nature in performing the assay and also its high-throughput and cost-effective
applications. In this chapter, we review the evolution of optical imaging with specific
emphasis on fluorescent proteins, as well as an introduction regarding the general
approach of optical imaging in in vitro and in vivo applications. We explain this by
briefly introducing different optical imaging methods and fluorescent assays developed based on fluorescent dyes and fluorescent proteins followed by a detailed
review of different fluorescent proteins currently used for various assay developments and applications.
Keywords BRET, Fluorescence dyes, Fluorescent proteins, FRET, In vivo imaging
1 Introduction
Fluorescent proteins and fluorescent dyes are routinely used to monitor biological
processes of cells in culture or cells in living animals; hence they are called
fluorescent reporters (FR). This includes fluorescent proteins, organic dyes, and
inorganic photonic materials. Fluorescent reporters are commonly used for developing assays involving fluorescence spectroscopy, fluorescence microscopy, and
whole-body preclinical imaging and to some extent in human applications for imageguided surgery in the operating room [1, 2]. Fluorescent reporters in combination
with an optical imaging system can provide key information in clinical oncologic
research while providing the opportunity to develop transgenic animal models for
studying various diseases, including cancer. Fluorescent dyes are widely used in
various bioassay applications. Here, we mainly discuss the role of fluorescent protein
as reporters (FPR) in various sensor designs and applications in bioimaging, drug
delivery, and drug discovery systems. We also briefly discuss the role of fluorescent
dyes in imaging applications (Fig. 1).
2 Fluorescent Biosensors and Evolution of Fluorescent
Protein Palette
Fluorescent proteins are frequently used for studying molecular mechanisms of cells
and physiological processes involved in cellular biological pathways. A plethora of
fluorescent proteins with characteristic excitation and emission spectra offer enormous scope for researchers to “paint” living cells as they desire [3]. At present we
have gone a step further and created sophisticated biosensors engineered with single
or multiple fluorescent proteins, including Förster Resonance Energy Transfer
(FRET)-based biosensors for studying macromolecular interactions in cells [4].
These fluorescent proteins exhibit environment-dependent changes in fluorescent
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U. K. Sukumar et al.
applications. In this chapter, we review the evolution of optical imaging with specific
emphasis on fluorescent proteins, as well as an introduction regarding the general
approach of optical imaging in in vitro and in vivo applications. We explain this by
briefly introducing different optical imaging methods and fluorescent assays developed based on fluorescent dyes and fluorescent proteins followed by a detailed
review of different fluorescent proteins currently used for various assay developments and applications.
Keywords BRET, Fluorescence dyes, Fluorescent proteins, FRET, In vivo imaging
1 Introduction
Fluorescent proteins and fluorescent dyes are routinely used to monitor biological
processes of cells in culture or cells in living animals; hence they are called
fluorescent reporters (FR). This includes fluorescent proteins, organic dyes, and
inorganic photonic materials. Fluorescent reporters are commonly used for developing assays involving fluorescence spectroscopy, fluorescence microscopy, and
whole-body preclinical imaging and to some extent in human applications for imageguided surgery in the operating room [1, 2]. Fluorescent reporters in combination
with an optical imaging system can provide key information in clinical oncologic
research while providing the opportunity to develop transgenic animal models for
studying various diseases, including cancer. Fluorescent dyes are widely used in
various bioassay applications. Here, we mainly discuss the role of fluorescent protein
as reporters (FPR) in various sensor designs and applications in bioimaging, drug
delivery, and drug discovery systems. We also briefly discuss the role of fluorescent
dyes in imaging applications (Fig. 1).
2 Fluorescent Biosensors and Evolution of Fluorescent
Protein Palette
Fluorescent proteins are frequently used for studying molecular mechanisms of cells
and physiological processes involved in cellular biological pathways. A plethora of
fluorescent proteins with characteristic excitation and emission spectra offer enormous scope for researchers to “paint” living cells as they desire [3]. At present we
have gone a step further and created sophisticated biosensors engineered with single
or multiple fluorescent proteins, including Förster Resonance Energy Transfer
(FRET)-based biosensors for studying macromolecular interactions in cells [4].
These fluorescent proteins exhibit environment-dependent changes in fluorescent
150
U. K. Sukumar et al.