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Proc. Arthropod. Embryol. Soc. Jpn. 53, 1–7 (2021)Ⓒ 2021 Arthropodan Embryological Society of Japan
ISSN 1341–1527[REVIEW]

Bioluminescence Microscopy for the Visualization of Gene Expression
Patterns and the Study of Embryonic Development*
Hirobumi SUZUKI
Olympus Corporation, Kuboyama 2–3, Hachioji, Tokyo 192–8512, Japan
E-mail: hirobumi2_suzuki@ot.olympus.co.jp

Abstract
Gene expression and its regulation are fundamental processes in cellular proliferation and differentiation, especially inmorphogenesis and embryogenesis. Real-time gene expression analysis for live cells has been performed using a gene promoter
assay with luciferase as the luminescent reporter. However the imaging of the promoter activity by microscopy is limited, since theintensity of the luminescence emitted from cells is too weak. To resolve this problem, a bioluminescence microscope was developedthat was optimized for low-light imaging. The real-time imaging of gene expression during embryogenesis using bioluminescencemicroscopy contributes substantially to the elucidation of the phenomena and the interpretation of the mechanisms of developmental
biology. For instance, it was elucidated that anisotropic tissue deformation along the proximal-distal axis occurred independently ofcell proliferation in the chick limb development based on tissue morphology, cell proliferation, and sonic hedgehog signaling activity
using bright field, fluorescence, and bioluminescence microscopies, respectively. However, the number of genes shown is limitedby bioluminescence microscopy. Recently, a multi-omics method based on the workflow of systems biology has allowed for the
integrated analysis of multiple-layer of organisms, from genes and expression to signaling pathways and metabolism. If image data of
the phenotype layer can be incorporated into multi-omics analysis, bioluminescence microscopy could also be used for the integrated
analysis of transcriptome and phenotype (morphogenesis or embryogenesis) layers.

Introduction
An organism composed of cells with various morphologies
and functions is made from a single fertilized egg during
embryogenesis. Therefore, gene expression and its regulation,
including signal transduction, are fundamental processes
in cellular proliferation and differentiation, especially inmorphogenesis and embryogenesis (e.g. Davidson, 1976;
Slack, 2001). In Drosophila, the mutation of homeotic genes
results in morphological abnormalities were known. Studies
on these genes have elucidated the relationships between theregulation of gene expression and morphogenesis related to thedetermination of the anterior-posterior axis, somitogenesis,
and appendage limb formation. Furthermore, these genes havebeen demonstrated to be involved in morphogenesis and areconserved in mammals and other animals (e.g., Gehring, 1998).
Microscopic technologies have been used in thesestudies. While morphometry uses conventional brightfield observation, three-dimensional (3D) reconstruction is
performed using a laser confocal fluorescence microscope.
The structural and functional protein localization is imaged
using immunostaining and fluorescence techniques. The studyof cellular signal transduction and second messenger, such as
Ca2+ or cyclic adenosine monophosphate (cAMP), dynamics
is measured in live cells using real-time imaging with afluorescent sensor probe. Molecular interactions and thetranslocation of signal transduction factors, such as thetranslocation of protein kinase C (PKC) from the cytoplasm
to the plasma membrane for nuclear factor kappa-light
polypeptide B (NF-κB) pathway activation, can be imaged
using fluorescence microscopy with green fluorescent protein(GFP) (Goda et al., 2015). In gene expression analysis, however,
the whole mount in situ hybridization method is performed
using immunostaining and/or fluorescence microscopy. Thismethod can be used to elucidate the detailed localization ofcells expressing specific genes, and is applicable for fixed
specimens. Therefore, the real-time imaging of gene
expression activity in live specimens is required, as it isessential to describe the phenomena and its interpretation
for morphogenesis or embryogenesis. Real-time gene
expression analysis for live cells has been performed using
a gene promoter assay with luciferase as the luminescentreporter. However the imaging of the promoter activity by* Review of a special lecture presented at the 55th Annual Meeting of the Arthropodan Embryological Society of Japan, May 31– June 1, 2019 (Fuchu, Tokyo).

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H. SUZUKI

microscopy is limited, since the intensity of the luminescenceemitted from cells is too weak. To resolve this problem,a bioluminescence microscope was developed that was
optimized for low-light imaging. This system has been widelyused for gene expression analysis in chronobiology (Yagita etal., 2010), neurobiology (Chang et al., 2016), developmental
biology (Morishita et al., 2015), medical research (Sramek etal., 2011), signal transduction analysis (Sugiyama et al., 2014),
molecular interaction (Compan et al., 2015), and radiation
biology (Kiru et al., 2018).
This review briefly summarizes the history of lowlight imaging of the promoter assay using luciferase and thespecification of a bioluminescence microscope developedwith bright luciferase development to improve luminescenceimaging. The recent applications of bioluminescencemicroscopy in developmental biology are also discussed.
Bioluminescence microscopy
for visualizing gene expression
In the late 1980s, the luciferase gene was first cloned
from the American firefly, Photinus pyralis (de Wet et al.,1987). Luciferase is commonly used as a reporter enzyme forthe promoter assays of gene expression (Brasier et al., 1989; deWet et al., 1989; Alam and Cook, 1990). In the luciferase reporter
assay, since the intensity of emitted light from cell populationor cell lysate is measured as total luminescence with a photoncounting luminometer, it is impossible to simultaneously
monitor the promoter activity of gene expression and cellularcharacteristics in the same cell as the image. Therefore,
microscopic imaging is essential for the gene expression
studies accompanied with cellular differentiation in embryonicdevelopment.
Advances in detector and digital imaging processing
systems have made it possible for low-light imaging to be
carried out using a high-sensitivity charge-coupled device(CCD) camera, such as liquid nitrogen cooled CCD cameras,
photon-counting CCD cameras, or image-intensifying CCD
cameras (Frawley et al., 1994; Thompson et al. (1995); Maire etal., 2000). This technology has allowed for the real-time image
analysis of gene expression dynamics within a single cell andelucidated the heterogeneity of gene expression in cultured
cells population for the prolactin gene promoter (Castaño etal., 1996; Takasuka et al., 1998), the L-pyruvate kinase genepromoter (Kennedy et al., 1997), and several other promoters(Rutter et al., 1995; White et al., 1995). In particular, theeffectiveness of microscopic imaging has been demonstrated
in clock genes analysis, namely in the oscillation of the clock
and its phase shift at the single cell level. Circadian oscillationsin the expression of clock genes are found not only in thecentral circadian pacemaker, suprachiasmatic nucleus (SCN),
but also in peripheral tissues. Welsh et al. (2004) found thatsingle fibroblasts are able to oscillate robustly, similar to SCN
neurons, but lacked coupling in dissociated cell cultures, which
led to a loss of synchrony among individual cells. The Notch
signaling component gene, Hes 1, is cyclically expressedin the presomitic mesoderm and constitutes the somite
segmentation clock. Masamizu et al. (2006) performed realtime bioluminescence microscopy of Hes 1 expression activity
in the presomitic mesoderm and embryo of mice and found thatcell-cell communication was essential for the synchronizationand stabilization of cellular oscillators.
However, temporal and spatial resolution was notenough to observe of cellular biological events or detect single
cells compared to conventional CCD cameras. Therefore,satisfactory analysis using bioluminescence microscopy islacking at the single-cell level. Subsequently, an electronmultiplying CCD (EM-CCD) camera, which yields higher
sensitivity and image quality, has been commercially released
and used for bioluminescence microscopy (Kwon et al., 2010;
Muranaka et al., 2013).
Development of a microscope optimized
for bioluminescence imaging
Although the image sensors of low-light imaging cameras
have been greatly improved over time, these improvements
have not been made commercially available for microscopes.
Because a microscope that has been designed with image
quality prioritized over image brightness. In the mid-2000s,
microscopes optimized for bioluminescence imaging werecommercially released by Olympus (Tokyo, Japan) and Atto
(Tokyo, Japan).
Generally, the degree of brightness (I) of an image isdirectly proportional to the square of the numerical aperture(NA) of the objective lens and inversely proportional to thesquare of the magnification (M) of the image. This can be
represented as I∝(NA/M)2. Therefore, a higher NA and lower
M yields brighter images. However, it is difficult to obtain bothconditions. A higher NA objective lens has a shorter focallength and, therefore, has a higher M. Thus, a high NA anda low M are mutual trade-offs. However, the value of NA/M
is the same as the NA value of the imaging lens (tube lens),
geometrically denoted as NAʼ. Therefore, a microscope with ahigh NAʼ (short focal-length imaging lens) allows for a higher
NA and lower M without requiring further improvements tothe objective lens. Thus, it was found that a higher value of I
(I > 0.01) was required for the bioluminescence microscopy ofsingle live cells (Suzuki et al., 2007; Ogoh et al., 2014; Suzuki
et al., 2016a). This is the concept on which the design of thebioluminescence microscope was based. Fig. 1 shows theinverted bioluminescence microscope constructed in ourstudies (Luminoview LV200; Olympus). A stage-top incubatorwith temperature and CO2 gas controllers is added to thesample stage. The observation area is covered with a dark box.
Figure 2 shows the bioluminescence images of U2OS
cells expressing beetle luciferase CBG99, CBR, and Luc2
(Promega, Madison, WI, USA) at 37 C captured using LV200
and IX70 microscopes (Olympus) with a UPlanFLN 40×
oil objective lens (Olympus) and DP70 color CCD camera(Olympus). The exposure times were 2 and 10 min for LV200(M = 8, I = 0.026) and IX70 (M = 40, I = 0.001), respectively.

GENE EXPRESSION AND BIOLUMINESCENCE MICROSCOPY

Thus, the bioluminescence images of cells expressing theluciferase gene could be captured using an LV200 microscopewith a conventional color CCD camera. In this case, the M of
the image was reduced by a power of 8 owing to the short focallength imaging lens with a I value of 0.026. To equalize the Ivalue between the LV200 and IX70 microscopes, a low M andhigh NA objective lens (e.g. 8×, NA 1.3) was required for IX70
(Ogoh et al., 2014; Suzuki et al., 2016a).
To show the spatial resolution of the bioluminescence
images acquired using LV200, organelle targeted images were
captured using an UPlanFLN 100× oil objective lens (Olympus)
and an ImagEM EM-CCD camera (C9100-13; Hamamatsu
Photonics, Shizuoka, Japan). NanoLuc luciferase (Promega),which is 150-fold brighter than firefly luciferase (Hall et al.,2012), was used as a tag for organelle localization, similar to afluorescent protein. Fig. 3 shows the bioluminescence imagesof NanoLuc fused with the nuclear localization sequence (NLS)

3(Fig. 3A), the mitochondrial targeting sequence (subunit VIII
of human cytochrome C oxidase, CoxVIII) (Fig. 3B), theendoplasmic reticulum resident protein, and the calreticulinwith KDEL retrieval sequence (Fig. 3C) or no targeting
sequence (Fig. 3D) with an exposure time from 300 msec to
1 sec in U2OS cells. Thus, the nucleus and cytoplasm werediscriminated clearly, and mitochondria and the endoplasmic
reticulum were observed in the cytoplasm (Ogoh et al., 2014;
Suzuki et al., 2016a).
Development of bright and multi-color luciferase
In addition to the developments of low-light imaging
cameras and microscopes, the development of bright
luciferases also greatly contributed to bioluminescencemicroscopy, and color variants of luciferase further allowed
to diversify bioluminescence microscopy in a manner similar
to fluorescence microscopy. Since the initial cloning of thefirefly luciferase gene, luciferase genes from many kinds ofbeetles (Coleoptera) have been cloned. The Jamaican click
beetle, Pyrophorus plagiophalam has two sets of light organs.
A pair of light organs on the dorsal surface of the prothorax
and a single light organ in the ventral cleft of the abdomen
emit yellowish green and orange luminescence, respectively.
Four types of luciferase clones were isolated using the colorof luminescence (green to orange) emitted by the light organs(Wood et al., 1989). Dual-color luciferase vectors (green andred color) were developed and released by Promega (ChromaLuc vectors). Viviani et al. (1999a) also cloned luciferase genes

Fig. 1 Bioluminescence microscope, LV200 (Olympus). A stage-top
incubator with temperature and CO2 gas controllers is added tothe sample stage. The observation area is covered with a darkbox.

Fig. 2 Bioluminescence images of U2OS cells expressing beetle
luciferase CBG99 (arrow 1), CBR (arrow 2), and Luc2 (arrow3) at 37 C captured by LV200 and IX70 microscopes with a
UPlanFLN 40× oil objective lens and DP70 color CCD camera.
The exposure times were 2 and 10 min for LV200 (M = 8, I =0.026) and IX70 (M = 40, I = 0.001), respectively. D-Luciferin,
1 mM. Scales bar = 100 μm (A) and 20 μm (B). This figure wasquoted and modified from Ogoh et al. (2014) and Suzuki et al.
(2016a) with open access terms and conditions of Wiley andIntechOpen.

Fig. 3 Bioluminescence images of NanoLuc fused with NLS (A),CoxVIII (B), calreticulin (C), and no targeting sequence (D)in U2OS cells at 37 C. Images were captured using an LV200
microscope with an UPlanFLN 100× oil objective lens andan ImagEM EM-CCD camera. The binning of the EM-CCD
camera was 1 × 1 (512 × 512 pixels), EM-gain was 1024, andexposure time was 300 msec (A, D), 500 msec (B), and 1 sec(C). Furimazine, 12.5 μM. Scale bar = 20 μm. This figure wasquoted from Ogoh et al. (2014) and Suzuki et al. (2016a) with
open access terms and conditions of Wiley and IntechOpen.

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H. SUZUKI

that emit green and red color luminescence from the Brazilian
railroad warms, Phrixothrix vivianii and P. hirtus, respectively.
Moreover, triple-color luciferase vectors (green, orange, andred color) were developed and released by Toyobo (Tripluc
vectors; Osaka, Japan). Furthermore, a luciferase gene that
displayed the most blue-shifted spectrum (λmax=538 nm)
among the beetles was cloned from the Brazilian click beetle,
Pyrearinus termitilluminans (Viviani et al., 1999b). It was
optimized for bioluminescence imaging (Nakajima et al., 2010),
which was 10-fold brighter than firefly luciferase and released
by Toyobo (Emerald Luc vectors). Five luciferase genes werecloned from Japanese and Malaysian fireflies and modified
for bioluminescence imaging, which emit a yellowish green
to orange color luminescence and were 2- to 12-fold brighter
than firefly luciferase, Luc2 (Promega). These were deposited
at RIKEN BioResource Research Center (Tsukuba, Japan)
under deposition numbers RDB14359–14363. Meanwhile, redshifted firefly luciferase was developed and used to accomplisha video-rate in vivo bioluminescence imaging from neurons inthe striatum of marmoset brain with D-luciferin substrate
modification (Iwano et al., 2018).
In contrast to the beetle luciferase, which requires
D-luciferin as a substrate, a luciferase originating from marineorganisms requires coelenterazine and emits blue color
luminescence. Renilla luciferase from a sea pansy was fused
to yellow fluorescent protein (YFP) and used to construct a
bioluminescence resonance energy transmittance (BRET)
system (Hoshino et al., 2007). The fusion protein emitted
yellow luminescence by BRET and was 4-fold brighter thanthe original Renilla luciferase by improving the quantum yieldof light emission. Saito et al. (2012) constructed Nano-lantern,which is a chimera of enhanced Renilla luciferase and Venus(enhanced YFP) with a high BRET efficiency. It was 12-foldbrighter than the Renilla luciferase, and its color derivatives
allowed for the bioluminescent sensing of Ca2+, cAMP andadenosine 5ʼ-triphosphate (ATP) within live single cells in thesame manner as in fluorescence microscopy (Takai et al., 2015;
Suzuki et al., 2016b).
NanoLuc luciferase (Promega), obtained from the deepsea shrimp, Oplophorus gracilirostris was 150-fold brighter
than firefly luciferase with the modified coelenterazine,
furimazine (Hall et al., 2012). Based on the NanoLuc, Robers etal. (2015) and Dixon et al. (2016) developed a protein-fragmentcomplementation (PCA) system using circular permutatedlarge and small fragments (NanoBRET, NanoBiT, HiBit;
Promega). It allowed not only for low-level gene expression

imaging but also for protein-protein interaction imaging in live
cells.
Advantages and limitationsof bioluminescence microscopy
Since bioluminescence microscopy requires no light
excitation, it is substantively different from fluorescencemicroscopy. Firstly, bioluminescence microscopy has none of
the autofluorescence problems associated with fluorescencemicroscopy which include a high background noise, a low
signal/noise ratio, and low quantitativity. In fact, it wasdemonstrated that the autofluorescence of Drosophila embryo
can cause problems during fluorescence microscopy (Akiyoshi
et al., 2014). Secondly, bioluminescence microscopy is lessphototoxic to live cells during their observation, since oneof causes of the phototoxicity is due to generation of reactive
oxygen species from fluorescent dyes by light irradiation (Ichaet al., 2017). Therefore, bioluminescence microscopy permits
non-lethal and long-term observation of live cells and issuitable for cell differentiation and embryonic studies. Thirdly,
bioluminescence microscopy is suitable for the observation oflight-sensitive cells, such as opsin-expressing cells (Sugiyama
et al., 2014) or retina cells, as there is no need to consider theeffect of fluorescent dye excitation light on photo-stimulation.
However, bioluminescence microscopy requires a
substrate. Although the substrate is derived from the biologicalcomponents of luminous organisms (firefly or sea pansy),
a high concentration of substrate can affect cell viability. As
such, it is necessary to consider the optimal concentration ofsubstrate according to cell types and luciferase characteristics(Suzuki et al., 2017).
Although bioluminescence microscopy has been achievedby improving the imaging devices and luciferases, the emission
intensity from cells remains lower than that obtained using
fluorescence microscopy. Therefore, the spatio-temporal
resolution of bioluminescence microscopy is limited compared
to fluorescence microscopy.
Table 1 summarizes the characteristics ofbioluminescence and fluorescence microscopies andhighlights the use of bioluminescence microscopy as a
powerful tool in cellular biology as well as its use as acomplementary strategy to fluorescence microscopy.
Recent applications in developmental biology
Drosophila
Akiyoshi et al. (2014) constructed a transgenic Drosophila

Table 1 Substantive differences between bioluminescence and fluorescence microscopy.
Excitation energy
Auto-fluorescence
Photo-toxicity
Long term observation
Observation of light-sensitive cells
Brightness of image
Spatio-temporal resolution

Bioluminescence
Chemical reaction
None
None
Excellent
Excellent
Acceptable
Acceptable

Fluorescence
PhotonAffected
Affected
Acceptable
Acceptable
Excellent
ExcellentGENE EXPRESSION AND BIOLUMINESCENCE MICROSCOPY

carrying armadillo (arm) and luciferase fusion gene using a
Gal4-UAS system. The arm is a segment polarity gene andencodes the mammalian homologue of β-catenin in mammals,
which is a key mediator of the Wnt signaling pathway (Peifer
et al., 1991) as well as being involved in cell–cell adhesion(Peifer et al., 1993). Although the arm expression has beencharacterized during the early stage of embryogenesis usingmRNA in situ hybridization (Riggleman et al., 1989), Akiyoshi
et al. (2014) demonstrated the continuous expression ofarm throughout the entire process of embryogenesis using
bioluminescence microscopy, and found that its expression was
dramatically increased in the anterior midgut rudiment, themyoblasts of the dorsal/lateral musculature, and the posterior
spiracle after stage 13 (Fig. 4), as well as in the cephalic region
just before hatching at stage 17. Furthermore, they also foundan arm expression pattern in embryos treated with ionomycin
or 6-bromoindirubin-3-oxime (BIO), an inhibitor and an
activator of Wnt/β-catenin signaling, respectively.
Chick
Sonic hedgehog (SHH) is expressed in the polarizing
region of the vertebrate embryo and is essential for theanterior-posterior patterning of the limb bud (Tickle
and Towers, 2017). Morishita et al. (2015) constructed atissue deformation map of the chick limb development tocharacterize the geometrical tissue growth modes basedon tissue morphology, cell proliferation, and SHH signaling
activity using bright field, fluorescence, and bioluminescencemicroscopies, respectively. They found that SHH signaling
activity changed dynamically through the developmental stage
and growth mode shift, and demonstrated that anisotropic
tissue deformation along the proximal-distal axis occurred
independently of cell proliferation.
Zebrafish
The Wnt/β-catenin signaling gradient acts as a morphogen
to determine the embryonic anterior-posterior axis in
mammals (Huelsken et al., 2000). Akieda et al. (2019) identified
unfit cells that appear spontaneously with abnormal Wnt/βcatenin activity and produce noise in the signaling gradient inzebrafish. These unfit cells were eliminated by apoptosis with
Smad signaling and reactive oxygen species production via theFig. 4 Merged bright field and bioluminescence images of armadillo
expression at stage 14 (9 h 20 min after fertilization) from lateral(A) and dorsal (B) side. Increased armadillo expression wasobserved in the anterior midgut rudiment (1), myoblasts of thedorsal/lateral musculature (2), and the posterior spiracle (3).
Scale bar = 100 μm. This figure was quoted from Akiyoshi etal. (2014) with open access terms and conditions of Springer.

5communication of unfit cells with neighbouring fit cells using
cadherin. Thus, the Wnt/β-catenin signaling gradient wasestablished by elimination of the unfit cells. In this study, thereal-time imaging of β-catenin gene expression in live embryos
using bioluminescence microscopy contributed substantiallyto these findings.
Perspectives
As shown in the previous “Recent applications indevelopmental biology” section, the real-time imaging ofgene expression (promoter activity) during embryogenesis
in a single specimen using bioluminescence microscopy
contributes substantially to the elucidation of the phenomena
and the interpretation of the mechanisms of developmental
biology. Since the spatio-temporal resolution of real-time
bioluminescence microscopy is limited, it is compensated byfluorescence microscopy using fixed tissue as the end point
analysis. Therefore, the combination of bioluminescence andfluorescence microscopy in live cells or embryo is required.
Goda et al. (2015, 2017) developed a method that combines
bioluminescence and fluorescence microscopy by avoiding
autofluorescence from D-luciferin, and showed the sequential
imaging of the same cells from a signal transduction event(PKC translocation from cytosol to plasma membrane)using fluorescence microscopy to gene expression regulated
by nuclear factor kappa-light polypeptide B (NF-κB)
using bioluminescence microscopy, together with the cellmorphology using bright field microscopy. However, problems
with autofluorescence remained in the sample.
The transcriptome analysis using microarray or RNA
sequencing methods has revealed that a large number ofgenes being expressed, and even the expression of all genes
at once, as well as the activation of signal pathways can be
specified based on the specific expressing gene groups, with ahuge amount of data in biological systems analysis (Luo et al.,2009). Meanwhile, bioluminescence microscopy allows for thevisualization of real-time genes expression patterns, together
with the morphological changes of cells or tissues as images.
However, the number of genes shown is limited. Recently,
a multi-omics method based on the workflow of systems
biology has allowed for the integrated analysis of multiplelayer of organisms, from genes and expression to signaling
pathways and metabolism (Pinu et al., 2019). If image dataof the phenotype layer can be incorporated into multi-omics
analysis, bioluminescence microscopy could also be used
for the integrated analysis of transcriptome and phenotype(morphogenesis or embryogenesis) layers.
Acknowledgements: I would like to thank Emeritus
Professor Dr. Y. Kobayashi (Tokyo Metropolitan University)
for providing me with the opportunity to present a special
lecture in the 55th Annual Meeting of the Arthropodan
Embryological Society of Japan at Fuchu in Tokyo in 2019. I
would also like to thank my colleagues at Olympus Corporation
for their technical assistance and fruitful discussions during

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H. SUZUKI

the development of the bioluminescence microscope.

Protocols in Cell Biology, 77, 4.35.1–4.35.16.
Hall, M.P., J. Unch, B.F. Binkowski, M.P. Valley, B.L. Butler, M.G. Wood,

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