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Laser-Scanning Microscopy as a Tool to Study the
Spatio-Temporal Organization of InsP3-Mediated Ca 2+ signaling
Video-rate confocal microscopy in conjunction with UV photolysis of caged-IP3 and Ca2+
sensitive dyes reveals a high degree of spatio-temporal organization of Ca2+
release in the oocyte
The spread of Ca2+ during elementary events is consistent with passive diffusion from a point source
Optical schematic of the piezo z-scan unit and representative images of Ca2+ release events in the z-axis
Practical theory of 2-photon microscopy1. Near simultaneous absorption of the energy of two infrared photons results in
excitation of a fluorochrome that would normally be excited by a single photon of twice the energy.
2. The probability of excitation depends on the square of the infrared intensity and decreases as the inverse 4th power of the distance from the focus volume.
Advantages of 2-Photon microscopy
1. Increased penetration of infrared light allows deeper imaging.
2. No out-of-focus fluorescence, thus increased signal to noise.
3. Photo-damage and bleaching are confined to diffraction limited spot.
4. Multiple fluorochrome excitation allows simultaneous, diffraction limited, co-localization.
5. Imaging of UV-excited compounds with conventional optics.