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Comprehensive Course on Fluorescence Microscopy

In two-photon microscopy, a pulsed infra-red laser scans across the sample to produce a confocal-like (i.e., optically sectioned) image of a fluorescent specimen. Two-photon microscopy can have several advantages over confocal microscopy for thick specimens, including deeper penetration, reduced photobleaching outside of the focal plane, and reduced phototoxicity for live specimens. One drawback of the two-photon technique is that the resolution, which is directly determined by the excitation wavelength, is somewhat worse than a comparable confocal.
Our Two-Photon microscope is also a fully-loaded confocal microscope. There are 3 "normal" (single-photon) lasers that together provide 6 excitation lines from the blue to red: 458, 476, 488, 514, 543, and 633nm. For two-photon excitation, our tuneable Chameleon laser can excite UV to green excitable dyes (ie. DAPI to YFP). The confocal / two-photon scan head is mounted on a fully motorized inverted microscope: together with a heated stage and objective heater the setup is ready for live-cell imaging experiments such as FRAP. The Two-Photon microscope also has a META detector, allowing for spectral un-mixing of overlapping emission spectra (eg., CFP and YFP), which may be particularly useful for colocalization studies and techniques such as FRET microscopy.

Training Courses Available
Confocal/Multi-photon Microscope Training
Advanced Confocal/Multi-photon Techniques
Live Cell Imaging

Zeiss LSM 510 META NLO
Beam Path and Channel Assignment

Objective Lenses
FLUAR 20x/0.75 NA
C-Apo 40x/1.2 NA
C-Apo 63x/1.2 NA(water)
C-Apo 63x/1.4 NA(oil)

Illumination Sources
Argon Laser - (458nm, 476, 488, 514nm)
HeNe Laser - (543nm)
HeNe Laser - (633nm)
Chameleon - (720-930nm)

Advantages and Techniques
High resolution confocal and two-photon fluorescence imaging
Optical sectioning of thick fluorescent specimens
Live-cell imaging experiments (timelapse, FRAP, FRET)
Deep penetration into thick specimens as compared to Confocal
Less photobleaching outside of the focal plane as compared to Confocal
Spectral separation using META detector

Copyright 2006 Advanced Optical Microscopy Facility. All rights reserved.