Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 81

fluorescent semiconductor nanocrystals, widely known as quantum dots (QDs), has
provided a new avenue for fluorescence microscopy imaging [ 26 - 33 ]. QDs contain
a semiconductor core, e.g., Cd and Se, which is surrounded by a semiconductor
shell (ZnS) to improve their optical properties. The wide range of QD fluorescence
emission peaks stems from their tunable core sizes (1-10 nm). QDs show broad
absorption patterns but very narrow emission spectra, making QDs very useful
for fluorescence multiplexing and high-throughput screening [ 34 ]. Even though
majority of biological samples visualized by fluorescence microscopy imaging are
treated with extrinsic fluorescent probes, there are endogenous fluorophores in living
organisms which have been utilized in life sciences and clinical applications. For
example, FLIM measurements of endogenous NADH in tissues show promise for
the study and diagnosis of precancer conditions and cancers because the fluores-
cence properties of NADH are altered by cellular metabolism [ 35 ] (see Sect. 3.4.3 ).

3.3 Fluorescence Microscopy Techniques

Numerous fluorescence microscopy techniques have been developed, and many
of them are well described with interactive JAVA tutorials at the Molecular
Expressions website: http://www.micro.magnet.fsu.edu/primer/techniques . Several
commonly used and advanced fluorescence microscopy techniques are introduced
below, including widefield microscopy, single-photon excitation (SPE) confo-
cal microscopy, two-photon excitation (TPE) microscopy, total internal reflec-
tion fluorescence (TIRF) microscopy, Forster (or fluorescence) resonance energy
transfer (FRET) microscopy, fluorescence recovery after photobleaching (FRAP)
microscopy, fluorescence correlation spectroscopy (FCS), image correlation spec-
troscopy (ICS), stimulated emission depletion (STED) microscopy, photoactivated
localization microscopy (PALM), and stochastic optical reconstruction microscopy
(STORM). All these fluorescence microscopy techniques are now available in
commercial microscope systems.

3.3.1 Widefield Epifluorescence Microscopy

Widefield microscopy is the most widely used fluorescence microscopy technique.
The basic setup of an inverted widefield epifluorescence microscope is illustrated
in Fig. 3.2 , which also explains the basic principle of epifluorescence microscopy
imaging. Any colored (from violet to red) fluorescent probe can be imaged by a
widefield microscope equipped with a mercury or xenon arc lamp or X-Cite ( www.
ldgi-xcite.com ) light source, given a correct combination of the excitation and
emission filters and a dichroic mirror (see Fig. 3.2 ). Multicolor time-lapse imaging
can be achieved in a widefield microscope equipped with software-controlled
excitation and emission filter wheels and shutters, perfect for monitoring signaling