Total internal reflection fluorescence microscopy for high-resolution imaging of cell-surface events.
The wavelength of light imposes a physical limit of approximately 400 nm on the maximum resolution that can be achieved using light microscopy. This unit will describe the use of total internal reflection fluorescence microscopy (TIR-FM), or evanescent wave microscopy, an approach that partially overcomes this physical limit and permits one to selectively image just those fluorophores in the optical plane (along the z axis) within 50 nm of the cell surface. TIR-FM works by means of limiting the depth of penetration of the excitation light within this narrow region. This narrow excitatory plane not only provides a high signal-to-noise ratio but also minimizes the photodamage to the cell.
Jaiswal
Jyoti K
JK
Rockefeller University, New York, New York, USA.
Simon
Sanford M
SM
D000818:Animals; D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011817:Rabbits; D012680:Sensitivity and Specificity
D049452:Green Fluorescent Proteins