CCD based acquisition setup

The cooled CCD camera based acquisition setup consists of a cooled CCD camera MicroMax (Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. The excitation and emission filter wheels (Sutter Instruments, Co., Novato, CA) are driven using a computer controlled shutter instrument (Lambda 10-2, Sutter Instruments).

The computer (Pentium III processor and not less than 512 mb RAM are recommended) is equipped with MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA, USA) which controlls CCD, two filter weels, and two shatters. The same programm than is used for processing of the acquired images.

To see scheme or photo of the setup

Table of filter settings used for acquisition and for photoactivation of preinjected caged compounds

Mode	Exitation filter	Dichroic mirror	Emission filter
Acquisition Green	BP 485	<505	BP 515-565
Flash-photolysis	BP 290-390	<505	-

The following acquisition protocols are available on our CCD setup:

• Green
• Green+Visible
• Green+Red
• Green+Red+Visible
• Stream acquisition
• Green/Red splitter
• Green/Red splitter+Visible

Flash-photolysis of caged compounds can be combined with any of acquisition protocols.

Similar acquisition setup based on a cooled CCD camera CoolSnap (which is very similar to Micromax) is coupled with an electrophysiological setup. Thus we have possibility of simultaneous measurement of cytoplasmic Ca2+ changes and membrane currents.

Confocal microscopy

To exclude the influence of light emitted by off-focus planes, for cytosolic Ca2+ changes we use a confocal laser scanning microscope Olympus FVX-ZM-IL (Olympus Optical Co., LTD., Japan), equipped with an UplanApo 20x/0.70, and UplanApo 40x/0.85 objectives. Photolysis of the caged compounds was performed by irradiating the oocyte with the UV light (330 nm) emitted by a shutter-controlled (Uniblitz, Vincent Associate, Rochester, NY, USA) mercury lamp and directed onto the cell through a dichroic mirror together with the laser scanning line. Fluorescence images were acquired with a Fluoview Personal Confocal Microscope System, version 1.2.

Confocal microscope in our laboratory is also used for morphological studies of cell organells, such as nucleus, ER, lysosomes and others.