Equipment

Filters

Instructions for starting the system

Instructions for using the software

Instructions for shutting down the system

Problems







Equipment:

Olympus microscope:

upright
fixed stage
water immersion (dipping) lenses: 10X NA0.3, 40X NA0.8, and 60X NA0.9
differential interference contrast (DIC)
IR viewing

Princeton Instruments PentaMax 512EFT camera:

cooled (-40oC)
frame transfer
15.7 X 15.7 microns pixel size
512 X 512
12 bit
A/D

TILL monochromater

Filters:

Excitation: TILL monochromater provides wavelengths from 260-680 nm. with a bandwidth of 15nm.

Dichroic and Emission filters:

Note: All excitation and emission filters from Chroma are marked with an arrow. The arrow should point to the inside of the cube. Dichroic filters are oriented with the coated side toward the excitation filter.

Fluorophore Dichroic (Beamsplitter) Emission Notes
FURA 400DCLP - -
MEQ 400DCLP 420LP -
Lucifer Yellow 460DCLP 500LP -
Calcium Green 515DCLP 520LP 400LP *
Calcium Crimson 595DCLP 600LP -

*A 400LP filter is installed in the excitor filter position. This prevents illuminating the sample when shuttering to a wavelength <400nm.


IR bandpass filters: 775/150 or 770/40

Instructions for starting the system


1. The xenon lamp must be ignited before anything else is turned on.

Turn on the red lamp switch on the Polychrome II.

Wait 30 seconds (you will hear the lamp ignite).

Turn on the green DCPower switch.

All of the voltage lights should come on.

The lamp should be changed at approx. 500 hours.

2. Turn on the power strip behind the video monitor. This will start the cameras, shutter and video monitor.

3. Turn on the on the power strip by the computer monitor to start the computer and monitor - this switch is also for the transmitted light.

4. A password is necessary to use the computer.

5. Select Windows95 from the System Commander menu.

6. A password is not necessary for Windows Networking.

7. Start the "Use this MetaFluor". Type in your name to set up a configuration file.




Instructions for using the software


Instructions for shutting down the system


Problems

Polychrome II monochromater: "There is certainly some stray light with the Polychrome II monochrometer. This is due mostly to light scattered off the grating, and the background level has little to do with which end the monochrometer is parked at. Tests have shown that about 2000 seconds of exposure at the resting wavelength of 250nm produces a FURA-emission equivalent to 1 second of exposure at 380nm."

Solution:

1. a 400LP cut-off filter was installed in the excitation slot of the calcium green filter set
2. use the manual shutter behind the microscope between experiments
3. a shutter was installed in the monochrometer that can be run with experiments.




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