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Imaging of calcium wave propagation in guinea-pig ventricular cell pairs by confocal laser scanning microscopy technology (CAT#: STEM-MIT-0132-LJX)

Introduction

The service uses confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in isolated ventricular cell pairs. The scanning apparatus of our system, paired galvanometer mirrors, can perform narrow band scanning of an area of interest at a high temporal resolution of less than 70 msec per image. The actual [Ca2+]i is obtained directly through the fluorescence intensity of injected fluo-3, which responds to changes of [Ca2+]i in optically sectioned unit volumes of the cell. Images of the calcium wave obtained during propagation between paired cells revealed that the wavefront is constant in shape and propagates at constant velocity without any delay at the cell-to-cell junction. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool for taking pictures of the sequence of biological events in living cells.




Principle

Laser scanning confocal microscope is a high-tech microscope. It is based on fluorescence microscope imaging and equipped with a laser scanning device, which uses ultraviolet or visible light to excite the fluorescence probe, thereby obtaining fluorescence images of the internal microstructure of cells or tissues.
The laser beam is used as the light source in the laser scanning confocal microscope. The laser beam passes through the illuminating pinhole and is reflected to the objective lens through the spectroscope. The laser beam is focused on the sample, and every point on the focal plane of the specimen is scanned. If there is a fluorescent substance that can be excited in the tissue sample, the fluorescence emitted after excitation is directly reversed back to the spectroscope through the original incident light path, and is first focused when passing through the detection pinhole. The focused light is detected and collected by the photomultiplier tube (PMT), and the signal is sent to the computer, and the image is displayed on the computer monitor after processing.

Applications

Imaging and analysis in the fields of morphology, molecular cell biology, neuroscience, pharmacology, genetics

Procedure

1. Sampling
2. Preparation of slices
3. Staining (Select according to the specific experimental situation)
4. Observation

Materials

• Sample Type:
Guinea-pig ventricular cells

Notes

Operate in strict accordance with the operating procedures, and shall not arbitrarily change the operating procedures
In the starting sequence of the switch and in the scanning process, try to do fast and orderly, to protect the laser
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