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S. P. Singh and S. Siddhanta
fluorophores [4]. The extinction coefficient (ability to absorb light in relation to the
concentration) and quantum yield (efficacy of fluorescence remission) are the two
main parameters used to characterize the brightness of fluorophores. A large variety
of organic dyes, nanoparticles (quantum dots), and fluorescent proteins have been
utilized as fluorophores. Among these, the organic dyes are very common as they
cover the entire visible and NIR spectrum. Also, the fact that these are water-soluble
and consist of active functional groups (amino, carboxy, thiol, azide, etc.) that can
facilitate covalent interaction with target molecules makes them more desirable. The
targeting ability of these dyes is enhanced through the conjugation of antibodies. The
utility of these fluorophores in understanding the intricacies of biological systems
has been evaluated through multiple studies ranging from cells to tissues. Another
type of commonly employed fluorophores includes fluorescent proteins (FP) from
natural sources. Green fluorescent protein (GFP) from jellyfish Aequoria victoria is
one of the most commonly utilized FP in biological imaging [13, 14]. Their applications are wide-ranging and relatively easy as spectral and optical properties of these
molecules are very well characterized. The original GFP has been modified extensively through mutagenesis to generate a palette of visible fluorescent proteins with
different colors, lifetimes, and photoactivation properties. The absorption spectrum
of GFP has a peak around 398 nm and a minor band at 475 nm. Also, the GFP
has a high quantum yield of 0.79 [13]. These proteins have been most commonly
utilized in monitoring protein localization and trafficking. FPs also can sense the
environmental pH [15] and metabolites [16]. The different category of fluorophores
has been shown to produce almost any color of light. Imaging of a variety of transparent specimens such as cells, bacteria, tissue components, or other pathogens has
been made possible due to these fluorophores. The fluorescence microscopy technique with the attributes of a high degree of specificity has become an essential
component of biomedical and material science research [17]. In a typical fluorescent
microscope, the fluorophore-labeled samples are illuminated with excitation light
to produce fluorescence and yields high contrast images. By employing multiple
labels together, several target molecules in a specimen can be imaged simultaneously. Although the spatial resolution of fluorescence microscopy cannot go beyond
the diffraction limit of specimens, few studies have revealed its ability in identifying
signals originating from single molecules as well [18]. In the subsequent sections, a
brief discussion about several advancements in the fluorescence imaging methods is
presented.
4.1 Fluorescence Lifetime Imaging Microscopy (FLIM)
Fluorescence lifetime imaging microscopy (FLIM) is a variant of fluorescence
imaging where the total amount of time (lifetime) an electron spends in the excited
state before releasing a photon and coming back to the ground state is measured
S. P. Singh and S. Siddhanta
fluorophores [4]. The extinction coefficient (ability to absorb light in relation to the
concentration) and quantum yield (efficacy of fluorescence remission) are the two
main parameters used to characterize the brightness of fluorophores. A large variety
of organic dyes, nanoparticles (quantum dots), and fluorescent proteins have been
utilized as fluorophores. Among these, the organic dyes are very common as they
cover the entire visible and NIR spectrum. Also, the fact that these are water-soluble
and consist of active functional groups (amino, carboxy, thiol, azide, etc.) that can
facilitate covalent interaction with target molecules makes them more desirable. The
targeting ability of these dyes is enhanced through the conjugation of antibodies. The
utility of these fluorophores in understanding the intricacies of biological systems
has been evaluated through multiple studies ranging from cells to tissues. Another
type of commonly employed fluorophores includes fluorescent proteins (FP) from
natural sources. Green fluorescent protein (GFP) from jellyfish Aequoria victoria is
one of the most commonly utilized FP in biological imaging [13, 14]. Their applications are wide-ranging and relatively easy as spectral and optical properties of these
molecules are very well characterized. The original GFP has been modified extensively through mutagenesis to generate a palette of visible fluorescent proteins with
different colors, lifetimes, and photoactivation properties. The absorption spectrum
of GFP has a peak around 398 nm and a minor band at 475 nm. Also, the GFP
has a high quantum yield of 0.79 [13]. These proteins have been most commonly
utilized in monitoring protein localization and trafficking. FPs also can sense the
environmental pH [15] and metabolites [16]. The different category of fluorophores
has been shown to produce almost any color of light. Imaging of a variety of transparent specimens such as cells, bacteria, tissue components, or other pathogens has
been made possible due to these fluorophores. The fluorescence microscopy technique with the attributes of a high degree of specificity has become an essential
component of biomedical and material science research [17]. In a typical fluorescent
microscope, the fluorophore-labeled samples are illuminated with excitation light
to produce fluorescence and yields high contrast images. By employing multiple
labels together, several target molecules in a specimen can be imaged simultaneously. Although the spatial resolution of fluorescence microscopy cannot go beyond
the diffraction limit of specimens, few studies have revealed its ability in identifying
signals originating from single molecules as well [18]. In the subsequent sections, a
brief discussion about several advancements in the fluorescence imaging methods is
presented.
4.1 Fluorescence Lifetime Imaging Microscopy (FLIM)
Fluorescence lifetime imaging microscopy (FLIM) is a variant of fluorescence
imaging where the total amount of time (lifetime) an electron spends in the excited
state before releasing a photon and coming back to the ground state is measured