Transcript Laser-Scanning Microscopy as a Tool to Study the Spatio

Laser-Scanning Microscopy as a
Tool to Study the
Spatio-Temporal Organization
of InsP3-Mediated Ca 2+ signaling
Xenopus laevis and its oocyte
IP3/Ca2+ signaling pathway in the oocyte
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
Optical Schematic of Confocal Line-Scan
Microscope (CLSM)
Comparative resolution of the system
Optical Schematic and comparative resolution of the
Video-Rate CLSM
Do elementary events arise
from the activity of a
single IP3R?
Line-scan images of elementary events evoked by the
photorelease of InsP3
The spread of Ca2+ during elementary events is
consistent with passive diffusion from a point source
Does CICR and Ca2+ diffusion
between sites give rise to
global waves?
Clustering of release sites gives rise to
salutatory wave propagation
EGTA spatially decouples individual release
sites to block wave propagation
What is the radial organization
of release sites?
Optical schematic of the piezo z-scan unit and
representative images of Ca2+ release events in the zaxis
Rapid localized Ca2+ transients as resolved with
real-time x-z scanning confocal microscopy
Model of InsP3-mediated Ca2+ release in the oocyte
Practical theory of 2-photon microscopy
1. 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.
Optical schematic of video-rate 2-photon linescan microscope
3-D pollen grain
2-photon imaging of pyramidal cells in acute cortical
slices
2-Photon Ca 2+ imaging in cortical slices following
antidromic stimulation