Fluorescence Microscopy Imaging in Biomedical Sciences - Biomedical Optical Imaging Technologies: Design and Applications - page 80

Fig. 3.1 Fluorescence illustrated by Jablonski diagram (Adapted from Fig. 1.5 in Chapter 1 of
Ref. [ 13 ]). S 0 ,S 1 ,andS 2 are the singlet ground, first, and second electronic states, respectively.
At each of these electronic energy levels, the fluorophores can exist in a number of vibrational
energy levels, depicted by 0, 1, and 2. Following light absorption (A, solid ), a fluorophore is
usually excited to a higher vibrational level of S 1 or S 2 . Through an internal conversion process
( dotted ), the excited molecule will rapidly (10 12 s) relax to the lowest vibrational level of S 1 ,
where fluorescence emission (F, dashed ) typically results from. The molecule usually returns to
a higher excited vibrational ground state (S 0 ) level and then quickly (10 12 s) reaches thermal
equilibrium. Thus, the energy of the emitted photon is typically less than that of the absorbed
photon. The excess of the excitation energy is typically converted to the thermal energy. T 1 is
the first triplet state, from which emission is termed as phosphorescence (P, dotted dash )andis
generally shifted to longer wavelengths (lower energy) relative to fluorescence
extinction coefficient which is a direct measure of the ability of the fluorophore
to absorb the light and is typically determined by measuring the absorbance at its
maximum absorption wavelength for a molar concentration in a defined optical
path length. It is important to recognize that the photophysical properties (e.g.,
fluorescence lifetime and quantum yield) of some fluorescent molecules can be
influenced by their local microenvironmental factors, such as temperature, metallic
ion concentration, pH, and solvent polarity.
Fluorescent probes are essential to fluorescence microscopy, and hundreds of
them have been discovered or developed for scientific applications, especially in
the life sciences. Many classical organic dyes, such as DAPI (4 0 ,6-diamidino-2-
phenylindole), FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine
isothiocyanate), and Texas Red, have been widely used, and the novel ones (e.g.,
Alexa and cyanine dyes) exhibiting improved photo- and pH-stability as well as
excellent spectral characteristics provide additional choices for fluorescent labeling
(see www.introgen.com , www.gelifesciences.com , www.sigmaaldrich.com ) . Espe-
cially, advances in immunology and molecular biology have provided insight
into the molecular design of fluorescent probes targeted at specific subcellular
regions, which can then be visualized by fluorescence microscopy. The greatest
revolution in fluorescence microscopy is perhaps led by the discovery of GFP
isolated from jellyfish, Aequorea victoria [ 2 - 5 ]. Over the past 20 years, a broad
range of FP genetic variants have been developed, spanning almost the entire visible
light spectrum [ 6 , 7 , 14 - 21 ]. The usage of FPs in combination with fluorescence
microscopy imaging has provided scientists with the ability to visualize and track
protein dynamics in living cells, tissues, and even entire organism with high
spatial and temporal resolution [ 6 , 7 , 17 , 22 - 25 ]. More recently, the development of