protein translocation in response to an agonist or external stimuli. A combination of
high-resolution microscopy and advanced image recognition software enables quantitative analysis of translocation events with reliable information on the efficiency
of the influencing stimulus. Recently Fetz et al. have developed three classes of
modular protein translocation biosensors tailored to investigate (1) signal-mediated
nucleocytoplasmic transport, (2) protease activity, and (3) protein-protein interactions [168]. Besides the mapping of protein function, the biosensors can also be
applied to identify chemicals and/or (nano)materials modulating the respective
protein activities and be used for RNAi-mediated genetic screens. In general, the
rapidly developing field of translocation sensors appears very promising both for
basic science studies and in drug development applications.
4 Advances in Biosensors for Animal Imaging
Whole-body animal imaging with fluorescent proteins has been shown to be a
powerful technology to develop various disease models. The red-shifted proteins
with brighter emission wavelengths are preferred candidates for in vivo models as
they are more sensitive owing to the reduced light absorption by tissue with much
lower scattering. For example, a protein called Katushka driven by the hybrid CAG
promoter activated upon Cre-mediated recombination has been developed by
Hurtado et al., for deep tissue imaging in mice models. This group successfully
demonstrated the expression of Katushka exclusively in a specific cell population
within the deep animal body such as pancreatic beta cells which can be monitored by
noninvasive whole-body imaging [169]. The implication of imaging biosensors in
animal studies is severely affected by the level of biosensors expression and the
wavelength of fluorescent proteins used for imaging [170]. For example, Audet et al.
[171] have developed a double transgenic mouse line co-expressing the beta-2
adrenergic receptor fused to Renilla luciferase (beta(2)AR-Rluc) and beta-arrestin2 fused to a green fluorescent protein (GFP2-beta arr2). Although the two halves of a
bimolecular reporter are driven by the same ostensibly ubiquitous reporter, the first
reporter was expressed reasonably brighter in a number of tissue types, whereas the
second reporter appeared only in testes. In addition, the low-level expression of
sensors with tissue-specific promoters further hampers the in vivo imaging ability of
sensors constructed using fluorescent proteins. The effective FRET studies also
cannot be carried out in vivo because of extremely low SNRs. The implication of
a fluorescent protein for whole-body imaging is largely determined by the emission
region and the brightness. Transgenic animal models with fluorescent proteins have
been utilized for tracking tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection even at subcellular resolution depending on the position
of the cells in the animal [172]. Deep tissue imaging in animal models has been
severely limited by the interference by skin autofluorescence. Apart from this, in few
instances, overexpression of biosensors also leads to unintended changes like
embryonic lethality or even perturbation of the physiological relevance of the sensor
Applications of Fluorescent Protein-Based Sensors in Bioimaging
171
high-resolution microscopy and advanced image recognition software enables quantitative analysis of translocation events with reliable information on the efficiency
of the influencing stimulus. Recently Fetz et al. have developed three classes of
modular protein translocation biosensors tailored to investigate (1) signal-mediated
nucleocytoplasmic transport, (2) protease activity, and (3) protein-protein interactions [168]. Besides the mapping of protein function, the biosensors can also be
applied to identify chemicals and/or (nano)materials modulating the respective
protein activities and be used for RNAi-mediated genetic screens. In general, the
rapidly developing field of translocation sensors appears very promising both for
basic science studies and in drug development applications.
4 Advances in Biosensors for Animal Imaging
Whole-body animal imaging with fluorescent proteins has been shown to be a
powerful technology to develop various disease models. The red-shifted proteins
with brighter emission wavelengths are preferred candidates for in vivo models as
they are more sensitive owing to the reduced light absorption by tissue with much
lower scattering. For example, a protein called Katushka driven by the hybrid CAG
promoter activated upon Cre-mediated recombination has been developed by
Hurtado et al., for deep tissue imaging in mice models. This group successfully
demonstrated the expression of Katushka exclusively in a specific cell population
within the deep animal body such as pancreatic beta cells which can be monitored by
noninvasive whole-body imaging [169]. The implication of imaging biosensors in
animal studies is severely affected by the level of biosensors expression and the
wavelength of fluorescent proteins used for imaging [170]. For example, Audet et al.
[171] have developed a double transgenic mouse line co-expressing the beta-2
adrenergic receptor fused to Renilla luciferase (beta(2)AR-Rluc) and beta-arrestin2 fused to a green fluorescent protein (GFP2-beta arr2). Although the two halves of a
bimolecular reporter are driven by the same ostensibly ubiquitous reporter, the first
reporter was expressed reasonably brighter in a number of tissue types, whereas the
second reporter appeared only in testes. In addition, the low-level expression of
sensors with tissue-specific promoters further hampers the in vivo imaging ability of
sensors constructed using fluorescent proteins. The effective FRET studies also
cannot be carried out in vivo because of extremely low SNRs. The implication of
a fluorescent protein for whole-body imaging is largely determined by the emission
region and the brightness. Transgenic animal models with fluorescent proteins have
been utilized for tracking tumor growth and metastasis, gene expression, angiogenesis, and bacterial infection even at subcellular resolution depending on the position
of the cells in the animal [172]. Deep tissue imaging in animal models has been
severely limited by the interference by skin autofluorescence. Apart from this, in few
instances, overexpression of biosensors also leads to unintended changes like
embryonic lethality or even perturbation of the physiological relevance of the sensor
Applications of Fluorescent Protein-Based Sensors in Bioimaging
171