LSM 510 Duo Vario Instructions For General Operation and Z-Stack Setup

Before you Begin: 1. You should already have an experimental configuration

created by one of our staff during your initial training. If not, please get assistance.

2. Your samples should be prepared according to our document “Before You Start Using the Confocal”. This is a VERY picky instrument and sample prep is key.

Start Up Procedure: If you find the computer on when you arrive but the

ZEN software isn’t running, turn it off before you begin or the lasers may fail.

1. Turn on “System/PC” (Switch #1) on the remote control unit. (Fig. 1-A).

2. Wait for the small TFT screen by the microscope to FULLY boot up to the screen shown. (Fig. 1-B).

3. Start Computer (Switch #2) on the main PC. 4. Wait for Windows to start and login as “LSM User” with

no password. 5. Turn on Switch #3 (Fig. 1-A) to activate the Real Time

Computer for laser operations. Wait for the secondary monitor to fully boot up to the blue screen shown (Fig. 1-C) BEFORE launching the software. This is critical.

6. Turn on Switch#4 (Fig. 1-D) to warm up the ocular/visual lamp.

7. Start the Zen software. Once it launches, open the dropdown arrow on the pop-up window (Figure 1-E) and click “Start System” (Fig. 1-F). Wait for the launch before touching ANY buttons or controls on the microscope.

8. If you see any errors report them immediately to staff. Figure 1, ZEN Start up Procedure

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Set up Microscope and Focus:

1. Select the Objective you wish to use-either on the TFT screen (Fig. 2-A) or in the dropdown on the Ocular Tab in ZEN (Fig. 2-A). Add immersion product if required. (Note we have 3 kinds-be sure you choose the correct one)

2. Load your sample on the universal stage, and centre it using the joystick. You can tilt back the microscope head if needed.

3. For fluorescent samples, select the appropriate colour of sub stage filter (Figure 3-A) and click “Online” (Fig. 3-B) to open the visual lamp shutter.

4. Either the DAPI filter or the DIC filter may be located in the “Analyser D” position -ask staff to change it if needed.

5. For bright field and phase viewing, turn on the halogen lamp and open the shutter to the sample- it should show the light beam as in the image (Fig. 3-C). Ensure the sub stage filter is set to “none” (Fig. 3-A). Press the “Online” button (Fig. 3-B) to view.

6. Focus the microscope manually using the oculars. Relax-this uses a standard visual lamp. It is NOT using the laser.

7. Centre whatever you want to see in the field of view as you will only capture the middle area.

8. Immediately click “offline” (Fig. 3-B) to close the visual lamp shutter when done so you don’t bleach the sample. The lamp should shut off.

Figure 3, ZEN Ocular Tab

Figure 2, TFT Screen

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Configuration Setup: 1. From “Ocular”, go to “Acquisition” Tab (Fig. 3) 2. Activate the dropdown arrow next to

“Configuration” (Fig. 4-A) and load your configuration either from the “Recent” or alphabetical list (Fig. 4-B). Please see staff if you don’t have one.

3. A Pop-Up window should appear asking if you’d like to switch on the lasers. Agree to start them.

4. You should see yellow warning triangles appear next to the lasers (Fig. 5). Some lasers start in “Standby” mode for a warmup before running, and others go directly on.

5. If you are using the green argon laser: Highlight the Argon laser (Fig. 5-A) and open the “Laser Properties” Panel in the Laser Window (Fig. 5-B). Turn the tube output up to exactly 50% (Fig. 5-C) to achieve optimum brightness from this laser. (Do not exceed 50% or premature wear of the laser will occur)

Image Setup: 1. Open the “Acquisition Mode” and “Channels”

window panels and activate the “Show All” mode (Fig. 6-A).

2. Click on “Optimal” (Fig. 6-B) to select the correct number of pixels for the lens (Nyquist setting). This should be repeated whenever you change lens or zoom.

3. Begin with speed (Fig. 6-C) about 1.6 µSec. 4. Begin with Averaging at 1 (Fig. 6-D). Use 8 bit

depth for routine imaging. Use 12 or 16 bit for intensity analysis to provide greater numerical range.

5. Check/reset Zoom to 1.0 to begin (Fig. 6-E).

Figure 4, ZEN Acquisition Tab-Load Configuration

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C- Figure 5, ZEN Laser Window & Properties

Figure 6, ZEN Acquisition & Channels Setup

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6. On the “Channels” window, deselect all but one channel (Fig. 7-A). Use the best one for finding and focusing on the cells/sample.

7. Keep the pinhole open (Fig. 7-B) and ensure the Master Gain (brightness-white level) is up at least halfway (Fig. 7-C).

8. On top left of the screen use “Live” (Fig. 8-A) to bring up the image. Live mode is a rough sketch used for focus and movement ONLY. It “bins” together 4 pixels so it is faster, rougher and brighter than the final image.

9. Use the fine focus knob on TFT screen to ensure you are at the brightest position.

10. Centre the sample using the fine XY controls on the joystick module.

11. Close the pinhole (Fig. 7-D) by clicking the 1 AU (airy unit) button, but make no further adjustments on “Live”.

12. Switch to “Continuous” Mode (Fig. 8-B) to see the actual image quality for fine tuning.

13. On “Channels” panel, activate and highlight one channel at a time (Fig. 9-A) and work on their setup individually. (You can deselect channels in the light path window too)

14. At the bottom of the screen on the “Dimensions” tab click right in the coloured channel square (Figure 9-B) to activate the “Range Indicator Mode.” This will show you the saturation: Red indicates too bright and Blue indicates too dark. (Fig. 9-C)

Figure 7, ZEN Channels Setup

Figure 8, ZEN Scan Operation Controls

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Figure 9, ZEN Channel and Dimension Controls

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Adjusting Illumination: (see next page for tips) 1. Acquisition Mode Panel (Figure 10): These settings

will affect all of your channels: a. Scan mode: Choose “frame” b. Frame size: Begin with “Optimal”.

Decreasing frame size will sacrifice resolution but can increase brightness

c. Scan speed: Begin about 1.6 µsec. Slow to increase brightness. Use Max if high speed is necessary.

d. Bit Depth: Use 8-bit for routine imaging. Use 12-16 bit for image analysis as it gives exponentially more grey values.

e. Averaging: Use averaging on “line mode” and “mean method” to reduce noise from high gain. “Sum method” can enhance brightness.

f. Direction: Leave the raster scan on one direction. g. Scan area (zoom/tilt): Adjusts the zoom and tilt of the image. After you

zoom, click “optimal” again. Less image size uses less pixels. 2. Channels window (Fig. 11) These settings affect only one channel at a time. You

must activate and highlight the channel as shown to access its controls. a. Laser Power: adjusts image brightness. Avoid too much or bleaching will

occur. (Fig. 11-A) b. Pinhole: closing the pinhole to 1AU makes

the image confocal. Readjust this for each magnification (lens specific). (Fig. 11-B)

c. Master Gain: is the primary way of adjusting brightness/white level. If above 700 you need averaging. (Fig. 11-C)

d. Digital Offset: is the black level. It provides contrast and fine detail. (Fig. 11-D)

e. Digital Gain: is used as a last resort to enhance brightness on dim signals if other methods don’t work. (Fig. 11-E)

Figure 10, ZEN Acquisition Mode Controls

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Figure 11, ZEN Channel Controls

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Proper Illumination Setup Tips: Avoiding saturation is critical for proper measurements, analysis, and comparison of signals between samples. You can make post-capture adjustments if you want more contrast or brightness later. You CANNOT recover image data lost from saturation

1. ALWAYS use the range indicator mode (Fig. 12-1) if you want good images.

2. Set the “master gain” first to get bright whites with few or no red pixels.

3. Then set the “offset” until you see blue pixels in the background. Back off one step just until they disappear.

4. Proper setup results in images with pure black background, bright white foreground, and high detail as shown.

5. If you are turning the “master gain” much above 700 and the image looks really noisy, try any of the following tips to brighten it, but be aware of the negative implications:

a. Increase the laser power (Fig. 12-A). The sample will bleach if you go over 5 mAmps (Laser properties power x setting).

b. Decrease the number of pixels (Fig. 12-B). You lose some XY resolution. c. Slow the Scan Speed (Fig.12-C). It could bleach and imaging is slower. d. Add image averaging in either mean or sum mode (Fig. 12-D). The sample

could bleach and imaging is slower. e. Open the pinhole slightly to 2 or 3 AU. (Fig. 12-E). You lose some Z-

resolution and may have some out of focus light if you go too large. f. Use digital gain (Fig. 12-F). The image will lose some sharpness.

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Figure 12, ZEN Optimization

Capturing images: 1. When all of the separate channels have been

adjusted, click “Snap” (Fig. 13-A) to capture the image.

2. If you are capturing Z-Stacks or other complex images, activate the checkboxes and use “Start Experiment” (Fig. 13-B) instead.

3. A progress bar will appear at the bottom of the screen to indicate the time required.

4. To avoid over-writing your image, either save it or press the “New” button (Fig. 13-C) before doing any further scanning.

Z-Stack setup: 1. To use the Z-Stack function, activate “Z-Stack”

on the Scan control section of the Acquisition panel. (Figure 14-D)

2. You should now be able to see the Z-Stack window. Activate “Show All” (Fig. 14-A).

3. Use “First-Last” mode (Fig. 14-B) and “Keep Interval” (Fig. 14-C) for best results if you plan analysis or animations.

4. Set scanner to “Live”. Dial fine focus knob until the structure just goes out of focus but is still visible. Click “Set First” (Fig. 14-D). Move focus to opposite side of structure. “Set Last” (Fig. 14-E).

5. Click “Optimal” to set the best number of slices for optimal distance between the stack layers. (Fig. 14-F)

6. Before you begin the capture, click on the centre navigation button (Fig. 14-G). This will show you an image of the centre of your structure so you can verify your settings once more. (Discuss optimizations at bottom of Z-stack panel with staff)

7. Click “Start Experiment” (Fig. 13-B) to begin the capture 8. The progress bar at page bottom will indicate total capture time. If it is too long,

you can change the averaging, scan speed, number of pixels, or number of slices to decrease the time. Less slices may lose information between layers.

Figure 13, ZEN Acquisition Tab- Scanner Controls

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Figure 14, ZEN Channel Controls

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Saving and Exporting Images: 1. Thumbnail images appear on the right side of

the screen as you capture. (Fig. 15) 2. Always save Z-Stack images before manipulating

them in 3D or Topography modes. 3. Click the save icon (Fig. 15-A). 4. Always save images as “.lsm” format-not tiff or

jpeg. This saves the metadata including scaling, calibration, and full channel illumination setup data for later publication or reuse.

5. At any time, the contents of the image window can be exported via the main File-Export menu. This is useful for renderings and colocalization.

6. Please refer to our document on exporting images (see staff) for creating scale bars and exporting tiffs or jpegs. There is a free software you can download to do this. We do not recommend this 2009 version of the ZEN software for doing this as it has some bugs.

Shut Down (If another User is Coming Soon): DO NOT QUIT THE SOFTWARE! If you do, a full shut down and restart will be required.

1. Save all images. 2. Close any images you have open. 3. Deactivate the lasers you had on in the laser panel

(Fig. 16). Some lasers require a cool down and must be set to standby (Fig. 16-A) for 60 seconds before they can be turned off. Leave them off.

4. Clean any oil lenses with a dry Kimwipe. 5. Return the objective lens to the 5x to 20x (low

magnification) position 6. See next page for full/last user shut down.

Figure 15, ZEN Images and Documents Window A-

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Figure 16, ZEN Laser Panel

Full Shut Down-End of Day/Last user: 1. Save any unsaved images 2. Turn off switch 4 (Fig. 17-A). (This can be done at any point-it is not connected

to the system) 3. Quit the ZEN software- the lasers will go into auto cool down mode. They will

require a full 5 minutes to cool before proceeding or you can damage them. 4. This is a good time to copy images to Dropbox and clean up the area. Place

any glass in glass waste. Close oil containers tightly. 5. Remove oil from lenses with a dry Kimwipe. Oil is a solvent and destroys the

lens adhesives if left on. 6. Set the objective to 5x to 20x low magnifications position. Do not leave

expensive oil lenses upright or they may get damaged. 7. After 5 full minutes, or when the fan clicks down to low, turn off switches 1

and 2 on the control panel (Fig. 17-B). 8. Once your Dropbox folder in the main lab changes to a green checkmark (Fig.

17-C) indicating file transfer is complete, you may shut down the ZEN computer. (If the transfer is large, leave the computer running and see staff so we can finish the shut down).

9. Cover the microscope with the dust cover. 10. Sign out on the log sheet.

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Figure 17, ZEN Shut Down

APPENDIX 1: Troubleshooting Faint, Noisy or Uneven Images: This is often the result of poor staining, poor sample preparation, or poor image setup. Refer to illumination set up or our document “Before You Use the Confocal” for tips. Check the following:

1. Pinhole is correct size for lens: Always click 1AU button after each lens change. 2. Coverslip Thickness: Use only a #1.5 coverslip. #1.0 will also work, but with less

brightness. All other thicknesses are incorrect and will greatly compromise the image quality. Biotron sells coverslips and confocal dishes if required.

3. Slides must be spotlessly clean: If not, clean with 70% ethanol. All confocal slides should either be sealed with nail polish or mounted with hard-set media, otherwise cleaning can damage the slide.

4. Noise is caused by High Gain: If you are using gain above 650-700, use averaging. If it is above 800-900, also try other methods to improve signal (See Illumination Setup)

5. Check your Image Capture Configuration with staff: You may be using the wrong laser/filter combination for your dye.

6. Uneven Images: are caused by a loose stage insert, uneven sample, or uneven pinhole alignment. See staff for help.

7. On the Live Cell Imager: Collimations must be done daily-weekly for proper signal-see staff for training. Book this in advance.

APPENDIX 2: Troubleshooting No Laser Signal: Check the following in order:

1. Activate the range indicator-you can see better in black and white. 2. Turn up master gain to about 700-900 3. Set the offset all the way left (no offset/ no black) 4. Open the pinhole to MAX. 5. Ensure Laser power is up to at least 1-4 mWatt (Look in “Laser properties” and

multiply original Laser mWatts x % attenuation)

6. Go back to the ocular panel and ensure your sample is focussed and in the middle of the field of view. Out of focus samples are BLACK in the confocal.

7. Check in “Dimensions” at the bottom of the screen that you haven’t left the channel visually turned off (Fig 18-A). Click the button with the channel to turn it back on (shown here as CH2)

If the image appears at any point, re-establish proper illumination as per protocol and proceed with imaging 8. If this still fails, check to see if the laser is showing at the sample when running.

(Look quickly from a distance-do not stare closely at lasers). If not, proceed to a full shut down and restart. (Be sure to cool lasers 5 minutes)

9. If you see the laser but don’t hear the scanner mirrors (sounds like a mosquito buzzing when the scan is running), proceed to a full shut down and restart. (Be sure to cool lasers 5 minutes)

APPENDIX 3: Troubleshooting Blurry Images: Please read our document “Before You Use the Confocal”. Confocal images are never blurry unless something is obscuring light in the sample path such as dirt, bad sample preparation, or poor set up. Check these items:

1. Pinhole must be closed to 1AU. An open pinhole lets in out of focus light 2. Slide must be spotlessly clean. If not, clean with 70% ethanol. All confocal slides

should either be sealed with nail polish or mounted with hard-set media, otherwise cleaning can damage the slide.

3. Clean the lens gently with lens paper and lens cleaner. Make sure you always clean slides of oil before returning to air immersion lenses.

4. Coverslip Thickness- for confocal use only a #1.5 coverslip. #1.0 will work with less brightness. All other thicknesses are incorrect. Biotron sells both types if required.

5. Correct the Lens: Biotron is equipped with a wide variety of lenses that work with and without coverslips and have varying depths of focus they are capable of. Are you trying to focus beyond the capability of the lens? On the 63x multi

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Figure 18, ZEN Dimensions Panel

glycerine/water lens, have you checked its correction collar? Refer to our “Choose the Correct Objective” chart or see staff for guidance.

6. Correct the Immersion Product-we have several kinds of lenses that use oil, water, glycerine, or no immersion, and these lenses are ground for these specific refractive indices. Incorrect combinations cause blur.

a. 63x “Gly” multi lens: This has a correction collar with a dial that goes from glycerine to water. This must be adjusted for your sample. “Water” for samples in cell culture media, water of PBS, “Glycerine” for most aqueous mounting medias.

b. Oil Immersion: is used with our 40x and 63x and 100x oil lenses. c. Air Immersion: is used with our standard 5x, 10x and 20x/0.8 NA lenses.

Don’t use these without a coverslip. d. NO COVERSLIP + AIR: is used with our 20x/0.5NA and 50x LD lenses.

Don’t use these with a coverslip. 7. Correct the Mounting Media: cloudiness may be caused by poor choice of

mounting media, or samples with lipids, salts, cell culture media or dirt in them. We DO NOT recommend mounting medias with DAPI or other counterstains in them as they frequently cause haze.

8. Thick samples: or piled-up layers of cells between the focal plane and the lens create blurry results. Outside of your focal plane, light is being scattered by the intervening material. Refocus gently on “Live” as close to the coverslip as possible where you see the greatest brightness. If you must image thick samples, physical sectioning may be needed

9. Test the imaging using one of our control samples. If you get a clean image, you sample preparation is the problem.