Array
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Widefield microscopes for ratiometric [ion}i and/or electrophysiological measurements

System 1: Widefield microscope for single and dual wavelength fluorescence measurements in live cells.
Zeiss Axiovert 10 epifluorescence microscope (inverted) with 40x long-working distance and 63x oil immersion objectives. Interfaced with Atto Biosciences intensified CCD camera (8 bit, 512 x 480 pixels), camera controller, filter changer, high speed shutter and RatioVision software. Temperature-controlled superfusion chamber (accepts 18 or 22 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).

This system is designed for [ion]i measurements in live cells using single excitation (e.g. fluo-3 for [Ca2+]i) or dual excitation (e.g. fura-2 for [Ca2+]i, BCECF for pHi, SBFI for [Na+]i, PBFI for [K+]i) probes. Although very sensitive, the camera on this system has limited spatial resolution (reliable recordings can be obtained only from structures and/or cells >5 µm diam.).

System 2: Widefield microscope for single wavelength, dual wavelength and dual emission fluorescence measurements in live cells.
Zeiss Axiovert 135 epifluorescence microscope (inverted) with 40x long-working distance and 63x oil immersion objectives. Interfaced with two Atto Biosciences intensified CCD cameras (each 8 bit, 512 x 480 pixels), camera controller, filter changer, high speed shutter and RatioVision software. Temperature-controlled superfusion chamber (accepts 18 or 22 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).

This system can perform the same [ion]i measurements (and has the same limited spatial resolution) as System #1. The addition of a second camera and filters allows the use of dual emission (e.g. indo for [Ca2+], SNARF for pH) as well as dual excitation probes. A particular advantage of this system is the ability to measure the intracellular concentrations of more than one ionic species simultaneously (e.g. Sheldon et al., J. Neurosci. 24, 11057-11069, 2004). Automated routines are available for the simultaneous measurement of [Na+]i or [K+]i + pHi; [Ca2+]i + pHi; [Cl-]i + pHi; [Na+]i or [K+]i + [Ca2+]i; [Ca2+]i + [cAMP]i; and [Ca2+]i + mitochondrial membrane potential (e.g. Fernandes et al., J. Neurosci. 27, 13614-13623, 2007). Other routines are easily developed and electrophysiological recordings can be performed simultaneously with dual [ion]i measurements (e.g. Kelly & Church, J. Neurophysiol. 96, 2342-2353, 2006).

System 3: Widefield microscope for single and dual wavelength fluorescence measurements in live cells.
Zeiss Axioskop 2 FSPlus microscope (upright) with 20x, 40x, 63x and 100x water-dipping objectives. Interfaced with a QImaging Retiga EXi cooled CCD camera (12 bit, 1360 x 1036 pixels), Sutter DG5 high speed filter changer and shutter, and Intelligent Imaging Innovations software (Slidebook). Temperature-controlled superfusion chamber (accepts 15 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).

This system can perform the same [ion]i measurements as System #1, with the added advantage of high spatial resolution. DIC images can be captured during the course of ratiometric [ion]i measurements, making this system particularly useful for examining changes in [ion]i at the leading edge of migrating cells or neuronal growth cones.

Although none of the above systems are part of the core facility systems, Dr John Church is very pleased to accommodate other users. Please contact him by email or call at 604-822-2751 to discuss potential experiments, set up training on the systems and/or to reserve a time slot.

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