MICROSCOPY: IMAGING OF BIOLOGICAL SPECIMENS - A Guide to Methods in the Biomedical Sciences - page 93

than optimal for resolving proteins that are expressed at relatively low
abundance.
Two-photon excitation microscopy
Two photon microscopy is used for imaging relatively thick tissue
samples rather than individual cells. It allows for the collection of a 3-
dimensional image of a section with absorption of light either above or
below the focal plane, and therefore there is little photobleaching of the
out-of-focus images within the sample. Two photon microscopy allows
for the imaging of events at some distance from the surface of tissues
and has been used to examine intact viable tissues, therefore providing
the opportunity to visualize cell organization and movement within a live
tissue.
Two-photon microscopy is based on the principle that fluorophores
that can be excited at a particular wavelength can also be excited by two
red photons at twice the wavelength. High energy lasers are needed to
generate the energy required for this type of microscopy.
Deconvolution Microscopy
Deconvolution microscopy is computationally intense image process-
ing technique that can be used to generate optically sectioned images
from standard widefield fluorescent microscopy (37). Its advantage over
laser confocal microscopy is that it can be used to collect images at
lower light than using confocal microscopy, and it does not result in the
intense photobleaching common with confocal beams. Deconvolution
acquires images from multiple focal planes in the cell and uses iterative
mathematical algorithms to reassign the light to its original fluorescent
source. The resulting image (on the computer, not in the microscope!) is
an accurate rendering of the cell which can be viewed as a single focal
plane or as a 3-dimensional structure, similar to images generated by
confocal microscopy.
Total internal reflection (TIR)
fluorescence microscopy
TIR fluorescence microscopy is a method that can be used to analyze
events that occur just below the plasma membrane of a cell, to a depth
of approximately 50 nm. It is often used to visualize the docking and
fusion of secretory vesicles to the plasma membrane. TIR functions
by reducing the background fluorescence within the cell. In standard