Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 86

Tabl e 3. 1 Comparison of two-photon excitation (TPE) imaging and single-photon excitation
(SPE) confocal imaging
Characteristics

Two-photon Confocal

Excitation source Femtosecond or picosecond pulsed

IR lasers with 80-100 MHz
repetition rates; tunable
650-1300 nm; CW also
possible

CW or pulsed UV/visible lasers;
white-light or supercontinuum
lasers (tunable in the visible
and IR spectrum range) are
available

Excitation/emission
separation

Fluorescent emissions of UV and
visible fluorophores can be
well separated from a TPE
wavelength

Usually close, depending on the
Stokes shift

Detection The detector can be a PMT

(typically), an APD, or a CCD
camera. No pinhole

The detector can be a PMT
(typically), an APD, or a CCD
camera. Pinhole(s) required

Excitation
volume
selectivity

Intrinsic (fraction of femtoliter) Determined by the pinhole

dimension

Imaging
penetration

TPE can go deeper than 400 m,
although problems can be
caused by pulse shape
modifications and scattering

About 50-100 m, depending on
specimens

Spatial resolution Diffraction limited, depending on

the objective lens.
Theoretically, spatial resolution
inTPEislessthanthatin
confocal because of the IR
excitation wavelength.
However, TPE often gives a
higher signal-to-noise ratio

Diffraction limited, depending on
the objective lens

Photobleaching
and photo
damage

TPE typically causes less photo
damage and photobleaching
since only the fluorophores
within the small TPE volume
are excited and infrared light is
less toxic

Photobleaching is usually a
concern for time-lapse confocal
imaging. Photo damage can be
an issue in confocal imaging of
UV fluorophores

provides a sensitive tool for investigating a variety of phenomena that produce
changes in molecular proximity. Counting the number of FRET-related publications
in many diverse fields of the life sciences has shown an exponential growth in FRET
applications since the early 1990s [ 43 ] - the list of publications in each category is
available at http://www.kcci.virginia.edu/Literature .
FRET depopulates the excited state of the donor, resulting in a decreased
probability of the photon emission from the donor and a shortening in the fluo-
rescence lifetime of the donor; meanwhile, the probability of the photon emission
from the acceptor increases (sensitizes). Measurement of each event can provide
direct proof of the energy transfer. Various FRET microscopy methodologies have
been developed using widefield, SPE confocal, and TPE microscopy [ 43 , 72 -
76 ]. Acceptor photobleaching or donor dequenching FRET microscopy methods