sets the dyeing method which

was set last time by clicking

the <Apply> button.

Fig. 1-6 [Dyes] Sub-panel

[Assign dyes manually] check box

Checking this enables the manual
setting. Dragging the dyeing method in
the list directly to the [Ch] group box
assigns the dye to the desired channel.

<Apply> button

pplies the dyeing method dragged in
the [Selected Dyes] group box to the
[Ch] group in the [Acquire] panel.

3. Click the <Apply> button to apply the selected dyeing method to the [Ch] group box on

the upper part of the [Acquire] panel.

OPERATION INSTRUCTIONS

1-20

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

TIP

TIP

When the dyeing method is selected from the [Available Dyes] list box and the

<Apply> button is clicked, a channel for acquiring fluorescence is set

automatically according to the switched filter. And the dyeing method is shown

in the [Ch] group box.

The Confocal Aperture value is also set automatically according to the

wavelength and the objective for every channel.

If you have changed the objective, click the <Apply> button in the [Dyes] sub

panel. The Conforcal Aperture value is set appropriately.

For detailes, see section 1-3-2-4 “Configuring the Filters” for automatic

Confocal Aperture setting with switching the objective.

OPERATION INSTRUCTIONS

Page

1-21

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

One Point!

The [Assign dyes manually] check box can also be used to set the dyeing method to the

desired channel.

1. Check the [Assign dyes manually] check box in the [Dyes] sub-panel.

2. Select the dyeing method in the [Available Dyes] list box and drag it directly to the
field of the [Ch] check box.

3. After dragging, the icon appears on the right of the [Ch] check box and the dyeing

Dragging the icon to the out of the [Ch] check box field cancels the setting of the dyeing

method.

OPERATION INSTRUCTIONS

[Assign dyes manually]
check box

method is set.

The dyeing method is set

Icon

1-22

Page

1-2-5 Selecting the Filters

The excitation dichroic mirror, beam splitter and barrier filters are set automatically to the light path according to the dyeing method

selected for the specimen.

To change filters, see section 1-3-2-4, “Configuring the Filters” in this volume and follow instructions in the [Optical System

Configuration] window.

The following table shows the possible combinations of the barrier and excitation filters.

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

Laser Combination

Multiline Ar. HeNe(G),
HeNe(R)
458/488/515, 543, 633
(3 channels)

Multiline Ar. HeNe(G),
HeNe(R)
458/488/515, 543, 633
(3 channels)
SPD system

Multiline Ar,
Kr, HeNe(R)
458/488/515, 568, 633
(3 channels)

Multiline Ar,
Kr, HeNe(R)
458/488/515, 568, 633
(3 channels)
SPD system

Excitation

Dichroic Mirror

(1) BS20/80
(2) DM405/488
(3) DM488/543/633
(4) DM458/515
(5) ­(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/543/633
(4) DM458/515
(5) ­(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/568/633
(4) DM458/515
(5) ­(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/568/633
(4) DM458/515
(5) ­(6) -

Beam

Splitter 1

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

Beam

Splitter 2

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

Beam

Splitter 3

Barrier Filter 1 Barrier Filter 2

(1) BA505IF
(2) BA505-525
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA505-525
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA505-550
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA505-550
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA560IF
(2) BA560-620
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA560IF
(2) BA560-620
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA585IF
(2) BA585-615
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA585IF
(2) BA585-615
(3) BA535-565
(4) ­(5) ­(6) -

Barrier
Filter 3

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) BA560IF
(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) BA560IF
(3) ­(4) ­(5) ­(6) -

Barrier
Filter 4

OPERATION INSTRUCTIONS

Page

1-23

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

Laser Combination

LD440, Multiline Ar,
HeNe(G), HeNe(R)
440, 458/488/515,
543, 633
(3 channels)

LD440, Multiline Ar, Kr,
HeNe(R)
440, 458/488/515,
568,633
(3 channels)

LD405, Multiline Ar,
HeNe(G), HeNe(R)
405, 458/488/515, 543,
633
(3 channels)

LD405, Multiline Ar, Kr,
HeNe(R)
405, 458/488/515, 568,
633
(3 channels)

LD405, Multiline Ar,
HeNe(G), HeNe(R)
405, 458/488/515, 543,
633
(4 channels)

LD405, Multiline Ar, Kr,
HeNe(R)
405, 458/488/515, 568,
633
(4 channels)

Excitation

Dichroic Mirror

(1) BS20/80
(2) DM405/488
(3) DM488/543/633
(4) DM405-440/515
(5) DM458/515
(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/568/633
(4) DM405-440/515
(5) DM458/515
(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/543/633
(4) DM405-440/515
(5) DM405/488/543
(6) DM458/515

(1) BS20/80
(2) DM405/488
(3) DM488/568/633
(4) DM405-440/515
(5) DM458/515
(6) -

(1) BS20/80
(2) DM405/488
(3) DM488/543/633
(4) DM405-440/515
(5) DM405/488/543
(6) DM458/515

(1) BS20/80
(2) DM405/488
(3) DM488/568/633
(4) DM405-440/515
(5) DM458/515
(6) -

Beam

Splitter 1

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) SDM490
(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) SDM490
(6) -

(1) Mirror
(2) Glass
(3) SDM490
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM490
(4) SDM510
(5) ­(6) -

Beam

Splitter 2

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) SDM560
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) SDM560
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) ­(5) ­(6) -

Beam

Splitter 3

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

Barrier Filter 1 Barrier Filter 2

(1) BA465-495
(2) BA505-525
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA465-495
(2) BA505-550
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA465-495
(2) BA505-525
(3) BA430-470
(4) BA480-495
(5) ­(6) -

(1) BA465-495
(2) BA505-550
(3) BA430-470
(4) BA480-495
(5) ­(6) -

(1) BA465-495
(2) BA430-470
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA465-495
(2) BA430-470
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA560-620
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA585-615
(3) BA535-565
(4)

-

(5) -
(6) -

(1) BA505IF
(2) BA560-620
(3) BA535-565
(4) BA505-525
(5) ­(6) -

(1) BA505IF
(2) BA585-615
(3) BA535-565
(4) BA505-550
(5) ­(6) -

(1) BA505IF
(2) BA505-525
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA505-550
(3) BA535-565
(4) ­(5) ­(6) -

Barrier
Filter 3

(1) BA650IF
(2) BA560IF
(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) BA585IF

-

(3)
(4) -
(5) -
(6) -
(1) BA650IF

(2) BA560IF
(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) BA585IF
(3) ­(4) ­(5) ­(6) -

(1) BA560IF
(2) BA560-620
(3) ­(4) ­(5) ­(6) -

(1) BA585IF
(2) BA585-615
(3) ­(4) ­(5) ­(6) -

Barrier
Filter 4

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

I

OPERATION INSTRUCTIONS

1-24

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

Laser Combination

UV, Multiline Ar,
HeNe(G), HeNe(R)
351, 458/488/515, 543,
633
(3 channels)

UV, Multiline Ar, Kr,
HeNe(R)
351, 458/488/515, 568,
633
(3 channels)

UV, Multiline Ar,
HeNe(G), HeNe(R)
351, 458/488/515, 543,
633
(4 channels)

UV, Multiline Ar, Kr,
HeNe(R)
351, 458/488/515, 568,
633
(4 channels)

Excitation

Dichroic Mirror

(1) BS20/80
(2) DM351/488
(3) DM488/543/633
(4) DM351/543
(5) DM458/515
(6) -

(1) BS20/80
(2) DM351/488
(3) DM488/568/633
(4) DM351/568
(5) DM458/515
(6) -

(1) BS20/80
(2) DM351/488
(3) DM488/543/633
(4) DM351/543
(5) DM458/515
(6) -

(1) BS20/80
(2) DM351/488
(3) DM488/568/633
(4) DM351/568
(5) DM458/515
(6) -

Beam

Splitter 1

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) SDM490
(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) SDM510
(5) SDM490
(6) -

(1) Mirror
(2) Glass
(3) SDM490
(4) SDM510
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM490
(4) SDM510
(5) ­(6) -

Beam

Splitter 2

(1) Mirror
(2) Glass
(3) SDM640
(4) SDM560
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) SDM560
(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM560
(4) ­(5) ­(6) -

Beam

Splitter 3

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

(1) Mirror
(2) Glass
(3) SDM640
(4) ­(5) ­(6) -

- :Invaild

Barrier Filter 1 Barrier Filter 2

(1) BA380-470
(2) BA505-525
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA380-470
(2) BA505-550
(3) BA480-495
(4) ­(5) ­(6) -

(1) BA380-470
(2) BA480-495
(3) ­(4) ­(5) ­(6) -

(1) BA380-470
(2) BA480-495
(3) ­(4) ­(5) ­(6) -

(1) BA505IF
(2) BA560-620
(3) BA535-565
(4) BA505-525
(5) ­(6) -

(1) BA505IF
(2) BA585-615
(3) BA535-565
(4) BA505-550
(5) ­(6) -

(1) BA505IF
(2) BA505-525
(3) BA535-565
(4) ­(5) ­(6) -

(1) BA505IF
(2) BA505-550
(3) BA535-565
(4) ­(5) ­(6) -

Barrier
Filter 3

(1) BA650IF
(2) BA560IF
(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) BA585IF
(3) ­(4) ­(5) ­(6) -

(1) BA560IF
(2) BA560-620
(3) ­(4) ­(5) ­(6) -

(1) BA585IF
(2) BA585-615
(3) ­(4) ­(5) ­(6) -

Barrier
Filter 4

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

(1) BA650IF
(2) ­(3) ­(4) ­(5) ­(6) -

(1), (2), and (3) of the spectral filters are equipped as the factory configuration.

A single type of excitation filter can be equipped. Replace it if necessary.

Up to two types of barrier filter can be equipped per channel.

If the equipment of another filter set for laser configuration is required, please contact your local Olympus representative.

OPERATION INSTRUCTIONS

Page

1-25

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

f

1-2-6 Setting the ND Filters

When you use the laser combiner, you can set the laser intensity by setting ND filter on the

laser combiner.

Display the [Acquire] panel.

Set each laser intensity by sliding the scale bar in the [Laser Intensity] group box of the

[Lasers] sub-panel, in accordance with specimen’s brightness, fluorescence crosstalk and

photo-bleaching.

[Laser Intensity] group box

Set the laser intensity value
by the scale bar..
The number of the displayed
laser intensity sliders varies
depending on that o
channels setting for the
acquisition.

OPERATION INSTRUCTIONS

1-26

Page

Fig. 1-7 [Lasers] Sub-panel

While using the HeNe green laser, try out the laser power 50% by setting the [Intensity] scale

bar in the [Laser Intensity] group box .

For other lasers, try the laser power 5% .

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

r

y

r

One Point!

After the dyeing method has been set with the [Dyes] sub-panel, the ND filters can be

set using the [Ch] group box in the upper part of the [Acquire] panel.

In the upper part of the [Acquire] sub-panel, open the [Ch] group box for the ND to be

1.

changed.

2. Click the <More> button.

The field as shown below is displayed below the [Ch] group box.

Clicking this button allows fine
adjustment of the ND value.

Display of the optimum lase
and ND value which are set
automatically according to the
selected dyeing method.

Clicking this field allows the
ND value to be changed on a
large scale.
The ND values which are
usually used are displayed in
green.

3. Vary the ND value using the Laser LED slider.

Each click of the laser ND <+> or <-> varies the laser ND value by 1%.

Each click of the laser ND slider varies the laser ND value by 5%.

1-2-7 Setting the Observation Condition

1-2-7-1 Setting the Objective Magnification

From the drop-down list on the center of the [Acquire] panel, select the objective being used

with the microscope.

TIP

If you change the objective, click the <Apply> button in the [Dyes] sub panel.

The Conforcal Aperture value is set appropriately.

Display of the ND value set by
clicking the <+> and <-> buttons o
the field. The set ND value can
also be changed by entering its
value directl

from the keyboard.

For detailes, see section 1-3-2-4 “Configuring the Filters” for automatic

Confocal Aperture setting with switching the objective.

OPERATION INSTRUCTIONS

Page

1-27

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-7-2 Setting the Zoom Ratio to 1X

Use the [Zoom] scale in the [Acquire] panel to set the zoom ratio to “X1”.

Using the UV-Ar laser, set the zoom ratio to “X2”.

1-2-7-3 Setting the Channels

1. In the [Ch] group box, make sure that the check boxes showing the applicable dyeing

methods are check-marked to indicate that the channels are ready for image

acquisition.

If the check box of a channel is not check-marked, check it to make the channel ready.

Channel check boxes, with which
dyeing methods are displayed

TIP

To display the information on all channels simultaneously, right-click the

boundary between channel display boxes.

Click the boundary again to return to the original display.

OPERATION INSTRUCTIONS

1-28

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

A

A

f

1-2-7-4 Setting the Highest Scan Speed

1. Set the scan speed to the fastest speed by using the scale in the [Scan Speed] group

box in the [Acquire] panel

[Scan Speed] group box

Set the scan speed by clicking a point on
the scale line.

TIP

The focus mode makes it possible to increase the line skipped scan speed.

From the page tabs on the bottom right of the [Acquire] panel, select the [Scan]

sub-panel.

Select either option button in the [Focus Mode] group box.

[Focus Mode] group box
[X2] option button

cquires image at twice the

highest speed.

[X4] option button

cquires image at 4 times the
highest speed.
Increasing the number o
divided images in the image
window, line skipped scan at 4
times (Focus) cannot be done.

The focus mode is enabled when acquiring images using the <Focus> button.

NOTE

The Focus function reduces the scanning time by line skipped scan. As a

result, the acquired images become coarse.

OPERATION INSTRUCTIONS

Page

1-29

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-7-5 Setting the XY Observation Mode

1. In the [Mode] group box in the [Acquire] panel, select the [Surface] option button.

2. In the [Mode] group box in the [Acquire] panel, select [800 by 600] from the [Scan Size]

drop-down list.

3. In the [Acquire] panel, select the XY observation mode option button.

1-2-7-6 Repeated Scanning Operation

1. Select the <XY Repeat> button. The acquired image will be displayed in the [Live]

panel.

<XY Repeat> button

TIP

Use the <FOCUS> button to acquire image at an even higher speed. If the

specimen is already being scanned, stop scanning with the <STOP

<Focus> button

NOTE

NOTE

SCAN> button before selecting the <XY Repeat> button.

The Focus function reduces the scanning time by line skipped scan.

As a result, the acquired images become coarse.

Do not move FLOUVIEW FV1000 Menu while acquiring an image.

OPERATION INSTRUCTIONS

1-30

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-7-7 Setting the Cross-section to be Observed

While acquiring image, move the Z stage to select the cross-section to be observed.

1. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the

[Z Stage] sub-panel.

<Z stage fine adjustment> buttons

Displace the Z stage by 0.1 µm per
step.

[Current Pos] text box

Shows the current position of the stage.
The value can also be entered directly
from the keyboard.

<Z stage coarse adjustment>
buttons

Displace the Z stage by 1.0 µm
per step.

[Locked] check box

Z-motor is engaged by checking this
box.

Fig. 1-8 [Z Stage] Sub-panel

2. Check the [Locked] check box in the [Z Stage] sub-panel.

Do not turn the fine focus adjustment knob while the [Locked] check box

is checked, for this may damage the Z motor.

3. While observing the image in the [Live] panel, locate the plane to be observed by

displacing the stage using the <Z stage coarse adjustment> and <Z stage fine

adjustment> buttons in the [Z Stage] sub-panel.

OPERATION INSTRUCTIONS

Page

1-31

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

r

f

1-2-7-8 Setting the Area to be Observed

When the observation targets are concentrated in a narrow area or when observation of a

specific area detail is required, the image of a limited area can be selected.

The 4 buttons represent directions, and
clicking a button moves the acquired image
area in the direction indicated by the button.
Clicking the square button on the cente
returns the acquired image area to the center.

Click a point inside the circle to change the
position of the acquired image area directly.

1-2-7-9 Setting a Lower Scan Speed

The scan speed can be decreased using the scale in the [Scan Speed] group box on the

[Acquire] panel.

TIP

In general, setting a lower scan speed allows the acquired image quality to be

improved.

However, a low scan speed also lengthens the time required for image

acquisition.

Clicking a point in the scale area
to change the value on a large
scale.
Clicking the top or bottom arrow
button allows fine adjustment o
the value.
Dragging the square knob allows
the value to be changed directly.

Fig. 1-9 [Pan]/[Zoom] Group Box

[Scan Speed] group box

Set the scan speed by clicking a
point on the scale line.

OPERATION INSTRUCTIONS

1-32

Page

NOTE

When the scan speed is decreased during fluorescence observation, the

saturation of fluorescence may darken the image of certain types of

specimens. In this case, increase the scan speed and increase the PMT

Voltage or use accumulation in scanning.

1-2-7-10 Stopping Repeated Scanning

After the brightness and gain have been adjusted, select the <STOP SCAN> button in the

[Acquire] panel to stop scanning temporarily.

1-2-8 Acquiring Image

Select the <Once> button. The acquired image will be displayed in the [Live] panel.

<Once> button

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

Fig. 1-10 Image Acquired in the [Live] Panel

OPERATION INSTRUCTIONS

Page

1-33

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

1-2-9 Saving Image

1. Display the [File I/O] panel.

<Display channel switch>

buttons

2. When saving images acquired with more than one channel, it is possible to select

whether images from more than one image are saved simultaneously or only one of the

images is saved.

Use the <Display channel switch> buttons to select the images to be saved. The selected

images will be saved under the conditions set for each channel.

Example)When only the image of Channel 1 is displayed, only the image of Channel 1

will be saved.

OPERATION INSTRUCTIONS

1-34

Page

Getting Started FLUOVIEW/Outline of LSM Observation Procedures

3. Click <Experiment> button in the [Save] group box. The [Save Experiment As] dialog

box will open.

Fig. 1-11 [Save Experiment As] Dialog Box

4. Enter the file name in the [File name:] text box.

5. Select “FLUOVIEW MultiTif” from [Save as type:]

6. Click the <Save> button.

1-2-10 Exiting from the Software, Turning Power Off

Exit from the software, then set the power switch of each unit to off.

For details, see section 1-3 “Exiting from the Software” and section 1-4 “Turning the Power

Off” in Volume II PREPARATION For OPERATION of [HARDWARE GUIDE] of the FV1000

User’s Manual.

OPERATION INSTRUCTIONS

Page

1-35

Getting Started FLUOVIEW/Online Help

1-3 Online Help

The FLUOVIEW application comes with two kinds of online help facility:

• function help for referencing the function and operation procedure description while

controlling the application, and

• microscope help providing information on the system setup.

1-3-1 Function Help

This section describes a simple method for displaying and consulting the online help on the

functions and operation procedures.

The figure below shows the initial display (table of contents) of the FLUOVIEW Online Help

window. To display this window, click the <Help> button in the toolbar at the bottom left of

the screen.

<Help> button

Finger pointer

Enhanced display

Some words in the displayed information are shown in enhanced display (underscored or

colored green). Clicking one of these words allows you to “jump” to the meaning of the word

or to more information about its meaning.

OPERATION INSTRUCTIONS

1-36

Page

Fig. 1-13 Initial Window

Getting Started FLUOVIEW/Online Help

A

1-3-2 Microscope Help

The microscope, scan unit and laser types can be set up from the FLUOVIEW software, by

selecting the dyeing method and following the displayed guidance information.

• Selecting the Dyeing Method

Place the pointer on the icon
displayed in the [Selected
Dyes], and the dyeing method
is shown in the pop-up display.

TIP

When the mouse pointer is placed on a word in enhanced display, the mouse

pointer turns into a “finger pointer”.

One Point!

Click the <Contents> button to the initial display. Click the <Back> button to

return to the previous information page.

1. From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes] sub-

panel.

[Assign dyes manually] check box

Checking this enables the manual
setting. Dragging the dyeing method
directly to the [Ch] group box assigns
the dyes to the desired channel.

[Available Dyes] list box

Lists the available dyes. Select the
desired items from this list and drag
them to the field above it to select the
dyeing method.

<Prev.> button

Sets the dyeing method which was
set last time by clicking the <Apply>
button.

2. Select the specimen dyeing method by dragging desired dye names in the [Available

Dyes] list box in the [Selected Dyes] group box to the field immediately above the list

box.

<Clear> button

Clear the set dyeing method.

<Apply> button

pplies the dyeing method
dragged in the [Selected Dyes]
group box to the [Ch] group in
the [Acquire] panel.

Fig. 1-14 [Dyes] Sub-panel

OPERATION INSTRUCTIONS

Page

1-37

Getting Started FLUOVIEW/Online Help

3. Click the <Apply> button to apply the selected dyeing method to the [Ch] group box

on the upper part of the [Acquire] panel.

TIP

When the dyeing method is selected from the [Available Dyes] list box and the

<Apply> button is clicked, a channel for acquiring fluorescence is set

automatically according to the changed filter. And the dyeing method is shown

in the [Ch] group box.

The Confocal Aperture value is also set automatically according to the

wavelength and the objective per channels.

OPERATION INSTRUCTIONS

1-38

Page

Getting Started FLUOVIEW/Online Help

One Point!

The [Assign dyes manually] check box can also be used to set the dyeing method to the

desired channel.

1. Check the [Assign dyes manually] check box in the [Dyes] sub-panel.

2. Select the dyeing method in the [Available Dyes] list box and drag it directly to the
field of the [Ch] check box.

[Assign dyes manually]
check box

3. After dragging, the icon appears on the right of the [Ch] check box and the dyeing
method is set.

The dyeing method is set

Dragging the icon to the out of the [Ch] check box field cancels the setting of the dyeing

method.

Icon

OPERATION INSTRUCTIONS

Page

1-39

Getting Started FLUOVIEW/Online Help

1-3-2-1 Configuring the Microscope

When the combination with BX61 or IX81 is in use, the microscope and scan unit can be

configured on the FLUOVIEW software.

Use the following procedure to configure the microscope.

1. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the

[Settings] sub-panel.

<Scope> button

Sets the system.

Fig. 1-15 [Settings] Sub-panel

OPERATION INSTRUCTIONS

1-40

Page

2. Select the <Scope> button on the bottom of the [Settings] sub-panel. The [Microscope

r

r

r

p

r

r

Control] window as shown below appears.

[Light Path] group box

Selects the light path. <BI> is fo
direct observation, <LSM> is fo
LSM observation and <TV> is fo
TV observation.

[Filter Turret] group box

Clicking the filter for visual
observation to be set switches the
turret automatically.

[Shutter] group box

Clicking inside the box switches the
EPI shutter to be closed/opened,

The shutter is opened.

The shutter is closed.

Getting Started FLUOVIEW/Online Help

[EPI lamp]

Indicates the EPI lamp.

[Filter Turret] group
box

Clicking the filter fo
reflected light
observation to be set
switches the turret
automatically.

[Mirror Unit] group box

Clicking the cube automatically
switches the turret.

[Nosepiece] group box

Click to change the objective.

[Top Lens] group box

Clicking inside the box switches the
top lens to be engaged into the light
path.

The top

lens is engaged into the light oath.

The top

lens is disengaged from the light

ath.

[Condenser Turret] group box

Clicking the universal condense
automatically switches the turret.

[Aperture Iris] group box

Changes the AS value.

<Link Setting> button

Selects the function to change settings corresponding to
the change of BX settings.

<Focus Setting> button

Enables parfocal corrections and adjustments of jogging
sensitivity for the objectives.

[Options] group box

Used for optional BX settings.

(Combination with BX)

[Lamp] group box

Clicking inside the box switches the
TD lamp ON/OFF.

The TD lamp is set to OFF.

The TD lamp is set to ON.

OPERATION INSTRUCTIONS

Page

1-41

Getting Started FLUOVIEW/Online Help

r

[Lamp] group box

Clicking inside the box switches the TD lamp ON/OFF.

The TD lamp is set to OFF.

The TD lamp is set to ON.

[Options] group box

Used for optional IX settings.

<Link Setting> button

Selects the function to change settings
corresponding to the change of IX
settings.

<Focus Setting> button

Enables parfocal corrections and
adjustments of jogging sensitivity for the
objectives.

[Light Path] group box

Selects the light path. <BI> is for direct
observation, <LSM> is for LSM
observation.

[Condenser Turret] group box

Clicking the universal condense
automatically switches the turret.

[Nosepiece] group box

Click to change the objective.

[Mirror Unit] group box

Clicking the cube automatically switches
the turret.

[Shutter] group box

Clicking inside the box switches the
EPI shutter to be closed/opened,

The shutter is opened.

[EPI lamp]

Indicates the EPI lamp.

OPERATION INSTRUCTIONS

1-42

Page

The shutter is closed.

(Combination with IX)

z Clicking the <Link Setting> button displays the [Link Setting] dialog box as shown

[

below.

Checking here links the objective in the [Nosepiece] group box
with the condenser turret.

Checking here escapes the stage or revolving nosepiece when
the objective is selected and changed in the [Nosepiece] group

Checking here disengages the top lens from the light path
when the objective of ×4 or lower magnification is selected in
the [Nosepiece] group box. (BX only)

Checking here enables parfocality correction when the
objective is selected and changed in the [NosePiece] group
box.

When objective is selected in [Nosepiece] group box while this
box is checked, close shutter of incident lighting lamp first and
then, change objective lens. After change, open the shutter.

Checking here sets the TD lamp to OFF when the fluorescent
cube is selected in the [Mirror Unit].

Checking here disengages the top lens from the light path
when the fluorescent cube is selected in the [Mirror Unit]. (BX
only)

Checking here closes the shutter when the Mirror Units are
switched in the [Mirror Unit].

Checking here closes the FL shutter and engages Analyzer and
Polarizer into the light path when the TD lamp is set to ON in

Lamp] group box.

the

<Import Setting> button

Loads the microscope
settings already
registered to reflect it.

Getting Started FLUOVIEW/Online Help

<Export
Setting>button

Saves the current

Click the desired check box to be checked.

Select the <Close> button to close the dialog box.

The microscope settings are automatically saved and read out when the software is

started up next time.

OPERATION INSTRUCTIONS

Page

1-43

Getting Started FLUOVIEW/Online Help

Open the setting

Open the file to which the microscope settings were exported with the <Export

Setting> button.

The Settings configured by another user or configured for another combination can

be applied.

Selecting the <Import Setting> button of the [Link Setting] dialog box displays the

[Open] dialog box as shown below.

When the setting file which you want to read is not displayed in the list box, use the

[Look in:] drop-down list and select the drive or directory where the file is saved.

Select “BX Setting File (*.ini)” in the [Files of type: ] drop-down list.

In the list box, select the setting file which you want to read.

Select the <Open> button to close the dialog box.

OPERATION INSTRUCTIONS

1-44

Page

Getting Started FLUOVIEW/Online Help

Save the setting

Settings in the [Link Setting] dialog box can be applied to other users or other

combinations.

Selecting the <Export Setting> button of the [Link Setting] dialog box displays the

[Save As] dialog box as shown below.

To change the save destination drive or directory, use the [Save in: ] drop-down list.

Enter the setting file name into the [File name] text box.

Select the <Save> button to close the dialog box.

z The <Focus Setting> button enables parfocal corrections and adjustments of jogging

sensitivity for the objectives.

See section 1-3-2-2, “Parfocality Correction and Jog Sensitivity Adjustment”.

Select the <Exit> button to close the dialog box.

3. After completing the system setup, click the <Close> button to close the window.

OPERATION INSTRUCTIONS

Page

1-45

Getting Started FLUOVIEW/Online Help

f

1-3-2-2 Parfocality Correction and Jog Sensitivity Adjustment

When the system use the BX or IX microscope, the parfocality correction and jog sensitivity

can be set per objective.

1. Open the [Microscope Control Panel] window.

For the method of displaying this window, see steps 1 and 2 in section 1-3-2-1,

“Configuring the Microscope ” in this section.

2. Click the <Focus Setting> button in the [Microscope Control Panel] window.

The [Focus Setting] dialog box as shown below opens.

[Objectives] group box

Click the button for the objective
subjected to parfocality correction so
that the button looks depressed.

[Pfcl Setup Wizard] group box

The Wizard for parfocality correction.

<Next> button

Click to execute the Parfocality Setup
(correction) Wizard.

<Import> button

Click to import the previously saved
parfocality correction and jog
sensitivity values for each objective.

<Pfcl> button

Depress to activate the
parfocality correction. To
deactivate, depress this
button once again.

[Jog Fine] group box

Select the jog sensitivity o
each objective from the
drop-down list.

Display of the parfocality
correction value of each
objective.

<Exit> button

Click to close the [Focus
Setting] dialog box.

OPERATION INSTRUCTIONS

1-46

Page

3. When the [Focus Setting] dialog box is displayed, the objective mounted on the

microscope is selected.

To use the setup wizard to begin the parfocality correction from the value of the

objective with maximum magnification in descending order, click the <Next> button in

the [Pfcl Setup Wizard] group box.

Getting Started FLUOVIEW/Online Help

r

4. The following option button menu is displayed in the [Pfcl Setup Wizard] group box. If

you want to begin the parfocality correction value setting with the objective with the

maximum magnification, click the [Switch to...] option button then click the <Next>

button.

Click to set the objective with the
maximum magnification in the list as
the reference for correction of othe
objectives.

Click to set the objective being
selected in the [Objectives] group box
as the reference for correction.

OPERATION INSTRUCTIONS

Page

1-47

Getting Started FLUOVIEW/Online Help

5. The objective set as the specified in the [Objectives] group box is selected, and the

[Pfcl Setup Wizard] group box displays the message shown below. Bring the specimen

in focus by observing it visually or on the scanned image, and then click the <Next>

button.

OPERATION INSTRUCTIONS

1-48

Page

6. The [Pfcl Setup Wizard:] group box displays the option button menu shown below. If

r

you want to set the parfocality correction values of objectives with other power values,

simply click the <Next> button.

Click to proceed to the setting of the
objective with next higher power to the
current objective.

Getting Started FLUOVIEW/Online Help

Click to select the desired objective
from the [Objectives] group box and
set the parfocality correction value fo
it.

Click to save the currently set
parfocality correction and jog
sensitivity values, then close the
dialog box.
(This option button is displayed when
the setting is changed.)
(Click the <Next> button to save the
setting and close the dialog box.)

OPERATION INSTRUCTIONS

Page

1-49

Getting Started FLUOVIEW/Online Help

7. The [Pfcl Setup Wizard] group box displays the option button menu shown below.

Adjust the focal position and click the <Next> button.

Click to set the current focal position
as the parfocality correction value for
the current objective and proceed to
the setting of the next objective.

Click to set the parfocality correction
value of the current objective to “0.00”
and proceed to the setting of the next
objective.

Click to proceed to the parfocality
correction setting of the next objective
without changing the setting for the
current objective and jog sensitivity
values.

OPERATION INSTRUCTIONS

1-50

Page

Getting Started FLUOVIEW/Online Help

8. Set the parfocality correction values of objectives by repeating steps 6 and 7 for each.

The [Pfcl Setup Wizard] group box displays the option button menu shown below.

Click the <Pfcl> button to activate the parfocality correction.

[Jog Fine] group box

Select the jog sensitivity
of each objective from
the drop-down list.
The * mark represents
the value recommended.

Click to save the currently set
parfocality correction and jog
sensitivity values, then close the
dialog box.
(This option button is displayed when
the setting is changed.)
(Click the <Next> button to save the
setting and close the dialog box.)

9. Set the jog sensitivity values of the objectives.

Select the jog sensitivity of each objective from the [Jog Fine] drop-down list.

The drop-down list appears when clicking each value in the [Jog Fine].

10. After completing the setting, click the [Save each Perfocal value and Jog Setting, then

Exit] option button and then click the <Next> button.

The setting will be registered and the dialog box will close.

If you do not want to register the setting, simply click the <Exit> button to close the

dialog box.

OPERATION INSTRUCTIONS

Page

1-51

Getting Started FLUOVIEW/Online Help

r

r

r

1-3-2-3 Configuring the Filters (When using a filter system)

The barrier filters, excitation filter and beam splitter are set automatically to the light path

according to the dyeing method selected for the specimen.

Use the following procedure to change these filters.

1. Select the [Settings] sub-panel.

Fig. 1-16 [Settings] Sub-panel

<Scan Unit> button

Display the filter and lase
types to be set.
(Combination with the lase
combiner sets up the lase
automatically.)

OPERATION INSTRUCTIONS

1-52

Page

Getting Started FLUOVIEW/Online Help

f

r

r

2. Click the <Scan Unit> button at the bottom of the panel. The window as shown below will

appear.

[Laser Unit] group box

Shows the type of laser to be used. (With the laser combiner operation, the laser type is set and
displayed automatically.) When the <On> button is depressed, the laser is oscillating the beam.
When the <Stby> button is depressed, the laser is not oscillating. When the [Auto standby]
check box is checked, the [After] text box appears below it. When not using laser for a long time,
in order to suppress useless electric-power consumption, we recommend you making <Stby>
mode. The laser oscillation stops when the time shown in this box has elapsed after the end o
laser scanning. The laser oscillation stops when the time shown in this box has elapsed afte
the end of laser scanning. The laser is suspended as standby mode (using Ar or Kr-laser) or the
laser oscillation stops (using UV-Ar laser) When the time shown in this box has elapsed afte
the end of laser scanning.

[Excitation DM] and [Beam
splitter] drop-down lists

Change the excitation filter and
beam splitter of each channel.

Clicking this area causes the [Microscope
Configuration] window to appear.

[Fit C.A. for change of Objective Lens]
Check box

In case of check this box, Confocal Aperture
diameter is set with switching the objective.

[TD Unit] group box

Shows the transmitted light detection.

[Barrier Filter] drop-down list

Changes the barrier filter of each channel.

[Dyes]

Shows the dyeing method set for
each channel.

[XY Resolution]

Shows lateral resolution for each
channel,

[Z Resolution]

Shows the Z resolution set for each
channel.

TIP

Fig. 1-17 [Optical System Configuration] Window

Virtual channels can be used. For the virtual channels, see section 2-2-8-1,

“Virtual Channel”.

3. To change the displayed filter types, use the [Excitation DM], [Beam splitter] and/or

[Barrier Filter] drop-down lists.

4. After completing the fitler setup, click the <Close> button to close the window.

OPERATION INSTRUCTIONS

Page

1-53

Getting Started FLUOVIEW/Online Help

The formulas of resolution of X and Y are as follows;

FWHM : Resolution of X and Y

L : The length of a diagonal line of Square Pinhole

NA = nSINθ :NA of Objective lens

MG : The objective Magnification

N : The refractive index of a medium

Air : 1.0

Water : 1.3

Oil : 1.5

X = L/(1.22*3.8*λ*MG/NA)

One Point!

The case where the variable x is as follows; 0 <= x < 0.5

FWHM = (0.5λ*XYPSF)/NA

XYPSF = -5.1459x6 + 1.0147*10x5 - 7.1668x4 + 2.2804x3 - 1.7696*10-1x2 + 1.9256*10

7.1915*10

-1

-02

x +

The case where the variable x is as follows; 0.5 <= x < 1.0

FWHM = (0.5λ*XYPSF)/NA

XYPSF = -3.05539x6 + 1.32031*10x5 - 2.25074*10x4 + 1.89617*10x3 - 7.89685x2 + 1.56587x

+ 6.20850*10

-1

The case where the variable x is as follows; 1.0 <= x < 1.7

FWHM = (0.5λ*XYPSF)/NA

XYPSF = -2.61786x6 + 2.14478*10x5 - 7.18428*10x4 + 1.25767*102x3 - 1.21445*102x2 +

6.17335*10x - 1.21495*10

The case where the variable x is as follows; 1.7 <= x < 2.0

FWHM = (0.5λ*XYPSF)/NA

XYPSF = -0.0078x3 + 0.0422x2 - 0.0689x + 1.0478

OPERATION INSTRUCTIONS

1-54

Page

The case where the variable x is as follows; 2.0 <= x

FWHM = (0.5λ*XYPSF)/NA

XYPSF = 1.0164

Getting Started FLUOVIEW/Online Help

One Point!

The formula of resolution of Z is as follows;

FWHM : Resolution of Z

L : The length of a diagonal line of Square Pinhole

NA = nSINθ :NA of Objective lens

MG : The objective Magnification

N : The refractive index of a medium

Air : 1.0

Water : 1.3

Oil : 1.5

X = L/(1.22*3.8*λ*MG/NA)

The case where the variable x is as follows; 0 <= x < 2.0

FWHM = (2λ*ZPSF)/(4n(1-COSθ))

ZPSF = -5.5158*10-2x5 + 2.0877*10-1x4 - 2.1172*10-1x3 + 2.0864*10-1x2 - 2.2669*10-2x +

6.9038*10

The case where the variable x is as follows; 2.0 <= x

FWHM = (2λ*ZPSF)/(4n(1-COSθ))

ZPSF = 0.6164x+0.1283

-1

OPERATION INSTRUCTIONS

Page

1-55

Getting Started FLUOVIEW/Online Help

r

r

r

r

1-3-2-4 Configuring the filters (When using a spectral detecting system)

When the system incorporates the spectral detector unit that is composed of a 2-channel

spectral detector and 1-channel filter, it is possible to set the detection conditions more

flexibly, acquire the fluorescence spectral data and use the fluorescence isolation function.

As the upper and lower limit wavelengths can be set while acquiring images, any desired

wavelength regions can be selected easily.

To acquire image with a spectral detecting system, set the wavelength regions in the

[Spectral-Control] sub panel.

Shows the graph of fluorescence
wavelength (Em_**) for each
dyes. When area inside the graph
is double clicked, a window to
change graph display method will
appear.

Set the upper limit (left) and lowe
limit (right) wavelengths of the
barrier filter.

Move the upper limit and lowe
limit wavelength of the barrier filte
while maintaining the width
between the upper limit and lowe
limit wavelengths.

The settings are interlocked with those in the [Optical System Configuration] window.

Fig. 1-18 [Spectral-Control] sub panel and [Dyes] sub panel

OPERATION INSTRUCTIONS

Page

1-56

Getting Started FLUOVIEW/Online Help

r

r

r

The parameters of the spectral detecting system can also be set in the [Optical System

Configuration] window as well as in the [Spectral-Control] sub panel.

1. Select the [Settings] sub panel.

<Scan Unit> button

Display the filter and laser
types to be set.
(Combination with the laser
combiner sets up the laser
automatically.)

2. Click the <Scan Unit> button on the bottom of the [Settings] sub-panel. When the

window shown below is displayed, set up the spectral detecting system in the [SPD]

group box.

Set the upper limit (left)
and lower limit (right)
wavelengths of the barrie
filter.

Move the upper limit o
lower limit wavelength
while maintaining the
width between the uppe
limit and lower limit
wavelengths.

Fig. 1-19 [Settings] sub panel

Fig. 1-20 [Optical System Configuration] window

OPERATION INSTRUCTIONS

Page

1-57

Getting Started FLUOVIEW/Online Help

For other functions available in the [Optical System Configuration] window, see section 1-3-

2-3, “Configuring the Filters (When using a filter system),”

3. After completing the setup, click the <Close> button to close the window.

OPERATION INSTRUCTIONS

1-58

Page

1-3-2-5 Setting the C.A. Diameters

f

The pinhole diameters are set automatically according to the selected dyeing method.

Use the following procedure to change the pinhole diameters.

1. Display the [Acquire] panel.

[Ch] group box

Sets whether the image o
each channel is to be
acquired or not, the PMT
voltage, Gain and Offset.
Right-clicking the mouse
inside this group box
displays the [Ch] group
boxes of all channels.

Getting Started FLUOVIEW/Online Help

OPERATION INSTRUCTIONS

Page

1-59

Getting Started FLUOVIEW/Online Help

r

2. Right-click the mouse inside the [Ch] group box to display the [Ch] group boxes of all

channels.

[Ch1], [Ch2], [Ch3], [Ch4] and
[Ch5] group boxes

Select the channels from which
you want to acquire

[PMT], [Gain] and [Offset] LED
sliders

Set these values independently.
Increasing the PMT Voltage
improves the sensitivity.
Increasing the Gain brightens
the image.
Increasing the Offset darkens
the image.

images.

< > button

Click to display the pinhole
diameter and the laser ND filte
setting.

OPERATION INSTRUCTIONS

1-60

Page

3. Click the < > button of the channel you want to change the pinhole diameter.

The group boxes for setting the pinhole diameters and laser ND filter values of the

channels are displayed below the [Ch] group box. (They are not displayed for channels

set for transmitted light observation.)

[Ch1], [Ch2], [Ch3], [Ch4] and [Ch5]

group boxes

Select the channels from which you want
to acquire images.

[PMT], [Gain] and [Offset] LED sliders

Set these values independently.
Increasing the PMT Voltage improves
the sensitivity.
Increasing the Gain brightens the image.
Increasing the Offset darkens the image.

< > button

Click to close each group box showing the
pinhole diameter and laser ND filter value.
The pinhole diameter and laser ND filter
value are valid even before the group box is
closed by clicking the <

> button.

Getting Started FLUOVIEW/Online Help

[C.A.] and
Laser ND LED sliders

Set these values
independently.
The laser type used with
each channel is
displayed below its
Laser ND LED
slider.

OPERATION INSTRUCTIONS

Page

1-61

Getting Started FLUOVIEW/Online Help

4. While observing the images in the [Live] panel, change their pinhole diameter

settings.

Clicking this button allows
fine adjustment of the
value.

Clicking this field allows the
value to be changed on a
large scale.
The ND value which are
usually used are displayed in
green.

Clicking the <+> or <-> button
or the field displays the set
value. The set value can be
varied by direct input from the
keyboard.

TIP

TIP

Each click of the <+> or <-> button of the [C.A.] LED slider increases or

decreases the pinhole diameter by 5 nm.

Each click of the slider section of the [C.A.] LED slider varies the pinhole

diameter by 25 nm.

Each click of the <+> or <-> button of the Laser ND LED slider increases or

decreases the laser ND value by 1%.

Each click of the slider section of the Laser ND LED slider varies the laser ND

value by 5%.

To set to the Confocal Aperture value to its primal value, click the <Apply>

button in the [Dyes] sub-panel of the [Acquire] panel.

OPERATION INSTRUCTIONS

1-62

Page

APPLIED OPERATIONS/General Operation Procedure

2 APPLIED OPERATIONS

2-1 General Operation Procedure

This section describes the general image acquisition procedure with the aim to get

accustomed with the operation.

Begin using the FLUOVIEW system by acquiring an image or opening an image from a

file. For the detailed operation method of each item in the procedure, see the section

specified in parentheses (( )).

NOTE

The following is the general operation procedure of FLUOVIEW. Many

other functions that are not shown in the following are also available.

Please also study their description.

OPERATION INSTRUCTIONS

Page

2-1

APPLIED OPERATIONS/General Operation Procedure

Turn power ON and start the FLUOVIEW software.

(Sections 1-2-1 & 1-2-2)

Acquire an image.

(A)

(According to the selected

observation mode)

For detailed operation procedures for image

acquisition, see section 2-1-1, “Image

Acquisition Procedure (Section A)”.

Process the image.

Filtering (Section 2-6-1) Contrast conversion (Section 2-6-2)

Observe the image.

z Observation of image shape

Image display in simulated colors

(Section 2-5-1)
LUT change (Section 2-5-2)
Simultaneous display of multi-channel
images (Section 2-5-4)
Side-by-side image display (Section 2-5-7)
Magnified/reduced image display

(Section 2-5-9)
Stereo 3D image display (Section 2-9-3)
Color-eyeglass 3D image display

(Section 2-9-4)
Animation display (Section 2-9-2)
Continuous display (Frame-by-frame
display) (Section 2-9-1)

Open an image in a file.

(Section 2-3-2)

Inter-image operation (Section 2-6-3)

z Observation of image change

over time

Side-by-side image display

(Section 2-10-1)
Continuous display (Frame-by-frame
display) (Section 2-10-2)

z Measurement of image shape

Length measurement(Section 2-7-3-1)
Area measurement (Section 2-7-3-2)

Exit from the FLUOVIEW software and turn power OFF.

OPERATION INSTRUCTIONS

2-2

Page

Measure the image.

z Measurement of image intensity

value and distribution

Intensity on a line (Line profile)

(Section 2-7-1-1)

Intensity on a plane (Bird’s eye view)

(Section 2-7-1-2)

Intensity distribution (Section 2-7-2)

Save the image.

(Section 2-3-1)

Compile the presentation data.

Drawing characters on image (Section 2-12-1)
Drawing pictures on image (Section 2-12-2)
Drawing scales on image (Section 2-12-3)

Output the image at the printer.

(Section 2-13)

(Sections 1-2-10)

APPLIED OPERATIONS/General Operation Procedure

(A)

(

)

g

2-1-1 Image Acquisition Procedure (Section (A))

This section describes the procedure for acquiring images. See sections 2-2 and after

for the actual operation methods. The detailed operation methods of each item in the

procedure are described in the section specified in parentheses (( )).

After “Turn power ON and start the FLUOVIEW software”,...

Configure the system and confirm the images to be observed.

The system configuration and image confirmation are described in details in section
1-2, “Outline of LSM Observation Procedures” in the [OPERATION] Volume. Refer
to this section as required.

(B)

XY observation
(Section 2-2-1)

Acquire an image in an observation mode.

(FLUOVIEW provides the following observation modes.)

TIME

XZ observation

(Section 2-2-2-1)

XT observation

(Section 2-2-2-2)

XZT observation
(Section 2-2-2-3)

When saving the

e in a file:

ima

Save the acquired data in a file.

Section 2-3-1

Go to “Process the image”.

TIME

XYZ observation
(Section 2-2-2-4)

TIME

XYT observation
(Section 2-2-2-5)

With this system, the observation
by XY scanning is called simply as
“XY observation”.

TIME

XYZT observation
(Section 2-2-2-6)

OPERATION INSTRUCTIONS

Page

2-3

APPLIED OPERATIONS/General Operation Procedure

2-1-2 Image Acquisition Procedure in an Observation Mode (Section (B))

As an example of “Acquire an image in an observation mode”, this section describes the

procedure in the XY observation mode. For the procedures in other observation modes,

see section 2-2-2, “Image Acquisition in Other Observation Modes” as well as the

following procedure.

After “Configure the system and confirm the images

to be observed”...

XY observation

(Section 2-2-1)

(B)

When noise is noticeable:

Perform accumulation.

(Section 2-2-1-6)

Set the acquisition parameters.

(Section 2-2-1-4)

z

Observation mode

z

Scanning speed

Image brightness

z

z

Multiple-section to be

observed

z

Area to be observed

Acquire an image in the XY observation mode.

(Section 2-2-1-5)

OPERATION INSTRUCTIONS

2-4

Page

Go to “Save the acquired data in a file.” or “Process the image”.

2-1-3 Examples of Operation Procedures

Begin using the FLUOVIEW system by acquiring an image or opening an image in a file.

For the detailed operation method of each item in the procedure, see the section

specified in parentheses (( )).

APPLIED OPERATIONS/General Operation Procedure

Example 1) To perform XYZ observation of a cell,

apply filter processing to the image,

display animation to identify the cell shape,

then calculate the area, and save the

obtained image data:

Turn power ON and start the

FLUOVIEW software.

(Sections 1-2-1)

Acquire an image.

(Section 2-2)

Apply filter processing.

(Section 2-6-1)

Display the animation.

(Section 2-9-2)

Measure area

(Section 2-7-3-2)

Example 2) To open a previously acquired image in a

file, measure the intensity distribution and

edit data with Excel:

Turn power ON and start the

FLUOVIEW software.

(Sections 1-2-1)

Open an image in a file.

(Section 2-3-2)

Specify the area to be measured

When
there is
another

Measure the intensity distribution.

(Section 2-7-2)

area to be
measured

Save the measurement data in

the Excel format.

(Section 2-11-1)

Save the image.

(Section 2-3-1)

Exit from the FLUOVIEW software

and turn power OFF.

(Sections 1-2-10)

Read the saved measurement data

with Excel and edit it.

Exit from the FLUOVIEW software

and turn power OFF.

(Sections 1-2-10)

OPERATION INSTRUCTIONS

Page

2-5

APPLIED OPERATIONS/General Operation Procedure

Example 3) To acquire an image and compared it with a

previously acquired image:

Turn power ON and start the

FLUOVIEW software.

(Sections 1-2-1)

Open an image in a file.

(Section 2-3-2)

Acquire an image.

Section

(

2-2)

Display the opened and acquired

images side by side and compare

them.

(Section 2-5-6)

Example 4) To open an image in a file, improve its

contrast and create a presentation image by

entering comment, etc.

Turn power ON and start the

FLUOVIEW software.

(Sections 1-2-1)

Open an image in a file.

(Section 2-3-2)

Convert the contrast.

(Section 2-6-2)

Draw comment text on the image.

(Section 2-12)

Exit from the FLUOVIEW software

and turn power OFF.

(Sections 1-2-10)

Save the image.

(Section 2-3-1)

Output the image at the printer.

(Section 2-13)

Exit from the FLUOVIEW software

and turn power OFF.

(Sections 1-2-10)

OPERATION INSTRUCTIONS

2–6

Page

2-2 Image Acquisition

Confirm the image to be acquired using the microscope, and acquire its image using

FLUOVIEW. The image can be saved in a file as required.

APPLIED OPERATIONS/Image Acquisition

NOTE

If FLUOVIEW window is moved while an image is being acquired, it may

cause image acquisition failure.

If at all possible, refrain from moving.

OPERATION INSTRUCTIONS

Page

2-7

APPLIED OPERATIONS/Image Acquisition

2-2-1 Image Acquisition in XY Observation Mode

This section describes the basic operation procedure from the system configuration to the

image acquisition in the XY observation mode and image saving in a file as shown in the

following chart. The details of each operation will be described in the subsequent sections.

Set the dyeing method.

(Section 2-2-1-1 in [OPERATION])

Configure the microscope and scan

(Sections 2-2-1-1 & 2-2-1-2 in [OPERATION])

unit.

Set the objective magnification.

(Section 2-2-1-4-1 in [OPERATION])

Set the zoom ratio to 1X.

(Section 2-2-1-4-2 in [OPERATION])

Set the channel to be acquired.

(Section 2-2-1-4-3 in [OPERATION])

Set the highest scan speed.

(Section 2-2-1-4-4 in [OPERATION])

Set the XY observation mode.

(Section 2-2-1-4-5 in [OPERATION])

Perform repeated scanning.

(Section 2-2-1-4-6 in [OPERATION])

Set a lower scan speed.

(Section 2-2-1-4-10 in [OPERATION])

If the image
becomes
clean

If the image
does not
become
clean

Re-adjust the image

brightness.

(Section 2-2-1-4-9 in [OPERATION])

Stop repeated scanning.

(Section 2-2-1-4-11 in [OPERATION])

Acquire the image.

(Section 2-2-1-5 in [OPERATION])

Adjust the Z-position to observe the

desired multiple sections.

(Section 2-2-1-4-7 in [OPERATION])

Set the observation range.

(Section 2-2-1-4-8 in [OPERATION])

Adjust the image brightness.

(Section 2-2-1-4-9 in [OPERATION])

OPERATION INSTRUCTIONS

2-8

Page

Save the image.

(Section 2-3-1 in [OPERATION])

2-2-1-1 Configuring the Microscope

A

y

Set the light path so that the image can be observed through the microscope.

1. Display the [Acquire] panel.

<Once> button

cquires an image in the currentl
selected observation mode and display
the image in the [Live] panel.

<XY Repeat> button

Repeats XY scanning to display images
successively in the [Live] panel.

[Ch1], [Ch2] and [Ch3] check boxes

Check to select the image acquisition
channel.

[PMT], [Gain] and [Offset] LED
sliders

These can be adjusted independently.

Increasing the PMT voltage improves
the sensitivity.

Increasing the Gain brightens the
image.

Increasing the Offset darkens the
image.

Option buttons

Check a button to select one of the
displayed observation modes.

[Scan Speed] group box

Sets the scan speed. Sliding
left of the scale increases the scan

speed and to the right decreases it.

to the

APPLIED OPERATIONS/Image Acquisition

<Focus> button

The repetition images can be acquired at a high speed.

Use this button to find an optimum image.

The acquired image is displayed.

<Load> button

Loads the observation conditions
at image acquisition.

<Save> button

Saves the observation conditions
at image acquisition.

[Scan Size] drop down list

Select the image acquisition size.

Select the magnification of the
objective on the microscope.

[Settings], [Z Stage], [Time Series],
[Dyes] and [Lasers] sub-panels

These panels are used to set the
information required for image
acquisition.

Fig. 2-1 [Acquire] Panel

OPERATION INSTRUCTIONS

Page

2-9

APPLIED OPERATIONS/Image Acquisition

A

2. Setting the dyeing method

1) From the page tabs on the bottom right of the [Acquire] panel, select the [Dyes]

sub-panel.

Place the pointer on the icon
displayed in the [Selected
Dyes], and the dyeing method
is shown in the pop-up display.

[Available Dyes] list box

Lists the available dyes. Select
the desired items from this list
and drag them to the field above
it to select the dyeing method.

[Assign dyes manually]
check box

Checking this enables the
manual setting. Dragging
the dyeing method in the
list directly to the [Ch]
group box assigns the dye
to the desired channel.

<Clear> button

Clear the set dyeing method.

<Prev.> button

Sets the dyeing method which
was set last time by clicking the
<Apply> button.

2) Select the specimen dyeing method by dragging desired dye names in the

[Available Dyes] list box in the [Selected Dyes] group box to the field immediately

above the list box.

<Apply> button

pplies the dyeing method
dragged in the [Selected
Dyes] group box to the
[Ch] group in the [Acquire]
panel.

Fig. 2-2 [Dyes] Sub-panel

Fig. 2-3 [Dyes] Sub-panel

OPERATION INSTRUCTIONS

2-10

Page

APPLIED OPERATIONS/Image Acquisition

3) Click the <Apply> button to apply the selected dyeing method to the [Ch] group box

on the upper part of the [Acquire] panel.

TIP

TIP

When the dyeing method is selected from the [Available Dyes] list box and the

<Apply> button is clicked, a channel for acquiring fluorescence is set

automatically according to the changed filter. And the dyeing method is shown

in the [Ch] group box.

The Confocal Aperture value is also set automatically according to the

wavelength and the objective per channels.

If you switch the objective, click the <Apply> button in the [Dyes] sub panel. The

Confocal Aperture value is set appropriately.

For detailes, see section 1-3-2-6 “Setting the Filters” for automatic Confocal

Aperture setting with switching the objective.

OPERATION INSTRUCTIONS

Page

2-11

APPLIED OPERATIONS/Image Acquisition

The [Assign dyes manually] check box can also be used to set the dyeing method to the

desired channel.

1. Check the [Assign dyes manually] check box in the [Dyes] sub-panel.

2. Select the dyeing method in the [Available Dyes] list box and drag it directly to the
field of the [Ch] check box.

One Point!

3. After dragging, the icon appears on the right of the [Ch] check box and the dyeing

Dragging the icon to the out of the [Ch] check box field cancels the setting of the dyeing

method.

OPERATION INSTRUCTIONS

[Assign dyes manually]
check box

method is set.

The dyeing method is set

Icon

2-12

Page

APPLIED OPERATIONS/Image Acquisition

3. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the

[Optics] sub-panel.

<Scope> button

Displays useful information for the
system setup.

Fig. 2-4 [Optics] Sub-panel

OPERATION INSTRUCTIONS

Page

2-13

APPLIED OPERATIONS/Image Acquisition

4. Select the <Scope > button at the bottom of the panel. The window as shown below will

appear (in case of a combination with the IX81).

OPERATION INSTRUCTIONS

2-14

Page

Fig. 2-5 [Microscope Control Panel] Window

5. Configure the microscope in [Microscope Control Panel] window.

6. After completing the setup, click the <Close> button to close the window.

TIP

7. While looking into the microscope, move the stage and check the observed image.

For details of [Microscope Control Panel] window, see section 1-3-2-1

“Configuring the Microscope” in this volume.

2-2-1-2 Setting the Filters

r

r

The excitation filter, spectral filter and barrier filters are set automatically to the light path

according to the dyeing method selected for the specimen.

To change filters, use the following procedure.

1. From the panel page tabs shown on the bottom right of the [Acquire] panel, select the

[Settings] sub-panel.

APPLIED OPERATIONS/Image Acquisition

Fig. 2-6 [Settings] Sub-panel

<Scan Unit> button

Click to display the filter and
laser types to be set.

(Combination with the lase
combiner sets up the lase
automatically.)

OPERATION INSTRUCTIONS

Page

2-15

APPLIED OPERATIONS/Image Acquisition

A

2. Click the <Scan Unit> button at the bottom of the panel. The window as shown below

will appear.

[Laser Unit] group box

Shows the type of laser to be used. (With the laser combiner operation, the laser type is set
and displayed automatically.) When the <On> button is pressed-in, the laser is oscillating the
beam. When the <Stby> button is pressed-in, the laser is not oscillating. When the [Auto
standby] check box is checked, the [After] text box appears below it. When not using laser for
a long time, in order to suppress useless electric-power consumption, we recommend you
making <Stby> mode. The laser oscillation stops when the time shown in this box has
elapsed after the end of laser scanning. The laser oscillation stops when the time shown in
this box has elapsed after the end of laser scanning. The laser is suspended as standby
mode (using Ar or Kr-laser) or the laser oscillation stops(using UV-Ar laser) When the time
shown in this box has elapsed after the end of laser scanning.

[Excitation DM] and [Beam splitter] drop-down
lists

Change the excitation filter and beam splitter of
each channel.

[Fit C.A. for change of Objective Lens]
Check box

In case of check this box, Confocal

perture diameter is set with switching the

objective.

[TD Unit] group box

Shows the transmitted light detection.

[Barrier Filter] drop-down list

Changes the barrier filter of each channel.

[Dyes]

Shows the dyeing method set for each channel.

[XY Resolution]

Shows lateral resolution for each channel.

[Z Resolution]

Shows the Z resolution set for each channel.

3. To change the displayed filter types, use the [Excitation DM], [Beam splitter] and/or

[Barrier Filter] drop-down lists.

Fig. 2-7 [Optical System Configuration] Window

4. After completing the filter setup, click the <Close> button.

OPERATION INSTRUCTIONS

2-16

Page