Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 83

Fig. 3.3 Demonstration of the homodimerization of the CCAAT/enhancer-binding protein alpha
.C=EBP'/ in live mouse pituitary cell nuclei using widefield FRET microscopy. Images from the
nucleus of a cell coexpressing CFP-C/EBP˛ (FRET donor) and YFP-C/EBP' (FRET acceptor)
were acquired in the donor (a), acceptor (b), and FRET (c) imaging channels using an Olympus
IX70 widefield epifluorescence microscope equipped with a 60/1.2NA water objective lens, a

Hamamatsu Orca2 CCD camera, and an X-Cite r 120 fluorescence illumination system ( www.
ldgi-xcite.com ) . The processed FRET (PFRET, d) and apparent FRET efficiency (E%, e) images
were obtained after processing the data with the PFRET algorithm in combination of the images
acquired from the single-label expressing cells (see Sect. 3.3.4 ). The interaction between CFP-
and YFP-tagged C/EBP˛ is demonstrated by the E% image, indicating the homodimerization of
C/EBP˛ in regions of centromeric heterochromatin of cell nucleus. For each imaging channel,
the same dichroic mirror designed for imaging both CFP (445-485 nm) and YFP (520-580 nm)
was used, and the excitation and emission filters were 436/20 nm and 470/30 nm (donor channel),
500/20 nm and 535/30 nm (acceptor channel), and 436/20 nm and 535/30 nm (FRET channel). See
Sect. 3.4.6 for more details about the C/EBP' biological model (Adapted from [ 43 ])

CAT scan) from a thick specimen [ 48 , 49 ]. For example, a laser scanning confocal
microscope (LSCM) focuses a laser beam on a single point of a specimen through an
objective lens and uses a point detector - usually a photomultiplier tube (PMT) and
occasionally an avalanche photodiode (APD); a pinhole is placed before the detector
to reject the out-of-focus light (light signal above and below the focal plane); a 2-D
image is obtained by moving the laser beam over a region of interest of the specimen
through the XY raster point-scanning mechanism; a stack of 2-D images at the
different depths of the specimen are acquired by moving the microscope objective
lens or stage along the optical axis. Compared to LSCM, a spinning-disk confocal
microscope can provide a much faster imaging speed since it employs a number
of pinholes designed in a specific pattern to focus laser beams at multiple points
of a specimen and uses a charge-coupled device (CCD) camera to acquire a 2-D
image [ 49 - 51 ]. Confocal microscopy has been applied to a wide range of biological
applications [ 46 ] - an example of applying an LSCM to study the receptor-ligand
binding and internalization based on FRET [ 52 - 54 ] is shown in Fig. 3.4 (see
Sect. 3.3.4 ). Many aspects related to confocal microscopy imaging techniques and
data analysis for various applications are described in the literature [ 55 ].

3.3.3 Two-Photon Excitation (TPE) Microscopy

Two-photon absorption was theoretically predicted by Goppert-Mayer in 1931.
TPE imaging experiments in a laser scanning confocal microscope were first
demonstrated in 1990 [ 56 ]. In a TPE event, two photons, each of which carries