Neuroscience Microscopy Service (NMS)2P summary ! pulsed infrared laser " less scattering " deeper...
Transcript of Neuroscience Microscopy Service (NMS)2P summary ! pulsed infrared laser " less scattering " deeper...
Neuroscience Microscopy Service (NMS)
Andrew Olson web: nisms.stanford.edu email: [email protected] Lokey Stem Cell basement: G0901
Three imaging techniques:
n Laser scanning confocal microscopy (LSCM) n Two-photon microscopy (2P) n Structured Illumination (SI) microscopy
2P summary
n pulsed infrared laser q less scattering q deeper penetration
n non-linear absorption q restricted excitation volume q useful for spatially restricted uncaging,
photoactivation q 2P excitation spectra generally broader than 1P
n non-descanned detection n resolution similar to confocal
Equipment: two-photon rigs
n dual laser system q two photon imaging q two photon
photoactivation
n dual PMT detectors n AOD fast-scanning
option (e.g. 25 fps, 512×512)
tissue slice rig in vivo rig
Structured illumination Microscopy (SIM)
n 3D- sinusoidally patterned illumination via laser diffraction
n Interference between patterned illumination (known) and the fluorescent sample (unknown) is used to computationally reconstruct a super-resolution image
n 5 phase shifts × 3 rotations = 15 images per z-plane
Structured illumination Microscopy (SIM)
n 2-fold increase in x, y and z resolution vs. confocal
n Sample must be near cover slip (10-20 µm)
n Uses standard immunofluorescent probes (blue, green, red)
References
n Davidson, et al., 1998-2008. Molecular Expressions Optical Microscopy Primer. http://micro.magnet.fsu.edu/primer/anatomy/kohler.html
n ibid. http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html n Invitrogen, 2007. The Handbook — A Guide to Fluorescent Probes
and Labeling Technologies, Tenth Edition. http://probes.invitrogen.com/handbook/
n Pawley, JB, ed., 2006. Handbook of Biological Confocal Microscopy, 3rd Edition, Springer:New York.
n Spring and Davidson, 2000-2008. Nikon MicroscopyU: Introduction to Fluorescence Microscopy. http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html
References
n Gustafsson, M.G.L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy J Microscopy 2000; 198:82-87.
n Helmchen, F. and Denk, W. Deep tissue two-photon microscopy Nature Methods 2005; 2:932–940.
n Rubart, M. Two-Photon Microscopy of Cells and Tissue http://www.biology-online.org/articles/two-photon_microscopy_cells_tissue/abstract.html
n Soeller C, Cannell MB. Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc Res Tech. 1999;47:182–195.