Am J Physiol Heart Circ Physiol 301: H663–H671, 2011.
First published June 10, 2011; doi:10.1152/ajpheart.00337.2011.
Review
In vivo bioluminescence for tracking cell fate and function
Patricia E. de Almeida,1,2 Juliaan R. M. van Rappard,4 and Joseph C. Wu1,2,3
Departments of 1Medicine and 2Radiology and 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University School of Medicine, Stanford, California; and 4Leiden University School of Medicine, Leiden, The Netherlands
Submitted 4 April 2011; accepted in final form 1 June 2011
bioluminescence imaging; heart; gene therapy; stem cell therapy; in vivo cell
tracking
THIS ARTICLE is part of a collection on Assessing Cardiovascular Function in Mice: New Developments and Methods.
Other articles appearing in this collection, as well as a full
archive of all collections, can be found online at http://ajpheart.
physiology.org/.
Cell-based therapies have rapidly emerged as a potential
therapeutic approach for heart disease. After the initial work to
characterize putative endothelial progenitor cells (1) and their
potential to promote cardiac neovascularization and to attenuate ischemic injury, a decade of intense research has examined
several novel approaches to promote cardiac repair in adult
life. A variety of adult stem and progenitor cells from different
sources have been examined for their potential to promote
cardiac repair and regeneration: bone marrow-derived cells
(55, 72), circulating and mobilized CD133⫹ and CD34⫹ stem
and progenitor cells (36), mesenchymal stem cells (56, 65, 83),
cardiac resident stem cells (43, 62), and skeletal myoblasts (54,
97). However, many questions such as the optimal type and
number of progenitor cells to be administered, the route of
administration, and the best time to administer cells after injury
remain unresolved. This is particularly true in vivo where it is
difficult to assess cellular activity in the heart in real time.
Therefore, it would be helpful to have practical tools to
accurately track cell location and their functional status over
time in vivo.
Address for reprint requests and other correspondence: J. C. Wu, Stanford
Univ. School of Medicine, Lokey Stem Cell Research Bldg., 265 Campus Dr.,
Rm. G1120B, Stanford, CA, 94305-5454 (e-mail: [email protected]).
http://www.ajpheart.org
Histopathological examination has been the main approach
for ex vivo evaluation of the distribution, the engraftment, and
the differentiation of injected cells. However, histopathology
precludes the evaluation and monitoring of these parameters in
real time and in vivo. Molecular and cellular imaging has
provided various techniques for identifying and tracking transplanted cells in cardiovascular research (15). Magnetic resonance imaging (MRI), computed tomography (CT), positron
emission tomography (PET), and single photon emission computed tomography (SPECT) offer deep tissue penetration and
high spatial resolution (64, 70, 94a). However, in small animal
studies, these techniques are more costly and time consuming
to implement compared with optical imaging. Among the
optical imaging tools, bioluminescence imaging (BLI) is a
promising technique that is especially useful in small animal
models and does not require the use of radionuclides with their
associated hazards. BLI is a high throughput technique that can
provide unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the
low background luminescence emanating from animals. BLI
can be very useful in evaluating the delivery efficiency of
therapeutic genes and their expression levels in vivo. By the
use of different cellular promoters to drive the expression of a
luminescent reporter gene, BLI allows monitoring of the transplanted cells’ differentiation status as well as their location and
functional characteristics in vivo (39, 51, 72, 78, 94).
Overall, BLI of reporters cloned into promoter/enhancer
sequences or engineered into fusion proteins has demonstrated
the modality’s ability to monitor fundamental processes such
as transcriptional regulation, signal transduction cascades, pro-
0363-6135/11 Copyright © 2011 the American Physiological Society
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de Almeida PE, van Rappard JR, Wu JC. In vivo bioluminescence for
tracking cell fate and function. Am J Physiol Heart Circ Physiol 301: H663–H671,
2011. First published June 10, 2011; doi:10.1152/ajpheart.00337.2011.—Tracking
the fate and function of cells in vivo is paramount for the development of rational
therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for
monitoring physiological processes in real time, ranging from cell survival to gene
expression to complex molecular processes. In mice and rats, BLI provides
unmatched sensitivity because of the absence of endogenous luciferase expression
in mammalian cells and the low background luminescence emanating from animals.
In the field of stem cell therapy, BLI provides an unprecedented means to monitor
the biology of these cells in vivo, giving researchers a greater understanding of their
survival, migration, immunogenicity, and potential tumorigenicity in a living
animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool
for semiquantitative measurements of gene expression in vivo, allowing a better
optimization of drug and gene therapies. Overall, this technology not only enables
rapid, reproducible, and quantitative monitoring of physiological processes in vivo
but also can measure the influences of therapeutic interventions on the outcome of
cardiac injuries.
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tein-protein interactions, protein degradation, oncogenic transformation, cell trafficking, and targeted drug action under in
vivo spatial registration. In this review, we will discuss recent
advances and applications of BLI, specifically as they apply to
cardiovascular research.
Principles of Bioluminescence
In nature, numerous luminous species exist in more than 700
genera, of which 80% are marine species (93). Luciferase
enzymes have been cloned from both marine (e.g., Renilla
luciferase) and terrestrial (e.g., firefly and click beetle luciferase) eukaryotic organisms and are commonly used as
reporters for in vitro and in vivo studies. They emit long
wavelengths of bioluminescence (⬎600 nm) in the red and
near-infrared regions of the spectrum and are efficiently transmitted through mammalian tissues (20, 86). These wavelengths
can avoid absorbing and scattering environment of mammalian
tissues (69), thus can be efficiently detected outside a small
animal’s body using BLI.
BLI is based on the detection of light emitted by cells that
express light-generating enzymes such as luciferase. In bioluminescent reactions, luciferase generates visible light through
the oxidation of enzyme-specific substrates such as D-luciferin
for terrestrial organisms (29, 30) and coelenterazine for marine
organisms (31, 53). Luciferases from different organisms can
be distinguished by their abbreviations: lux (bacterial), luc
(firefly), and lcf (dinoflagellate). To track cells in vivo by BLI,
the cells of interest need to be genetically modified to express
luciferase. The animal recipient of the cells receives the luciferase substrate either intraperitoneally or intravenously and
is placed in a light-tight dark box where luminescence is
detected (Fig. 1). In a bioluminescent reaction, the generation
of light depends on several factors. Firefly luciferase requires
ATP, Mg2⫹, and oxygen to catalyze the oxidation of its
substrate D-luciferin and generates CO2, AMP, inorganic pyrophosphate, oxyluciferin, and a yellow-green light at a wavelength that peaks at 562 nm (Fig. 2). By comparison, Renilla
luciferase catalyzes the oxidative decarboxylation of coelen-
Fig. 2. Representation of the bioluminescence reaction.
A cell expressing the firefly luciferase reporter gene
produces the luciferase enzyme that in the presence of
oxygen, ATP, and Mg2⫹ catalyzes D-luciferin into oxyluciferin, CO2, inorganic pyrophosphate (PPi), and light.
Light can then be detected, collected, and quantified by
a charge-coupled device camera.
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Fig. 1. Diagram illustrating steps required for bioluminescence imaging (BLI). A: therapeutic cells are transduced
with a luciferase (Luc) reporter construct and injected into
an animal. B: alternatively, luciferase-expressing cells can
be isolated from a luciferase transgenic animal and injected
to the experimental subject. C: after systemic substrate
injection, a charge-coupled device camera can be used to
localize the luciferase photon signals in vivo. Pseudo-colored images that represent signal intensity are overlaid with
grayscale reference images of the animal to facilitate localization of the signal. Cell survival, expansion, and homing,
as well as the expression of therapeutic genes, can be
monitored using this strategy.
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IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION
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emitted. A mutation of a single amino acid has been shown to
cause red-shifted luminescence and improve in vivo performance. Recently, a red-shifted mutant of luciferase from Photinus pyralis was created and described to have an emission
maximum of 612 nm at pH 7.0, a narrow emission bandwidth,
and to be thermostable (with a half-life 8.8 h at 37°C vs. 0.26
h for the wild-type luciferase). This Photinus pyrallis mutant,
called Ppy RE-TS reporter, has been successfully used in small
animals to visualize cancer progr ession and shown to have
superior in vivo imaging performance compared with the
wild-type photinus pyralis luciferase (6, 7).
The choice of reporter gene reporter probe pair to be used
should ultimately be based on the specific biological process to
be monitored, the duration and intensity of the signal needed,
and the tissue to be imaged (21, 100) (Fig. 3). As longer
wavelengths of light penetrate mammalian tissues with less
absorbance, luciferase from Photinus pyralis (⬎600 nm) is
more readily detected (17). Moreover, its substrate D-luciferin
remains in circulation longer than other substrates because it is
poorly catalyzed by mammalian tissues (101). However, Photinus pyralis luciferase has the disadvantage of having a longer
coding sequence (1,653 bp) compared with marine luciferases
such as Renilla and Gaussia. Renilla and Gaussia luciferases
have coding sequences of 936 and 558 bp, respectively, making them more suitable for studies that require compact transgene sequences, such as gene transfer and gene expression
studies. Another advantage of Renilla and Gaussia luciferases
over Photinus pyralis is the fact that they do not require ATP
as a cofactor during bioluminescent reaction and thus can be
used for imaging cells independent of their metabolic state (5,
66). One particular characteristic of Gaussia luciferase is that
it is naturally secreted (96) and therefore can be used as a
reporter for quantitative assessment of cells in vivo by measuring its concentration in blood (61). However, Renilla and
Gaussia luciferases produce shorter wavelengths of light
(peaking at 480 nm), which are not transmitted through tissues
as effectively as Photinus pyralis (100). Overall, the utility of
Renilla and Gaussia for in vivo BLI can benefit from improvements in sensitivity (46 – 48).
Codon-optimized humanized Gaussia (2, 79) has improved
its sensitivity in mammalian cells, but the pharmacokinetics of
its reporter probe coelenterazine are somewhat limiting in vivo.
Coelenterazine is prone to quick inactivation, including degradation through autoxidation. This substrate is also costly with
low solubility. Furthermore, coelenterazine binds to serum
proteins, is cleared rapidly from the bloodstream (101), and
decays rapidly with time (5). Therefore, when imaging in vivo
with coelenterazine, the signal needs to be acquired immediately after substrate administration. On the other hand, this
short half-life can be advantageous in cases in which sequential
imaging of two luciferases, such as Gaussia princeps and
Photinus pyralis, is required to monitor two biological processes in tandem in the same animal (5).
In summary, the development of reporter gene variants with
better emission spectra, brightness, and stability can ultimately
improve sensitivity and the overall performance of luciferases
in BLI imaging studies. Moreover, cloning new luciferases
from different organisms, such as those from Luciola italica
and Cratomorphus distinctus, can certainly improve current
applications and make novel ones possible (8, 88).
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terazine in the presence of dissolved oxygen to yield oxyluciferin, CO2, and blue light that peaks at 480 nm (49). Bioluminescence generated by the luciferase reporter reaction is
captured by a cooled charge-coupled device camera that can
detect very low levels of visible light emitted from internal
organs (19). Charge-coupled device camera-imaged bioluminescence can then be superimposed on photographic images of
the mouse to detect quantitatively and repetitively the bioluminescent signal from a given location (95). Imaging can be
conducted 10 to 15 min after intraperitoneal injection of
D-luciferin (reporter probe), with relatively stable light emission levels for 30 to 60 min, depending on the experimental
conditions. The sensitivity of detection depends on the wavelength of light emission, expression levels of the enzyme in the
target cells, the location of the source of bioluminescence in
the animal, the efficiency of the collection optics, and the
sensitivity of the detector (95).
Reporter genes commonly used for BLI. To track cells in
vivo, reporter genes and reporter probes must be able to reveal
cellular and molecular processes throughout an entire study
period, be highly sensitive to small changes in cell function and
distribution over time, and must not alter the labeled biological
process itself. A variety of reporter genes have been introduced
and validated for different imaging modalities, including various luciferases for BLI (5, 67, 74). Luciferase (Luc) is the only
one that produces light without requiring an external excitation
source, and they offer inherently low background signals
because animal tissues do not emit significant amounts of light.
Since the first report of the cDNA encoding Luc in 1985 (23),
DNA sequences have been optimized so that the Luc gene can
be expressed at high levels and its product localized in the
cytoplasm of the cells. For all reporter systems, the intensity of
light is proportional to the amount of luciferase expressed in
each individual cell or the number of cells in which a gene has
been transferred.
Of the many available luciferase enzymes, only a subset
have been developed and used as reporter genes. Luciferase
from the North American firefly Photinus pyralis is the most
common choice, but other luciferases such as the sea pansy
Renilla reniformis, the click beetle Pyrophorus plagiophalamus, and the copepod Gaussia princeps have also been investigated (48, 79). Gaussia and Renilla luciferase enzymes emit
in the blue/green region of the UV visible spectrum, where
light is strongly absorbed and scattered by tissues. Consequently, the imaging performance suffers from poor sensitivity
and spatial resolution. On the contrary, Photinus pyralis and
click beetle luciferase emit ⬃60% of their light at ⬎600 nm,
which enables great tissue penetration. Photinus pyralis has
emission at 620 nm when collected at 37°C, making it among
the longest emitting luciferases at mammalian body temperatures and the most sensitive for in vivo applications (100).
Bacterial luciferases (Lux) such as from Photorhabdus luminescens emit blue light that has been used for BLI of
bacterial infections. They are unique in that their lux operon
cassette codes for the luciferase and also for enzymes that
produce the substrate required for luminescence reaction,
thereby eliminating the need for exogenous substrate. Transferring this operon to mammalian cells would be advantageous
for in vivo imaging; however, limited research has been done
to determine whether this is possible (16). Mutations in luciferases can change the wavelength of the luminescence
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Vector-mediated expression of reporter genes. A fundamental requirement for BLI is the expression of a reporter gene
(e.g., luciferase) by the cells or tissues to be imaged, which
requires the introduction of genetically encoded imaging reporters into cells cultured in vitro (50). Alternatively, luciferase-expressing cells can be obtained from luciferase transgenic mice (Fig. 1). This can be achieved by using a reporter
vector that incorporates a luciferase gene driven by a promoter
that allows luciferase to be constitutively expressed by all cells
in the animal’s body (11, 83).
Delivery of bioluminescent reporter genes to cells has been
achieved through several means, but lentiviral-based gene
transfer has been the method of choice because of its effective
gene delivery and high expression levels of transgenes in
mammalian cells in culture as well as in vivo (22). Lentiviruses
have the capability to deliver target genes to both dividing and
nondividing cells and are capable of inserting genetic information into the host genome, ensuring prolonged gene expression
with a more limited host immune response (80). The safety of
the lentiviral vectors has been further improved with the
generation of self-inactivating vectors and the use of minimal
packaging systems. The efficiency of gene expression has been
improved by the introduction of a relatively strong internal
promoter such as cytomegalovirus. This promoter drives the
expression of the reporter gene and guarantees that the reporter
expression is always “on” under all conditions, in all tissue
types. Moreover, lentiviral vectors can be used to simultaneously induce the expression of multiple genes in a cell. Coupling the expression of a gene with a luciferase reporter gene
provides a simple yet effective mechanism for studying the
regulation of gene expression and monitoring it by BLI. This
provides exciting opportunities for transcriptional targeting,
double reporter labeling for BLI monitoring, as well as gene
therapy. For example, by linking the expression of luciferase to
the cardiac-specific promoter myosin light chain 2v, it is
possible to monitor cells undergoing cardiac differentiation
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over time via the detection of reporter gene expression by BLI
(27, 35).
Applications of BLI in Cardiovascular Research
Because physiological processes are dynamic in time and
space, end-point assays do not always provide a comprehensive understanding of biology in vivo. In cardiovascular research and many other scientific areas, BLI has been used for
noninvasive visualization of a variety of biological processes in
real time. In this section, we will review the many applications
of BLI in cardiovascular research.
Monitoring expression of therapeutic genes in the heart.
Gene therapy is a rapidly evolving field in cardiovascular
medicine. This technology allows for the correction of functional gene loss and enables the expression of a therapeutic
gene in a target tissue (52). Nevertheless, gene therapy studies
continue to be plagued by suboptimal delivery of genes, poor
survival of cells carrying the therapeutic gene(s), and the
inability to evaluate the levels of gene expression in vivo (28).
Thus the information gathered from BLI studies in small
animals can be used to design solid clinical trials that will help
improve gene therapy for cardiac repair. In cardiovascular
research, BLI has been used to address a variety of questions
that range from determining the effects of transgene expression
(e.g., BCL2) on cardiomyoblast survival and cardiac repair
(40) to testing the efficiency of anti-inflammatory drugs on the
expression of specific genes (e.g., inducible nitric oxide synthase) (99). Additionally, BLI has been used to optimize vector
systems (33) and treatment regimens (71) for delivery of
therapeutic genes (e.g., hypoxia-inducible factor-1␣) to the
heart.
The applications of BLI in research are constantly expanding
with the development of new therapeutic modalities. For instance, BLI enabled tracking the activity of a short hairpin
RNA plasmid in knocking down inhibitory factors of angio-
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Fig. 3. Emission wavelengths for the most commonly used reporter luciferase enzymes in BLI and their advantages and disadvantages. V, violet; B, blue; Y,
yellow; O, orange; R, red.
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IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION
BLI has demonstrated the poor survival of bone marrow
mononuclear cells (72), cardiac resident stem cells (43), mesenchymal stem cells (83), adipose stromal cells (82), ESCderived cardiac cells (10), and ESC-derived endothelial cells
(45) by 8 wk after cell injection. Conversely, the tumorigenic
potential of ESCs can also be uncovered by BLI. Lee et al. (42)
demonstrated that a minimum of 100,000 human ESCs are
necessary to form teratoma in the heart. Overall, BLI studies
have provided significant insights into the biology of stem cells
in vivo and how issues such as poor survival and potential
tumorigenicity must be overcome before translation into the
clinic.
Monitoring immune rejection of heart transplant and cell
grafts. Allogeneic heart transplantation is the most commonly
used therapy for end-stage heart disease. Transplant loss due to
immune rejection remains a significant problem in clinical
heart transplantation despite current immunosuppressive therapies (73). Acute rejection of heart transplants is an immune
response mediated by the coordinated infiltration and function
of host alloantigen-specific T cells in the allograft (57). In this
context, BLI has allowed monitoring of cardiac allograft over
time and revealed its rejection by day 12 after transplantation.
A decrease in the intensity of bioluminescence signals was
detected as early as day 4, suggesting acute graft rejection (78).
The correlation of bioluminescence intensity to other measures
of heart function such as beating score, fractional shortening,
and lymphocyte infiltration has provided additional means to
assess function and to determine the possible mechanisms
responsible for rejection.
Acute rejection can also be a major issue in human ESCbased therapy (25, 84). BLI has provided significant information for the development of therapeutic strategies to prevent the
rejection of stem cells. An immunosuppressant cocktail consisting of tacrolimus and sirolimus has been shown by BLI to
mitigate the rejection of human ESCs (77). Recently, Pearl et
al. (63) used BLI to demonstrate that a cocktail of costimulatory blockade agents (anti-CD40 ligand, cytotoxic T-lymphocyte antigen 4-immunoglobulin, and anti-lymphocyte functionassociated antigen-1) induced long-term allogeneic and xenogeneic human ESCs and induced pluripotent stem cell
engraftment. Overall, these studies elucidate the importance of
in vivo imaging for the development of therapies that may one
day enable stem cell therapy to become feasible.
Table 1. Cells types that have been investigated for their potential to promote cardiac repair using BLI
Cell Type
Bone marrow mononuclear cells
Adipose tissue-derived stem cells (ASCs)
Mesenchymal stem cells (MSCs)
Human CD34⫹ cells
Rat cardiomyoblasts
Embryonic stem cells
Skeletal myoblasts
ESC-derived endothelial cells (ESC-EC)
Resident cardiac stem cells
Information Gained from BLI
References
Comparison of different cell types for treatment of MI
Timing of cell delivery on acute vs. chronic MI
Systemic homing to injured heart
Long-term survival of cells in injured heart
Homing and survival of fresh versus cultured cells to injured heart
Comparison of ASCs vs. MSCs for treatment of MI
Growth factor-treated MSCs for treatment of MI
Cell fate in the heart using a MI model
Improvement of engraftment and survival with collagen matrix
Cell survival, proliferation, and migration
Cell fate in MI model
Cell fate in MI model
Effects of nicotine on the therapeutic effects ESC-ECs
Cell fate and function in MI model
83
76
72
3
4
82
24
89
38, 39
9, 42, 77
83
44, 45
98
43
BLI, bioluminescence imaging; MI, myocardial infarction.
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genic genes in the heart (32). The advances in reporter gene
technology and dual reporter labeling have enabled a simultaneous monitoring of distinct physiological events via BLI.
Dual-reporter labeling can address questions such as how the
expression of a gene affects the survival or differentiation of a
specific cell type or how it alters the expression of another
gene. However, challenges in designing dual-reporter systems,
such as finding adequate means for separating the two bioluminescent signals while retaining sensitivity, remain (60, 87).
Monitoring survival and homing of therapeutic cells. Stem
cell-based therapies are expected to have an enormous impact
in the treatment and cure of various diseases and disorders in
the future. These cells, given the right conditions, have the
ability to differentiate into the constituent cells of various
organs. When stem cells are used in therapies, it would be ideal
to track their migration or differentiation process in vivo. The
therapeutic use of bone marrow stem and progenitor cells
initially was popular and has been evaluated furthest in the
clinic setting. More recently, circulating stem and progenitor
cells, resident cardiac stem cells, and mesenchymal stem cells
have also been used in translational studies for clinical applications (15). Although small animal studies have provided
promising results, significant questions regarding the biology
of transplanted cells and their ability to promote cardiac repair
remain (92) (Table 1).
BLI is an important tool that can answer some of these
pressing questions by uncovering the dynamics of cell expansion, migration, and survival upon transplantation into the heart
(90, 102). Several studies have indicated that cardiomyocytes
purified from embryonic stem cells (ESCs) can benefit cardiac
function following myocardial ischemia (10, 12, 13, 41, 59)
with a low risk of tumor formation. However, one of the central
issues in cell therapy is the lack of long-term survival of these
therapeutic cells in vivo. BLI has shown that more than 90% of
transplanted adult stem cells die within the first 3 wk of
delivery (43, 45, 83). This could be the reason for the short
duration of improvement of cardiac function observed in multiple studies focused on cell therapy for cardiac repair. Thus the
problem of donor cell death continues to be troublesome and
may limit the overall efficacy of stem cell-based therapy,
making further studies to improve cell fate monitoring via
imaging technologies essential.
Failure of long-term survival of therapeutic cells has been
demonstrated for a variety of cell types (43, 45, 83) (Fig. 4).
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Limitations
BLI has been successfully used to obtain semiquantitative
measurements of biological processes because of a strong
correlation between the number of cells and the bioluminescence signal detected both in vitro (75) and in vivo (68).
However, a simple quantification of light emission may not
provide a true representation of biological processes. This is
because the firefly luciferase reaction is a complex interaction
of a variety of molecules (e.g., ATP, Mg2⫹, oxygen, and
luciferin) and because the intensity of the bioluminescence
signal depends on multiple factors. In particular, the number of
metabolically active luciferase-transfected cells, the concentration of luciferin, ATP and oxygen levels, the spectral emission
of bioluminescence probes, and the depth and optical properties of tissues are known to alter the intensity of bioluminescence signal (69). Another issue that should be considered
during quantitative BLI is the limited and wavelength-dependent transmission of light through animal tissues. Light sources
closer to the surface of the animal appear brighter compared
with deeper sources because of tissue attenuation properties
(91). It is estimated that for every centimeter of depth, there is
a 10-fold decrease in bioluminescence signal intensity (18).
Mathematical models can be used to predict in vivo imaging
signal levels and spatial resolution as a function of depth and
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to help define the requirements for imaging instrumentation.
However, the overall low spatial resolution (3–5 mm range)
and limited tissue penetrance restrict the use of BLI to small
animal studies (69).
Changes in tissue oxygenation can also alter bioluminescence signal. In rat gliosarcoma for instance, bioluminescence
signal has been shown to decrease by ⬃50% at 0.2% oxygen
(58). Thus, for reliable BLI measurements, it is important to
understand the effects of local niche in which the luciferaseexpressing cells of interest reside. This is especially the case in
cardiovascular studies involving hypoxia (e.g., myocardial infarction). Similarly, BLI quantification has to be carefully
interpreted in studies that involve surgical procedures. Changes
in tissue thickness because of the presence of inflammation,
edema, sutures, and animal growth can alter light absorption
and scattering as well as the bioluminescence signal.
Conclusions and Future Directions
Many different organisms, ranging from bacteria and fungi
to fireflies and fish, are endowed with the ability to emit light.
The discovery of new luminescence reporters and the use of
genetically modified reporters may further strengthen reporter
gene expression in mammalian cells, thus improving the
sensitivity and expanding the applications of this imaging
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Fig. 4. Monitoring survival and homing of cells via BLI. A: skeletal myoblasts (SkMb), bone marrow-derived mononuclear cells (MN), mesenchymal stem cells
(MSC), and fibroblasts (Fibro) were injected intramyocardially after myocardial infarction. All cell types demonstrated a decrease in bioluminescence signal
intensity starting at 4 wk postinjection. Yellow arrows indicate homing of MN to femur, spleen, and liver. Red arrows indicate cells retained in the heart and
lungs. Values in y-axis are in photons·s⫺1·cm⫺2·sr⫺1. Abbreviations in x-axis: d, day; w, week. Reprinted with permission (83). B: BLI demonstrating preferential
homing of bone marrow mononuclear cells to the ischemic myocardium in a model of ischemic reperfusion injury (I/R). Bioluminescence was also detected in
the spleen and bone marrow (yellow and red arrows, respectively). A progressive decrease in bioluminescence signal was observed starting at day 14
postinjection. Reprinted with permission (72). C: cardiac resident stem cells were injected in the heart following myocardial infarction. Robust bioluminescence
activity was detected on day 2 but mostly disappeared by 8 wk postinjection. Values in x-axis represent days post-cell injection. Reprinted with permission (43).
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IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION
ACKNOWLEDGMENTS
Because of space limitations, we were unable to cite all the important
papers relevant to bioluminescence imaging and cardiovascular research. We
apologize to investigators not mentioned here who have made significant
contributions to this field.
GRANTS
This work was supported in part by National Institutes of Health Grants
HL-093172, HL-099117, and EB-009689 (to J. C. Wu) and by the International Society for Heart and Lung Transplantation (to P. E. de Almeida).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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BLI allows real-time monitoring of survival and homing of
various therapeutic cells that are currently being investigated to
promote cardiac repair. By the use of cardiac-specific promoters linked to reporter genes, BLI can be used to monitor cell
differentiation in vivo. It also provides excellent opportunities
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