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APPENDIX
Filter cube specs:
exciter dichroic emission
DAPI (quad)
DAPI (oly MNU2)
380
360-370
Quad
400
450/65
420LP
CFP 436/20 455 480/40
FITC (wide) 480/40HQ 505 510LP
FITC(narrow) 480/40HQ quad 535/30HQ
YFP 500/20 515 535/30HQ
Reds (quad)
Reds (oly MNG2)
530-550
530-550
Quad
570
630/60
590LP
Cy5 (quad narrow)
Cy5 (wide)
640/20HQ
620/60HQ
Quad
660
682/22/75HQ
700/75HQ
Fura-2 340; 380 400 510/40
Cameleons 2 440/20 455 535/35; 485/40
DIC analyzer (polarizer)
Stage heater and perfusion.
We have a homemade heater block that accommodates a standard 35mm dish. The heavy aluminum shell maintains
the dish at set point with constant circulation of water from the bath. This is normally set to 37°C or as high as 39°C to account
for thermal loss between the bath and stage. Use a digital thermometer to check the actual dish temperature, if critical. Heating
the dish will increase the rate of evaporation, a headache for long timelapse studies. If you are doing DIC imaging, you need to
avoid plastic since it degrades the DIC effect. One solution is to use a dish that is fitted with a coverslip as the bottom. You will
need to seal the top for long experiments and you may then need a cover also fitted with glass windows. Moisture will
condense over time, so a double pane design may work better. Another possibility is to perfuse the dish using a pump or
gravity-feed system. It is critical that you consult us on how to set this up so as to avoid flooding the system. If you have a
spill, let us know right away so that it can be cleaned up before damage to the microscope can occur. Do not think you can still
finish the experiment—it’s finished! We need to track down leaks quickly since the salts will seep in and corrode/ruin
components. You can use a J-tube to maintain bath levels and to help safeguard against overflow leaks.
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