Maheshri:FluorescenceM

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Fluorescence Microscopy

Equipment

• Zeiss Axio Observer Z1
• Xenon white light source (usually connected, on bench in corner to the right).
• PRIOR Lumen 200 metal halide lamp (for FISH)
• Cascade II Camera (coming from the left of the microscope)
• Ludl control box (on bench to the left)
• Sutter filter wheels for excitation (on back of microscope) and emission (on left side going to camera) wavelength control
• MetaMorph Imaging Station for data collection and analysis (PC on left)
• ONIX Microfluidic Control Platform (back left of float table, behind camera)
• Old PC to run ONIX Function Generator
• Filter sets
  • CFP Ex 430 Em 470
  • YFP Ex 500 Em 535
  • mCherry Ex 577 Em 632
  • DAPI Ex 350 Em 460
  • Rhodamine Ex 535 Em 610

Setup

1. Turn system on beginning with the lamp.
2. Switch on Ludl controller, wait for "Busy" LED to turn off, then turn on microscope with push button on rear left side. (Ensure flat, white voltage source is ON.)
3. Switch on camera. Wait 10 minutes before imaging, as the interior must cool to ~-80°C.
4. Open MetaMorph software on newer PC.
5. If performing a microfluidic experiment using a CellAsic device, switch on blue ONIX control box. Connect vacuum line (from next to Varioskan) and pressurized air (from left of gel viewer) to the back of the control box. Be sure the pressurized air is being fed at no more than 15psi. Open the Microfluidic Function Generator on the old PC to control flow in the device.
6. If using the 63x or 100x objective, place a drop of oil on the objective before putting sample/microfluidic device on stage. (To clean oil from objective wipe excess away with lens paper being careful not to apply pressure to the lens itself. Make a cotton swab with a cotton ball and wooden stick, dip in 80:20 mixture of petroleum ether:isopropyl alcohol, and gently swab away oil residue. Use canned air to evaporate ether:alcohol reside, using the red tube to guide the air and keeping the can upright to avoid getting moisture on the objective.)
7. Insert either the slide or well plate holder into the stage. Slides should be mounted on the stage coverglass side DOWN. If using a microfluidic wellplate, insert a pipet tip into the gap between the plate and the stage mount just behind the silver lever to ensure the plate does not shift during the experiment. Taping the manifold tubing to the microscope with some slack will isolate the plate from the rest of the system. Then use the joystick to move the stage to position objective below the center of the elongated well in column D to make finding the viewing chambers easier.
8. In MetaMorph, use the Acquire window or a journal in a taskbar to view the sample in bright field.
9. In MetaMorph, go to Device>Device Control to quickly change the Z value. Start at ~8000 for a slide, and ~8400 for a well plate. Raise the objective by hand (large black knob on base) so that the oil just touches the coverglass, and focus the image. (If nothing is visible use the joystick to move the sample around to find something on which to focus. DO NOT RAISE OBJECTIVE ALL THE WAY TO THE SLIDE, ~8600 slide, ~9000 well plate.)
10. To obtain a flat bright field illumination: view sample through eyepiece (change light path on microscope touch screen), and close the trasmitted light iris (black dial on front top of scope). Adjust the black knob on the side of the condenser and silver pins until the field of view is bordered by an octagon (the iris opening). The black knob will "focus" the edge of the octagon and the pins will center it. If no octagon is visible when the iris is as small as possible, this means it is either very "unfocused" or centered out of the field of view. Adjust the iris, knob, and pins simultaneously until it becomes clear where the octagon border is.
11. The iris for the Xenon light source (black lever to the back right of scope) should be preset and should not need to be adjusted. It should be set to just illuminate the entire field of view to give a flat illumination. If the edges appear black when viewing fluorescence, the iris may need to be expanded. (If only one edge is black, it is likely the bar with a circular aperture below the objective wheel has been pushed out of place.)
12. If performing a long timelapse experiment, the microscope cover can be used to shield out late afternoon sunlight, monitor light, etc. Be sure to turn the microscope touchscreen display off (under "Display").