Biomedical Engineering Reference
In-Depth Information
Chapter 3
Fluorescence Microscopy Imaging
in Biomedical Sciences
Yuansheng Sun and Ammasi Periasamy
Fluorescence microscopy is an important tool in biological sciences which provides
excellent sensitivity for detecting very low concentrations of molecules over broad
spatial and temporal dimensions. With fast developments of new fluorescent probes,
advanced electronic and optical devices, and sophisticated data acquisition and anal-
ysis software, fluorescence microscopy resides on the central stage of life-sciences
research. This chapter covers several commonly used and advanced fluorescence
microscopy techniques and focuses on fluorescence lifetime imaging microscopy
(FLIM). A number of FLIM systems and their applications are reviewed. As an
example, we describe how we built and calibrated a two-photon excitation time-
correlated single-photon counting (TPE-TCSPC) FLIM system and employed the
system to investigate protein-protein interactions in living cells.
3.1
Introduction
The phenomenon of fluorescence was first reported by George G. Stokes in 1852,
and the fluorescence microscope was devised in the early part of the twentieth
century. The potential of the fluorescence microscopy technique was not realized
until Albert Coons introduced a technique for labeling antibodies with fluorescent
dye in the early 1940s [
1
], which has matured as the well-known immunofluores-
cence field. Since the early 1990s, fluorescence microscopy has been dramatically
altered and advanced because of the development of the green fluorescent protein
(GFP) and various fluorescent protein (FP) genetic variants [
2
-
7
] (see Sect.
3.2
).
Hand in hand with above, the rapid improvement in modern microscope designs
Y. S u n
A. Periasamy (
)
W.M. Keck Center for Cellular Imaging, Department of Biology and Biomedical Engineering,
University of Virginia, Charlottesville, VA 22904, USA
e-mail:
ap3t@virginia.edu
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