Life technologies EVOS FL, EVOS FL Color User Manual

USER GUIDE

EVOS® FL EVOS® FL Color

Imaging Systems for Fluorescence and TransmittedLight Applications

Catalog Numbers AMF4300, AMEFC4300

Publication Number MAN0007988

Revision 1.0

For Research Use Only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice.

DISCLAIMER

LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

NOTICE TO PURCHASER: LIMITED USE LABEL LICENSE: Research Use Only

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California 92008.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners.

Contents

About this Guide ............................................................................................................................................. 5

1. Quick Reference Diagrams ...................................................................................... 6

Hardware Controls ......................................................................................................................................... 6

Hardware Glossary ......................................................................................................................................... 7

User Interface (UI) Controls ........................................................................................................................... 8

Software Glossary ............................................................................................................................................ 9

Specifications .................................................................................................................................................. 11

2. Installing the EVOS® FL Imaging System ............................................................... 12

Standard Items Included .............................................................................................................................. 12

Unpacking ...................................................................................................................................................... 13

Operating Environment ................................................................................................................................ 14

Pins/Locks and Power Supply .................................................................................................................... 15

Ports and Shields ........................................................................................................................................... 16

3. Basic Operations ................................................................................................... 17

Brightfield/Phase Operations...................................................................................................................... 17

Fluorescence Operations .............................................................................................................................. 19

4. Advanced Operations............................................................................................. 20

Transfection .................................................................................................................................................... 20

Time Lapse ..................................................................................................................................................... 21

5. Working with Images ............................................................................................. 23

Live/Captured Zoom and Scalebar Option ............................................................................................... 23

Cell Counting ................................................................................................................................................. 24

Saving .............................................................................................................................................................. 26

Image Review ................................................................................................................................................. 29

6. Optional Settings ................................................................................................... 30

Date/Time Settings ....................................................................................................................................... 30

Network Settings ........................................................................................................................................... 31

Login Settings ................................................................................................................................................ 34

7. Maintaining the EVOS® FL Imaging System ........................................................... 35

General Care ................................................................................................................................................... 35

Sterilization .................................................................................................................................................... 35

Updating Software ........................................................................................................................................ 36

Changing LED Light Cubes ......................................................................................................................... 37

EVOS® FL and EVOS® FL Color User Guide

Appendix A: Troubleshooting...................................................................................... 40

Image Quality Issues ..................................................................................................................................... 40

Software Interface Issues .............................................................................................................................. 41

Mechanical Issues .......................................................................................................................................... 42

Appendix B: Safety ...................................................................................................... 43

Safety Conventions Used in this Document .............................................................................................. 44

Symbols on Instruments ............................................................................................................................... 45

Safety Labels on Instruments ....................................................................................................................... 47

General Instrument Safety ........................................................................................................................... 48

Chemical Safety ............................................................................................................................................. 49

Chemical Waste Safety ................................................................................................................................. 50

Electrical Safety .............................................................................................................................................. 51

Physical Hazard Safety ................................................................................................................................. 52

Biological Hazard Safety .............................................................................................................................. 52

Safety and Electromagnetic Compatibility (EMC) Standards ................................................................. 53

Documentation and Support ....................................................................................... 54

Obtaining Support ......................................................................................................................................... 54

EVOS

FL and EVOS® FL Color User Guide

About this Guide

Audience

User Attention Words

Safety Alert Words

Except for IMPORTANT! safety alerts, each safety alert word in a Life

This user guide is for laboratory staff operating, maintaining, and analyzing data

using the EVOS

FL or the EVOS® FL Color Imaging System.

Three user attention words appear in this user documentation. Each word implies a particular level of observation or action as described below.

Note: Provides information that may be of interest or help but is not critical to the use of the product.

IMPORTANT! Provides information that is necessary for proper instrument operation, accurate installation, or safe use of a chemical.

This symbol indicates information specific to the color camera version.

The EVOS (EVOS

FL Imaging System is factory-configured with a monochrome

FL) or color camera (EVOS® FL Color). Throughout this User Guide, any operational differences between the two versions of the instrument have been noted.

• Monochrome cameras are commonly used for high-performance

fluorescence applications, and provide the best sensitivity for detection of faint fluorescence signals.

• Color cameras have lower fluorescence sensitivity but have the

advantage of being able to differentiate structures by color in transmitted light (for example, imaging stained tissue samples).

Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards. Each alert word—IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of observation or action, as defined below:

IMPORTANT! – Provides information that is necessary for proper instrument operation, accurate installation, or safe use of a chemical.

CAUTION! – Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices.

WARNING! – Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.

DANGER! – Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations.

Technologies document appears with an open triangle figure that contains a hazard symbol. These hazard symbols are identical to the hazard symbols that are affixed to Life Technologies instruments (see “Safety Symbols” on page 46).

EVOS® FL and EVOS® FL Color User Guide

1. Quick Reference Diagrams

Hardware Controls

Power Input Jack Y-Axis Stage Brake

Power Switch Focusing Knobs

USB and DVI Ports Objective Selection Wheel

Coarse Stage Positioning Knobs Light Cube Selection Lever

Stage X-Axis Knob Phase Annuli Selector

X-Axis Stage Brake Condenser Slider Slot

Stage Y-Axis Knob

Figure 1 FL footprint

EVOS

FL and EVOS® FL Color User Guide

Hardware Glossary

Power Input Jack

Power Switch

USB and DVI Ports

Coarse Stage Positioning Knobs

Stage X-axis Knob

X-axis Stage Brake

Stage Y-axis Knob

Y-axis Stage Brake

Focusing Knobs

Objective Selection Wheel

Plug the power adapter into the power input jack.

Set the power switch to “—” to turn the instrument on or “O” to turn it off.

Plug the mouse and flash drive into the USB ports. Use the DVI port for digital output to a projector or other display.

Use the coarse stage positioning knobs to position the specimen within the field of view, particularly at low magnifications.

Use the stage X-axis knob for fine left-right movements to position the specimen within the field of view, particularly at high magnifications.

To secure the stage in place for time-lapse image captures, tighten the X-axis stage brake.

Use the stage Y-axis knob for fine front-back movements to position the specimen within the field of view, particularly at high magnifications.

To secure the stage in place for time-lapse image captures, tighten the Y-axis stage brake.

Use the focusing knobs to bring the specimen into focus.

Turn the objective selection wheel to change magnifications. The objective turret

will click into place at each position.

Light Cube Selection Lever

Phase Annuli Selector

Condenser Slider Slot

Move the light cube selection lever to change light channels. The lever will click into place for each of the 4 positions.

Set the phase annuli selector to the position that corresponds with your selected objective for phase or brightfield observations. The selector will click into place for each position.

Insert the appropriate slider to enhance image quality.

Note: An exclamation point will appear instead of the current objective’s

magnification if the objective selection wheel is not in position.

Figure 2 Objective Turret

Note: The channel indicator bar across the top of the screen displays the

active channels for each lever position.

Note: See “Basic Operations” (page 17) to learn more about EVOS

sliders.

EVOS® FL and EVOS® FL Color User Guide

User Interface (UI) Controls

UI controls are software-driven controls for using the FL menu system.

Note: Periodically, Life Technologies adds functionality and other improvements to the EVOS

FL Imaging System up to date with the latest software. If you have

EVOS

user interface. We recommend keeping your

any questions about software updates, contact your local distributor. If you

do not have your distributor information, refer to the EVOS

FL Imaging System product page at www.lifetechnologies.com/evosfl or contact Technical Support (see page 54) for instructions on how to perform the

software updates.

Channel Indicator Bar with the Active Channel highlighted

Control Bar Tabs

• Find & Focus

• Actual – Exposure Time Slider

• Overlay – Overlay Color Dialog

Light ON/OFF

Illumination Slider

Box

Figure 3 UI Controls Reference

Exposure Time Slider

Image Capture Button

Color Option

Scalebar/Toolbar Options

Settings Control Button

Save Image Button

Info Display Bar

Selected Objective

EVOS

FL and EVOS® FL Color User Guide

Software Glossary

Channel Indicator Bar

Control Bar Tabs

Light ON/OFF Button

Illumination Slider

Exposure Time Slider

Image Capture Button

Color Display

Scalebar/Toolbar Options

The channel indicator bar highlights the active channel (i.e., the light channel that is currently selected).

The Login button allows for logging in and creating or changing user profiles. This button also displays the current user profile (for example, “Guest”).

Find & Focus

Use the Find & Focus tab to view the sample with either transmitted light (to minimize photobleaching) or fluorescence, and to acquire images. This feature displays 10 frames per second for focusing and capturing images at longerexposure, high-quality settings.

Actual

Use the Actual tab with fluorescence channels to view the image at the actual exposure time used for high quality image capture. With Actual tab selected, the EVOS changes, depending on the user-selected exposure time for the camera.

• Exposure Time Slider: Use this slider to manually select the exposure time

that will be used during image capture

Overlay

Use the Overlay tab to select and overlay multiple fluorescence channels as a single multicolor image.

• Overlay Color Dialog Box: The Color Adjustment button (left control bar)

opens the Adjust Color dialog box where you can fine-tune your live image Brightness, Contrast, Saturation, and Hue prior to capture.

Use this button to turn on the light source selected (for example, GFP).

Use this slider to adjust the illumination. TIP: For quick adjustments, use the mouse wheel to brighten and dim.

Use to turn on the selected light source (for example, GFP).

Captures the image on the screen.

Use to add a pseudo color to your monochrome image.

Scalebar

The scalebar option is a toggle button available after an image is captured that will display/hide the scalebar. Once displayed, the scalebar can be selected and dragged on-screen to the desired position.

Toolbar

The toolbar option includes time lapse, transfection, count and image review tools; click the small gray triangle to open each tool.

FL Imaging System responds more slowly to stage position and focus

EVOS® FL and EVOS® FL Color User Guide

Settings Control

Button

Opens the Settings dialog box.

Note: See “Optional Settings” (page 30) for a complete overview of the

settings.

Save Image Button

Use to save the onscreen image to a USB flash drive or network folder. The USB icon turns from red to green once the image has been saved.

Info Display Bar

Shows the following context-sensitive data in the Find & Focus and Actual tabs:

Note: A red USB icon indicates that the current image has not been saved.

• Intensity (Max and Min)

• Ratio (Max to Min)

• Cursor position

Objective Selection

Displays the magnification of the currently selected objective.

Note: In Overlay, the info display shows the Quick Save option with the

Quick Save file name and the current objective selected.

The color camera version shows the QuickSave option instead of the info display bar.

Field

EVOS

FL and EVOS® FL Color User Guide

Specifications

Optics

Objectives

Infinity-corrected optical system; RMS-threaded objectives with 45 mm parfocal distance

Objectives included vary per order. Contact Technical Support (see page 54) for a full list of objectives supported.

Objective Turret

Light Cubes

Illumination

Contrast Methods

Condenser

Condenser Working Distance

Mechanical “Glide” Stage

LCD Display

5-position; front-mounted control

Light cubes included vary per order. Specifications for our most popular light cubes are listed below. Contact Technical Support (see page 54) for a full list of LED light cubes supported.

• DAPI: 360 nm excitation, 447 nm emission

• GFP: 470 nm excitation, 525 nm emission

• RFP: 530 nm excitation, 593 nm emission

LED (50,000-hour life); adjustable intensity

Fluorescence and transmitted light (brightfield and phase contrast)

Includes 3-position with brightfield and phase contrast annuli

60 mm

XXY axis fine-positioning controls;

28.3 mm (1.11”) per rotation. 110 mm × 110 mm (4.3” × 4.3”) range of motion

15” color, 1024 × 768 pixels; adjustable tilt

XXZ-axis focusing controls, 480 µm/rotation. XX-Interchangeable vessel

holders available for most common shapes and sizes

Image Acquisition

Camera

Captured Images

Output Ports

Power Supply

Dimensions

Weight

Onboard microprocessor; built-in software for image acquisition via mouse control

Monochrome:

Sony ICX445 monochrome CCD, 1/3” 1280 × 960, 1.3 Megapixels

Monochrome camera:

16-bit monochrome TIFF or PNG (12-bit dynamic range); 24-bit color TIFF or PNG; jpeg, bmp (1280 × 960 pixels)

3 USB and 1 DVI (digital output)

AC Adapter; Input 100-240V, 50-60Hz; Output 12 VDC/4.15A

Operating height: 57.8 cm (22.75”) Storage/transport height: 32.4 cm (12.75” ) Depth: 47.0 cm (18.5”); Width: 35.5 cm (14.0”)

15.3 kg (33.7 lbs)

Color:

Sony ICX285AQ color CCD, 2/3” 1360 × 1024, 1.4 Megapixels

Color camera:

16-bit color TIFF or PNG (12-bit dynamic range); 24-bit color TIFF or PNG; jpeg, bmp (1360 × 1024 pixels)

EVOS® FL and EVOS® FL Color User Guide

2. Installing the EVOS

Standard Items Included

Before setting up the EVOS® FL Imaging System, unpack the unit and accessories and verify all parts are present. Contact your distributor if anything is missing.

Note: If you do not have your distributor information, contact Technical Support (see page 54).

• EVOS

• Power Adapter and Power Cord

• Dust Cover

• USB Flash Drive (preconfigured with instrument documentation)

• Accessories Kit

• LED Light Cube Lock

• Condenser Shield

FL or EVOS® FL Color Imaging System

• Mouse

• Sliders

• Light Shield Box

• UV Shield Assembly (includes mount, shield, screws, and L-shaped hex

key)

• Light Cube Access Cover (includes LED light cube installation tool; must

be installed before using the EVOS)

FL Imaging System

Light Cube Access Cover

Sliders

Light Shield Box

LED Light Cube Lock

EVOS

UV Shield Assembly

Condenser Shield

FL and EVOS® FL Color User Guide

Unpacking

Note: All steps MUST be completed PRIOR to using the instrument.

To remove the instrument from the packaging, lift the instrument by grasping it firmly with both hands under the support and place on a level surface away from vibrations from other pieces of equipment.

Figure 4 Grasp under support arm with both hands to lift EVOS

Note: Be sure to set aside packaging and foam for future transport and storage. To move or transport the instrument, the stage pin and light cube lock must be re-installed. Always be sure the instrument is properly

cushioned and braced to prevent damage.

EVOS® FL and EVOS® FL Color User Guide

Operating Environment

• Allow at least 5 cm (2 in) free space at the back of the instrument to allow for

proper ventilation and to prevent overheating of electronic components.

• Operating temperature range: 4°–32°C (40°–90°F).

• Relative humidity range: 30–90%.

• For best image quality, avoid setting up the EVOS

direct light sources. Place the light shield box on the stage over the sample (Figure 5, below) to reduce the effects of ambient light and improve image quality.

FL Imaging System near

Figure 5 Light shield box on stage

Desktop Setup

1. Lift the EVOS® FL Imaging System as before (see page 13) and place it on the table.

2. Tilt the LCD monitor upright.

3. Remove the stage pin, connect the power cord, mouse and, if desired,

keyboard.

Hood Setup

1. Lift the EVOS® FL Imaging System as before (see page 13) and place it inside

the hood.

2. Tilt the LCD monitor upright.

3. Remove the stage pin, connect the power cord, mouse and, if desired,

keyboard.

IMPORTANT! The EVOS

FL Imaging System should not be subjected to UV sterilization. UV degrades many materials, including plastic. Damage from UV exposure is not covered under the manufacturer’s warranty.

Note: The EVOS

FL Imaging System will fit in cell culture hoods that are at least 20 ½ inches (520 mm) deep. If your cell culture hood is smaller, it may be possible to turn the itsrument at a slight angle to fit.

EVOS

FL and EVOS® FL Color User Guide

Pins/Locks and Power Supply

Now that you have unpacked the unit and accessories and verified all parts are present, it is time to set up the EVOS powering on for the first time.

Stage Lock Pin

Pull firmly to remove this pin, making sure to store the stage pin for future use. Always re-secure the mechanical stage with the stage pin before moving/transporting the instrument.

Light Cubes

1. Move the stage back to allow access to the light cube lock, which is centered

2. Loosen the thumbscrew to remove the light cube lock, making sure to store

3. Place the light cube access cover into the opening and tighten thumbscrew.

Note: Before using the mechanical stage for the first time, you must remove the stage lock pin from the back right-hand corner of the stage plate.

Note: Before using the mechanical stage for the first time, you must remove the stage lock pin from the back right-hand corner of the stage plate. For information about adding optional LED light cubes, see “Changing LED Light Cubes” (page 37).

under the back of the stage.

the light cube lock for future use. Always re-secure the light cube lock before moving/transporting the instrument.

FL Imaging System. Follow all steps prior to

Power Supply

2. Connect the power adapter to the power jack on the right side of the

1. Verify the power switch is in the “O” (OFF) position before connecting the

IMPORTANT! Before changing light channels, ALWAYS verify the light cube lock has been removed. Applying force to the light cube selection lever while the lock is in place may seriously damage the mechanism. This type of damage is not covered by the manufacturer’s warranty.

IMPORTANT! Always use the correct power supply. The power adapter specifications appear on the serial number label (front of LCD hinge) and in “Specifications” (page 10). Damage due to an incompatible power adapter is not covered by warranty.

power adapter.

instrument base, attach the cord to the adapter, and plug the cord into an outlet.

EVOS® FL and EVOS® FL Color User Guide

Ports and Shields

USB Ports

DVI Output Port

UV Shield

Plug the mouse and the USB flash drive into the USB ports located on the bottom right of the support arm. You may also plug in a USB keyboard (not included) for text input.

Connect the DVI cable to the DVI port on the instrument.

Secure the shield in place, as shown in Figure 6, below.

Note: A DVI port is available for output to a projector or other display (DVI cable not included). This port produces digital output only; EVOS Imaging System is compatible with either a DVI-D or DVI-I display.

Note: The UV Shield is provided as a safety feature and should always be installed whenever the unit is in operation. The UV shield is removable for access to the condenser sliders used in transmitted light mode. Simply unhook it from the screws on the UV mount.

Figure 6 UV shield in place

Condenser Shield

After completing all of the above steps (page 13 to 16), your instrument is ready to

The Condenser Shield is shield is pre-installed (Figure 7, below) and helps reduce the potential effects of overhead lighting on your image.

Figure 7 Condenser Shield, removable

power on. Continue to “Basic Operations” (page 17) to get started.

EVOS

FL and EVOS® FL Color User Guide

3. Basic Operations

Brightfield/Phase Operations

1. Set the magnification using the objective selection wheel on the front of the

2. Move light cube selection lever towards you until the active channel on screen

3. Turn the phase annuli selector to the position that corresponds to the selected

4. Insert the desired condenser slider into the slot on the condenser assembly for

instrument.

reflects “transmitted”.

objective and contrast method.

optimal image quality.

5. Turn on illumination using the LIGHT ON button.

6. Focus the sample using the focusing knobs.

7. Adjust the illumination intensity if necessary.

Note: A variety of sliders can be inserted into the instrument condenser to

improve image quality (Figure 8).

Figure 8 Insert slider into condenser

EVOS® FL and EVOS® FL Color User Guide

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