Zeiss 880 Training Notes

Zeiss 880

Training Notes

Zen 2.3

Turn on

Main Power

Switch

Do you need the Argon

Laser

458, 488, or 514 nm lines?

Yes

Turn on the Systems PC

Switch

Turn on the HXP 120V Lamp

Turn on the Components

Switch.

Turn on the PC and log into

your account.

Start Zeiss Zen via Elan

Tracking Software.

No

Create

scanning

configuration

880 Start-Up Sequence

Set up sample on microscope.

Go to the Acquisition Tab and

initiate the warm up

procedure for the laser.

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Logging into the 780

• Log into the computer with your UCHC or

CAM credentials; be sure you are logging
into the proper domain.

• Double click on the Zen icon, the one

labeled with “AUT-Zeiss LSM 880” or
similar text.

The idea elan tracking and billing software will
initialize after clicking on the Zen icon.

• Enter your UCHC email. Once it has been

recognized, it will bypass the password
requirement and proceed to the session
confirmation window.

Starting Zen with idea elan

• Select your scheduled appointment

and/or click the login button as directed
by the software.

• Confirm your appointment and the Zen

software will be initialized.

Starting Zen 2.3

Select Start System from the Zen 2012 login
window. This will start up the hardware and
software.

Image Processing is for using the system in an
offline mode and this will ignore all the
hardware components.

Introducing the Zen Workspace

The Zen workspace is designed in a

tabular fashion allowing you to
step you through initially looking
at your specimen via the Oculars
tab, in the control panel, to
acquiring images via the Acquire
tab. The remaining tabs add off­line Processing and
Maintenance options.

Panels can be moved around
to customize the workspace.

Introducing the Zen workspace

Your imaging configuration and your

work space configuration can be
stored separately.

In addition, your imaging configuration

and acquisition parameters can easily be

retrieved from the Reuse function when
you have a previously saved image loaded
in the workspace.

Getting Light to the Oculars

Locate Tab

1. Click on the objective icon to access the list
of possible objectives. Be sure to note the
immersion type; air, oil or water. Please do
not change from oil to air/dry or from
water to oil and oil to water without
cleaning your specimen first.

2. Select your light source from:

Transmitted ON
Transmitted OFF
DAPI
FITC
Rhodamine

3. Be sure to turn the Reflected Light “Off”
when you are finished looking at your
specimen.

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Getting all the tools

Acquisition Tab

In order to view all the options available via the software,
be sure to go to:

1. View>Show All (global)

2. Select >Show all Tools.

Make sure to do this each time you use the software
otherwise the required features, like the lasers, will not
be accessible to you.

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Open/close the
submenu with the arrow.

Click the arrow to detach/attach panel.

Acquisition Tab

1. Experiment Manager

The manager allows you to save your scan configuration
settings for your experiment and reload the settings for
a quick start. This method does not record the
acquisition parameters.

2. Scan Control Tool Bar

• AF (Find Focus) – computer determines focus based

on contrast settings.

• Set Exposure – the computer automatically

calculates the gain and black level settings.

• Live – initiates a fast scan regardless of the scan

speed and continuously scans until Stop is selected.

• Continuous – continuously scans according to the

selected scan speed until Stop is selected.

• Snap – performs a single scan according to the

selected scan speed.

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Using Smart Setup

Smart Setup

The best and easiest way to get started is to use

the Smart Setup tool.

1. Click on the down arrow to open the dye
selection menu.

2. Select your dye from the drop down menu.
Repeat this procedure to select additional
dyes.

Depending on your imaging needs, the Smart
Setup will offer you up to four different
configurations for imaging your sample:

• Fastest

• Best Signal

• Best Compromise

• Linear Unmixing.

Whatever configurations you are presented with,
you can always manually modify the configuration
in the Light Path panel.

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Smart Setup - Fastest

Acquires the image with the simultaneous

excitation and detection of one or
multiple probes. This option produces a
single image, with a single scan however
it may result in bleed through between
the different channels.

Smart Setup – Best Signal

Acquires the image with the sequential

excitation and detection of the different
probes. Each probe is scanned separately
and the final image is the result of an
overlay of the individual channels. This
option avoids bleed through between the
different channels however multiple
scans are require to produce the image
which can lead to bleaching or other laser
induced damage.

Smart Setup – Smartest (Line)

Smartest acquires multiple channels at the

same time while quickly toggling laser
lines to acquire a second track.

There are limitations to this option, especially

if you are acquiring 4 different probes.

a) You must you use the same

dichroics between the two tracks.

b) You can’t send two probes, of

different intensities to the ChS
de te ct or.

Smart Setup – Linear Unmixing

This option creates a confirmation for
computationally subtracting bleed through
between channels. This can be performed
after you acquire individual, pure spectral
profiles of the probes you are using.

Using the Advanced Scanning Modules

Advanced scanning modules are available as a
single option or they can be used in combination
with multiple modules. The modules are initiated
via the Start Experiment button.

1. Time Series - collect a series of images based
on a specific time interval and duration.

2. Z-Stack – collect a series of optical sections
that can be reconstructed as a 3D image.

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Using the Advanced Scanning Modules

(cont.)

Bleaching – module for
FRAP (fluorescent
recovery after
photobleaching.)

Regions – drawing tools used
to customize and restrict the
scan area.

Tile Scan – create larger
images by tiling/stitching
smaller images together.

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Using the lasers

The lasers that are available for use are:
Diode 405 nm
Argon 458,488,514 nm
DPSS (Diode-pumped solid state) 561 nm
HeNe (Helium-Neon) 633 nm

The Argon, has a start-up and shut-down procedure. This can
be monitored by opening the Laser Properties panel.

The red highlighted color indicates the laser is not available
for use. ( Please note this color will go away, it will not turn
green, when the laser is warmed up.)

Looking at the Light Path

1. Tracks

• Create and/or delete tracks which contain the

scanning configuration for each probe or
combination of probes that are scanned. The
configuration consists of the laser line, excitation
dichroic, detector and emission range that is
captured. If you use the Smart Setup application,
your tracks will automatically be configured for
you.

2. Switch track every

• Line – tracks are switched line by line. Laser line,

laser intensity and channels can be changed
between tracks.

• Frame – tracks are switched frame by frame.

Laser line and intensity, filters, beam splitters,
gain and offset, and pinhole setting can all be
changed between tracks.

• Frame Fast – scanning procedure can be made

faster. Only the laser line intensity and Amplifier
offset can be changed between tracks.

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Imaging Setup and Light Path

1. Track – defines the fluorophore(s) that is(are)
being detected. A maximum of four tracks can
be created which specify the dye, laser
excitation line and the emission detection
range.

2. Visual representation of excitation line(s) and
the emission profile(s) which you can set by
adjusting the slider bars or alternatively you can
enter the range at the drop down arrow.

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Imaging Setup and Light Path

1. Tracks are defined by dye, pseudo color,
detector, and emission range. The check mark
indicates the track is enabled and will scan upon
activation.

Detectors – select from standard PMTs, Ch1
and Ch2 or the QUASAR detector ChS 1-8, and
the transmitted light detector ChD (not
confocal).

2. Select laser wavelengths, either visible or
invisible (UV), and their attenuation values (%
transmission) and the main dichroic beam
splitters.

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Acquisition Mode

1. Objective – change objectives, this is an
alternative to going to the locate tab.

2. Scan Mode, Frame Size and Line Step –

• Scan Mode - Frame, Line, Spot

• Frame Size – defaults to 512 x 512. Select

Optimal for appropriate number of pixels
depending on numerical aperture and
excitation wavelength.

• Line Step – scan every line or less than

every line.

3. Speed – adjust scan speed. A higher speed with
averaging yields results with the best signal-to­noise ratio. Use slower scan speeds for better
images, but be aware of photobleaching.

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Acquisition Mode (cont.)

4. Averaging – improves image by increasing the
signal-to-noise ratio. Select from Line or Frame
averaging and select the number to average by.

Averaging helps to reduce photobleaching.

5. Bit Depth – select from 8 bit (256 gray levels), 12
bit (4096 gray levels), or 16 bit (65536 gray
levels). Be sure your software can handle the
information. 12 and 16 bit images are
recommended for publication and quantitative
measurements.

Direction – x-y scan direction or bi-directional

6. Scan Area – set optical zoom setting and /or
rotate laser orientation.

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Working with Channels

The channels dialog allows you to select an individual
track and adjust the corresponding settings to
improve the image quality.

1. Tracks – as defined from the light path. Be sure
to highlight the channel you wish to modify. If a
track has a “check mark”, it is active and it will
be scanned.

2. Lasers – enable or disable and modify laser
po we r.

3. Pinhole – set pinhole to 1 AU (airy unit) to
achieve best z resolution based on the
excitation wavelength and objective numerical
aperture. Be sure to match the section thickness
when scanning multiple channels.

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Working with Channels(cont.)

4. Gain, Digital Offset, Digital Gain

• Gain (Master) – amplifies the voltage on

the photomultiplier tube; regulates
brightness of the image.

• Digital Offset – sets background/threshold

level or dark current of the image.

• Digital Gain – post acquisition

enhancement of the gain signal.

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Using the Range Indicator

Remember, when acquiring your images, you

want to be within the dynamic range of the
detector. This is especially critical when
collecting images that will be used for
quantitative analysis.

• Use the Range Indicator to adjust the gain

and offset. Blue= minimum, Red= maximum
(0 and 255 for 8 bit image, 0 and 4095 for 12
image, 0 and 65536 for 16 bit image.)

• Adjust Detector Gain and Amplifier Offset to

remove red and blue pixels. Minimum should
be ~ 10-20 gray levels above 0 and Maximum
should be 10-20 gray levels below 255 for an
8 bit image. You can scale this accordingly for
12 and 16 bit images.

Using the Focus Tool

The 780 offers the ability to manually control the

focus or to use the software for more precise
control of the focus.

1. Step size – set a fixed step size, in microns.
Move up or down in the focal plane based on
that step size.

2. Z-position – specifies location of focal plane,
with regard to a user set zero position.

3. Home – zeros the focal position.

4. Work – returns the objective to the desired
focus, set with the previous sample.

5. Load – lowers the objective so the user can
change the sample.

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Using the Stage Tool

The 780 allows you to manually control the stage via

the joy stick or to use the software to control
the stage.

1. x-y coordinates – indicates stage position.

2. Step – move specified step size, in microns.

3. Set zero – establish a zero point (0,0) on the
stage

4. Move to zero – move to the zero point on the
stage.

5. Marks – generate a list of stored positions, with
regards to x, y, and z.

Moves according to
set step size.

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Click in the direction
you wish to move your
sample.

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Acquiring a Z-stack

Used to collect multiple images, of different focal

planes, within a single file for 3-D
analysis/display.

1. You can enable the z-stack dialog from advanced
features menu.

2. Scroll down to access and then open the Z-Stack
dialog.

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Z-stack Dialog

1. First/Last or Center

• Establish the stack acquisition parameters.

2. Optimize Sectioning and Step

• Ensure the pinholes are set to the same section

thickness for multiple wavelengths.

3. Correction

• Options for adjusting refractive index and/or

auto brightness to ensure even illumination
through the stack.

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Defining First/Last

Set the first and last starting positions for the stack
and acquire the slices within that range.

1. Set Last – marks z position furthest away from
the specimen, as mounted on the cover glass.

• Range – total thickness of stack, in microns.

• Slices – number of slices making up the stack

• Interval – distance between each slice, in

microns

• Smallest – calculates the slice to be ½ the z-

resolution for proper Nyquist sampling.

• Keep Interval/Slice – selection if the stack

should be based on the interval thickness or a
specified number of slices.

2. Set First - marks z position closest to the
specimen, as mounted on the cover glass.

3. Position - Current z position

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Optimize Sectioning and Step

1. Match Pinhole – for multiple wavelengths,
ensures that the pinholes are yielding the
same section thickness.

2. Optimal - calculates each slice to be ½
the z-resolution for proper Nyquist
sampling.

3. Undo – clears selection

4. X:Y:Z=1 – sets the z interval so the voxel is
the same for x, y, and z dimensions.

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Z-Stack Display Options

1. Ortho – orthogonal display, xy, yz and xz
options

2. Cut – display a particular slice of stack and
have options for manipulating that slice

3. 2.5D – layered depth display

4. 3D – reconstruction of z-stack

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Image Display Options

1. 2D – x-y view of image

2. Gallery – display of z-stack or time series images.

3. Split – view multi-channel images as individual
and overlay displays.

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Advanced Image Display Options

1. Profile – display pixel intensities along defined
regions or lines.

2. Co-localization –display spatial overlap of
fluorescent regions in different channels.

3. Histogram – display of image intensities and
their frequencies within the image.

4. Information – summary of image file.

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Image Display Features

Dimensions Tab

1. Z position – step through optical z-stack
slices

2. Zoom – digital zoom feature

3. Channels – range indicator option,
pseudo-color selection, turn on/off
channels that are displayed

4. Ruse – load image acquisition parameters
for image being displayed.

5. Crop – isolates a smaller region of the
image and optically zooms the image.

6. Positions – when you click on the image, a
crosshair will be displayed, this will
correspond to and generate a list of
positions within the stage tool.

7. Stage – cursor appears as a crosshair in a
rectangle. As you click and change
position, this will update the center
position of the stage for the next image.

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Image Display Features

Display Tab

1. Channel selection– modify display
settings for all or individual channels.

2. Min/Max, Best Fit – methods for
adjusting contrast settings.

3. Black/White, Gamma - set max and
minimum levels and non-linear histogram
adjustment.

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Image Display Features

1. Player - controller for reviewing z-stack or
time series data.

2. Graphics – image overlays include scale
bars, text and other various drawing
elements.

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Are you using the argon

laser?

Within the software, put the

Argon laser in Stand-by.

Yes

No

Exit Zen and log off the

computer.

Shut Down Sequence

Verified Next User Coming

Exit Zen and log off the

computer.

Are you using the argon

laser?

Within the software, Select

Stand-by and then select

Shut-Down.

Yes

No

Turn off the HXP 120V lamp.

Exit Zen and shut down the

computer.

Has the laser fan turned

off?

LET THE LASER COOL DOWN

Yes

No

WAIT

Complete Shut Down

Sequence

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Turn off the Components

Switch.

Turn off

Main Power

Switch

Turn off the Systems PC

Switch.

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