Confocal Microscopy - Cell Biology

One-photon laser scanning microscope: An Olympus Fluoview FV300 scan unit is mounted on a BX51W upright fluorescence microscope equipped with an argon laser. The single photon imaging can be combined with electrophysiological recordings and the equipment for this microscope includes a Multiclamp 700A-2, DigiData 1322A, PClamp 8 (Axon Instruments), a Master-8 pulse generator (A.M.P.I) and one micromanipulator (Sutter Instruments). Photolysis of caged compounds can be performed with a LSI 337 nm pulsed UV laser attached to single path photolysis head equipped with a pointing system (Prairie Technologies).

Multiphoton laser scanning microscopy: An Olympus Fluoview FV300 scan unit is mounted on a motorized BX61W upright fluorescence microscope equipped with a fentosecond-pulsed titanium-sapphire laser (Mai Tai, SpectraPhysics), tunable from 800 to 920 nm. The multi photon imaging can be combined with electrophysiological recordings and the equipment for this microscope includes a Multiclamp 700A-2, DigiData 1322A, PClamp 8 (Axon Instruments), Voltage Clamp Amplifier (Dagan Corp), a Master-8 pulse generator (A.M.P.I) and two micromanipulators (Sutter Instruments).

The confocal microscopy core facility is located in the Department of Cell Biology and Anatomy which is located in the Basic Science Building.

Facility Co-Directors:

Patric K. Stanton, PhD
Department of Cell Biology And Anatomy
patric_stanton@nymc.edu
914-594-4883

William Ross, PhD
Department of Physiology
william_ross@nymc.edu
(914) 594-4111

If you wish to become a user of the facility please contact Patric K. Stanton, PhD or William Ross, PhD, either by phone or email. Prospective users must meet briefly with a Facility Director to discuss their imaging methodologies and goals. Thereafter, they may register for a specific time to use the appropriate instrument.

Technical Assistance

In addition to access to the equipment, the facility provides consultation and collaborative assistance in research efforts. The degree of involvement by staff depends on the nature and difficulty of the project and also on the expertise of the investigators in confocal microscopy and image analysis. For some users, this involves instruction in the use of the equipment and occasional consultation. Alternatively, the Facility Director may provide advice in experimental design and assistance in data interpretation. Please contact a Facility Director (above) should you require assistance.

RULES FOR USE OF THE TWO-PHOTON CONFOCAL MICROSCOPE

The microscope is now working and ready for use by a variety of groups. The microscope is more complicated than the regular confocal and experiments are expected to be more elaborate. In order for these groups to work together in an efficient and cooperative way certain procedures have been adopted and should be followed by everyone.

CONFOCAL

The microscope is simple to turn on. There are two modules with on-off switches. One sits under the table; the other is on top of the frame. The power supply for the arc lamp should NOT be turned on unless the user explicitly wants to view the fluorescence of the sample by eye. Currently, there are no filter cubes in the microscope to support this use.

The two sliders in the microscope should be in the appropriate positions: the side slider should be pulled out to the camera position; the front slider should be pulled out to the LSM position. The DIC slider should be out of the light path.

Clicking on the Fluoview icon starts the confocal software. Once the software is started focusing should be controlled by the program and NOT manually.

If any filters are changed they should be returned to the original configuration at the end of the experiment. These changes should be entered in the logbook.

LASER

The laser is simple to use. Before operating the laser the user should check that the bath circulator is on and the water level at the top. If necessary add distilled water. The on-off switch on the laser power supply should always be on. The laser should be activated with the key.

Turn on the laser software by clicking the MaiTai icon. Click on the COM1 box. Click on the start icon. Then wait for the laser to warm up. This may take a few minutes. When the Ready message appears click the start icon again (depression for 5 sec required). After ignition the only parameter that can be adjusted is the wavelength. The shutter icon also requires depression for 5 sec.

The mirrors or apertures in the beam path should NEVER be touched. Only the wheel on the platform can be rotated to adjust the laser intensity entering the confocal. It is recommended that the shutter be closed when measurements are not being made. The laser beam can be dangerous.

Turn off the laser by closing the software and turning the key on the main power supply.

GENERAL PROCEDURES AND CLEANLINESS

To make it easy for different groups to use the microscope it is imperative that the apparatus be cleaned after each use. This means (a) flush distilled water through all tubing; (b) wash salt residues from the stage; (c) clean the lens with distilled water and lens paper; (d) remove all pipettes, forceps, etc. from the microscope area; (e) empty the used saline bottle.

The counter behind the microscope should NOT be used as a storage area. All equipment, solutions, boxes, etc. that is not meant for general use should be removed and stored someplace.

All manuals are kept together in a labeled box. If these are removed for reading or copying they should be replaced.

The floors, tables and counters should be kept clean. Throw away all garbage. Store empty boxes that are needed on shelves. Store common equipment in the appropriate locations.

Eating in the confocal room is not permitted.

LOGBOOK

Every user should record his/her use in the logbook.

The entries should include: (a) user name, (b) name of P.I. (if different), (c) time and date of use, (d) one sentence description of the measurements made, (e) laser wavelength and power, (f) any changes made to the apparatus during the experiments, (g) any problems encountered.