sample by using a plane of light instead of a point as in confocal microscopy,
LSFM can acquire images at speeds ranging from 100 to 1000 times faster than
those obtainable by point-scanning methods.
8.8 Super-Resolution Fluorescence Microscopy
Super-resolutionfluorescence microscopytechniques allow the capture of images
with a resolution of just tens of nanometers, which is better by an order of mag-
nitude than is possible with diffraction-limited microscopic methods. A number of
different methods have been proposed and implemented [ 34 – 41 ]. Two general
groups that categorize these methods are the following:
1.Deterministic super-resolution microscopyis based on the nonlinear depen-
dence of the emission rate offluorophores on the intensity of the excitation laser.
These techniques typically require the simultaneous application of several
high-intensity pulsed lasers with specialized modulationfilters to control the
excitation beam geometry. These methods are referred to asensemble focused
light imaging techniques. For example, by using two lasers, one laser provides
the energy for excitation of an ensemble offluorophores to theirfluorescent
state. The other laser is used for de-excitation of thefluorophores by means of
stimulated emission. The methods include the following:
a.Stimulated emission depletion microscopy(STED)
b. Saturated structured illumination microscopy(SSIM)
2.Stochastic super-resolution microscopyis a single-molecule approach in com-
parison to the ensemble methods of deterministic super-resolution microscopy.
It functions by making several localizedfluorophores emit light at separate times
and thereby allowing thesefluorophores to become resolvable in time. The
methods include the following:
a.Photo activated localization microscopy(PALM)
b. Stochastic optical reconstruction microscopy(STORM)8.9 Summary
Many developments in microscopy have appeared in recent years to enhance
imaging performance and instrument versatility for biophotonics and biomedical
applications. These developments include increasing the penetration depth in
scattering media, improving image resolution beyond the diffraction limit,
increasing image acquisition speed, enhancing instrument sensitivity, and devel-
oping better contrast mechanisms. Different embodiments of these optical
8.7 Light Sheet Fluorescence Microscopy 255