Bioluminescent and fluorescent reporters
in circadian rhythm studies
Takako Noguchi and Susan Golden
*This review article assumes basic knowledge of gene expression and light wavelengths
Abstract
Various organisms create fascinating “glow-in-the-dark” proteins in nature. Scientists introduce these
special proteins into organisms that they study and use the emitted light to report where, when, and
how much genes or proteins of interest are expressed. Two major kinds of reporters, luciferases and
fluorescent proteins, are often confused with each other because they have common characteristics: (1)
they can be used to make it easy to track the expression of genes or proteins of interest by fusing a
reporter gene to a gene of interest (genetically encoded reporters), and (2) they glow in the dark.
However, there are fundamental differences between these reporters. Generally, luciferases, which
catalyze a reaction with a substrate luciferin and generate light as a product (bioluminescence), are dim,
but highly quantitative reporters and suitable to measure how much and when a gene or protein is
expressed. Fluorescent proteins, which require an external light source to stimulate emission of a
different wavelength of light, are bright reporters and suitable to locate in which cells a gene is activated
or where in a cell a protein is expressed. In this review, we introduce basic mechanisms of
bioluminescence and fluorescence, and how they are used in circadian rhythm research.
Entry Level
Intermediate Level
Advanced Level
Mechanisms to glow in the dark
As you study biological science, you encounter experiments using “bioluminescence” and
“fluorescence.” Both bioluminescence and fluorescence appear as specimens glowing in the dark.
However, the light is generated by different mechanisms. Bioluminescence is generated by chemical
reactions, whereas fluorescence is the re-emission of transformed light that was absorbed by a
substance at a different wavelength. In other words, bioluminescence can glow by itself like stars, but
fluorescence needs an external light source to glow, like the moon needs the sun to shine. In this
section, we will introduce the mechanisms that generate bioluminescence and fluorescence.
Bioluminescence
1
Figure 1. (A) Firefly (genus species unidentified) emitting yellow bioluminescence. (B) A tide glowing in blue by
bioluminescence created by unicellular plankton, dinoflagellates. (C) Bioluminescent mushrooms (Mycena
chlorophos).
[Attribution and copyright information: (A) This picture is copyrighted by Radim Schreiber, whose permission is
required for any usage. (B) This picture was cropped from a picture taken by Mike at Carlsbad, CA. CC BY-SA 2.0 (C)
Lalalfdfa. CC BY-SA 3.0.]
Bioluminescence is light emitted by living organisms. Bioluminescent organisms are found in most
branches of the evolutionary tree, including bacteria, fungi, invertebrates, and vertebrates (Oba and
Schultz, 2014) (Fig. 1). Organisms use bioluminescence for various activities, such as mating, attracting
prey, and escaping from predators (Oba and Schultz, 2014). The bioluminescence is generated by
chemical reactions involving an enzyme, luciferase, and a substrate (a small chemical molecule), called
luciferin (Shimomura, 2006). There is no similarity in the sequence of luciferases or chemical structures
of luciferins of organisms in different taxa, suggesting that bioluminescence was reinvented multiple
times in evolution. However, there is one common mechanism that generates bioluminescence: a
luciferase catalyzes oxidation of a luciferin using molecular oxygen as a substrate (Wilson and Hastings,
1998). The reactions are well studied in some organisms, such as fireflies (Shimomura, 2006) (Fig. 2), but
in some organisms, such as mushrooms, the bioluminescence reactions are not understood well
(Oliveira et al., 2012; Purtov et al., 2015).
Bioluminescence can be emitted in complete darkness through chemical reactions. Bioluminescence
is very dim compared to sun light or room light and usually occurs in naturally dark environments, such
as at night or in the deep sea.
Figure 2. Luciferin-luciferase reaction in firefly. Light emitting reaction requires D-luciferin, adenosine triphosphate
(ATP), and oxygen (O2). A luciferase and a cofactor (Mg2+) catalyze the reaction to generate oxyluciferin, adenosine
monophosphate (AMP), pyrophosphate (PPi), carbon dioxide (CO2), and light.
[Attribution and copyright information: D-lucifein, Yikrazuul, Public domain; oxylucifein, modified from
Yikrazuul’s D-lucifein by Takako Noguchi, Public domain; Crystal structure of firefly luciferase was obtained from
Protein Data Bank in Europe, Sundlov et. Al, Biochemistry (2012) (Sundlov et al., 2012), Terms of Use.)]
Fluorescence
Fluorescent substances absorb a higher energy (or shorter wavelength) of light and emit light at a
lower energy (or longer wavelength) (Fig. 3A). The higher energy light absorbed by a fluorescent
substance is called excitation light. The re-emitted light is called fluorescence (Lichtman and Conchello,
2005). Because wavelengths of emitted and excitation light are different, we can filter out excitation
2
light so that only the fluorescence is visible (Wilson and Hastings, 1998) (Fig. 3B). Thus, fluorescence
appears to be glowing in the dark because of light filtration, but the light does not originate with the
fluorescent material.
Fluorescent compounds are called fluorophores. At the atomic level the energy of excitation light is
absorbed by an electron of a fluorophore, causing it to move to a higher energy (excited) state. As the
electron returns to the lower energy (ground) state, the energy difference between states is released as
light (fluorescence) (Fig. 3C). Because a part of the energy is lost during this process, the wavelength of
fluorescence is longer than that of the excitation light. For example, blue excitation light turns into
green, and yellow light becomes red. This transition of energy states is also the basis of emission in
bioluminescence, although the energy that excites an electron is supplied by chemical reactions in
bioluminescence. Thus, bioluminescence is fluorescence in a strict physics definition. However, in the
biological field, scientists conventionally distinguish bioluminescence and fluorescence by the source of
energy that excites an electron, which is a chemical reaction (oxidation of luciferins) in bioluminescence
and external excitation light in fluorescence. This distinction dramatically affects the design of
experiments.
Figure 3. Basics of fluorescence. (A) Spectrum of visible light. White light consists of light with a wide range of
wavelengths. Photons related to shorter wavelengths (violet and blue) have higher energy than longer
wavelengths (orange and red). (B) Filtering fluorescence. A light source sends a broad range of light. An excitation
filter selectively passes the wavelength that is absorbed by a fluorescent compound. In this case, blue excitation
light is used for green fluorescent protein (GFP). A dichroic mirror reflects and passes selective wavelengths of
light. In this case, it reflects blue light and passes green light. An emission filter selectively passes the wavelength
of the fluorescence: this case, green light from GFP. Finally, filtered fluorescence is captured by a detector. (C)
Generation of fluorescence at the atomic level. Excitation light moves an electron to an unstable excited state. An
excited electron instantaneously relaxes and returns to ground status, emitting fluorescence as the difference in
energy between the two states, with some energy lost to entropy.
[Attribution and copyright information: (A) Picture was modified by Takako Noguchi based on Philip Ronan’s, CC
BY-SA 3.0 (B) Crystal structure of GPF was obtained from Protein Data Bank in Europe, Arpino JAJ, et. al, Biol.
Crystallogr. (2014) (Arpino et al., 2014), Terms of Use. The other part of diagram was created by Takako Noguchi.
CC BY 4.0. (C) Picture was created by Takako Noguchi. CC BY 4.0.]
3
Fluorescence is emitted by various substances including natural minerals and synthetic chemical
compounds. Fluorescence generated by organisms (biofluorescence) can be derived from fluorescent
proteins, such as the green florescent protein (GFP) found in jellyfish (Aequorea Victoria) (Shimomura et
al., 1962; Shaner et al., 2005) or small molecules generated by or consumed by organisms (GandiaHerrero et al., 2005). Biofluorescence is found in various organisms including bacteria (Drepper et al.,
2007), corals (Matz et al., 1999), flowers (Gandia-Herrero et al., 2005), butterflies (Vukusic and Hooper,
2005), fish (Sparks et al., 2014), and birds (Arnold et al., 2002) (Fig. 4). Some biofluorescence is thought
to have ecological advantages. For example, it protects corals by transforming harmful ultraviolet (UV)
to lower energy light (Salih et al., 2000), or attracts insects for pollination in flowers (Gandia-Herrero et
al., 2005). However, there are many substances in organisms that fluoresce without obvious ecological
reasons, such as heme in red blood cells, and riboflavin, a vitamin.
Figure 4. Biofluorescence in marine animals. (A-D) A coral and shark with or without fluorescent lighting
conditions.
[Attribution and copyright information: (A-B) Mikhail Matz, University of Texas, Joerg Wiedenmann, National
Oceanography Centre, Southampton, National Science Foundation, Conditions of use. Link to source of A and B. (C)
The picture was cropped from Rennett Stowe’s, CC BY-SA 2.0. (D) The picture was cropped from figure 1 of Sparks
et al. 2014 (Sparks et al., 2014), CC BY 4.0.]
Characteristics of bioluminescent and fluorescent reporters
Genetically encoded reporters, such as luciferases and fluorescent proteins, are powerful tools to
detect expression of genes and proteins. By connecting a reporter gene to the promoter or coding
sequence of a gene of interest, scientists can tell where, when, and how much the gene or protein is
expressed. Generally, fluorescent protein reporters are suitable to locate in which cell or tissue the gene
is activated or where in a cell the protein is expressed. Luciferases are suitable to quantify how much
and when the gene is expressed, but are usually not sufficiently bright for pinpointing where the light is
coming from. In this section, we introduce characteristics of bioluminescent and fluorescent reporters.
4
Evaluating gene expression by reporters
In the biological field, the word “gene expression” is often used to describe transcriptional activity,
including promoter activity and mRNA level, but it can also include protein level as well. The DNA
sequence of a gene is transcribed into RNA, which is then translated into a protein. A promoter located
upstream of a gene is the DNA sequence that directly controls the amount of transcription (affecting
mRNA level), and indirectly controls the amount of translation (protein level). Thus, the combination of
promoter activity, mRNA level, and protein level contribute to overall gene expression (Adams, 2010).
Scientists can evaluate promoter activities – the frequency of a transcript arising from a promoter – by
measuring the signal from a reporter that is transcribed/translated under control of the promoter, or
can monitor the protein product by a signal from a fusion protein made up of the protein of interest
fused directly to a reporter protein. However, mRNA levels are generally measured by other molecular
biology techniques, such as q-RT-PCR (quantitative real-time polymerase chain reaction), and RNA
sequencing.
Bioluminescent reporters
Bioluminescent reporters, also referred to as luciferase reporters, have advantages for quantifying
the expression level of genes and proteins. The characteristics that make luciferases more quantitative
reporters than fluorescent proteins are the following. (1) Low background. Bioluminescence recording is
performed in complete darkness by a light detector. There is virtually no background noise because the
luciferin-luciferase reaction is the only light-emitting reaction in cells and tissues. Because of this low
background, luciferase reporters allow detection of a lower level of gene expression than fluorescent
proteins (Choy et al., 2003). (2) Large dynamic range. Dynamic range refers to how low and high a
reporter can quantify a variable and still be linear (i.e., the range where light emission accurately reflects
the amount of material being measured). A light detector can measure more than 108-fold changes in
the amount of luciferase (Promega Corporation, 2000). (3) Adequate instability. Real-time increases and
decreases of gene expression in living cells cannot be measured with highly stable reporter proteins
because of accumulation of residual reporters. It is important that reporters are degraded when the
gene is not being actively transcribed and the message translated. Stability of proteins is indicated by
half-life, the time taken for proteins to drop in amount to half of the original level. The half-life of wildtype GFP is about 26-54 h depending on the conditions (Sacchetti et al., 2001; Kitsera et al., 2007). In
contrast, the half-life of firefly luciferase is about 3-4 h (Leclerc et al., 2000). Thus, luciferase is typically
better for circadian studies, to be able to detect one trough before a gene turns back on in the next
cycle. To faithfully report rapid changes of gene expression, there are cases where the half-life of
luciferase is further shortened by addition a destabilization sequence, such as a “PEST” sequence
(ProGluSerThr), promoting proteolysis (Leclerc et al., 2000).
A disadvantage of luciferase reporters is the dimness of the light. Bioluminescence is commonly
detected by photomultiplier tubes (PMT), a type of light detector that is capable of amplifying the signal
of photons (Hamamatsu Photonics KK, 2006). In many applications in circadian rhythm studies PMTs
detect summed bioluminescence from all samples in a tube or a plate. In this case, luciferase reports
quantity or temporal gene and/or protein expression (how much or when the gene or protein is
expressed), but not spatial information. It is possible to capture spatial information (where the light is
coming from) by luciferases, but it requires high-end detectors, such as super-sensitive cooled CCD
(charge-coupled device) camera, or a CCD camera with a photon enhancement function (Welsh and
Noguchi, 2012). These bioluminescent images taken by high-end detectors carry all quantity, temporal,
and spatial information. But the spatial resolution for bioluminescent images is much courser than
fluorescent images. For example, bioluminescent images can only visualize the shape of a single
eukaryotic cell, but fluorescent images can visualize down to the organelle level (Figs. 5-6).
5
Figure 5. Spatiotemporal clock gene or protein expression quantified from neonatal mouse suprachiasmatic
nucleus, SCN. (A, C) Representative images of Period1(per1) promoter activity reported by destabilized Enhanced
Green Fluorescent Protein, EGFP (Per1-EGFP) (Kuhlman et al., 2000) around expression trough (A) and peak (C). (B,
D) Representative images of PERIOD2 (PER2) protein level reported by a luciferase (PER2::LUC) around expression
trough (B) and peak (D). Note the higher background in fluorescence images than bioluminescent images. (E-F)
Single SCN neurons expressing both fluorescent protein (E) and a luciferase (F). Yellow fluorescent protein, Venus,
was introduced into dispersed SCN neurons of PER2::LUC mice. Note the higher spatial resolution in fluorescent
images than in bioluminescent images.
[Attribution and copyright information: (A-F) Pictures were taken by Takako Noguchi in Dr. David Welsh’s
laboratory. CC BY 4.0. The PER2::LUC mouse was generated by Dr. Joseph Takahashi while at Northwestern
University (Welsh et al., 2004). The Per1-EGFP mouse was generated by Dr. Douglas McMahon while at the
University of Kentucky (Kuhlman et al., 2000). We thank Drs. Takahashi and McMahon for their generous donation
of these mouse lines.]
Figure 6. Time-dependent localization of KaiC, a clock protein of cyanobacteria, was studied in cyanobacteria
expressing a fusion protein of KaiC and YFP (Cohen et al., 2014) by fixing samples at various time points of a day.
YFP-KaiC and autofluorescence from the cell’s photosynthetic machinery are displayed in green and red,
respectively. Example of repeated one-timepoint protein localization in cyanobacteria.
[Attribution and copyright information: These pictures were taken by Dr. Susan E Cohen in Golden’s laboratory.
CC BY 4.0.]
6
Fluorescent protein reporters
Fluorescent proteins have advantages for visualizing the location of a protein in a cell (termed
subcellular localization). The characteristics that make fluorescent reporters more suitable for
visualization of location than luciferases are the following. (1) Brightness of the light signal. Because
fluorescence is bright, scientists can magnify cells and observe structures inside of cells, such as the
nucleus or other organelles. (2) Color variations. There are many color variants in fluorescent proteins
(Shaner et al., 2005). Scientists can express multiple genes that encode fluorescent proteins of different
colors in a cell, fused to different proteins of interest, at the same time and study where the different
proteins are expressed in the cells (termed co-localization). (3) Site-specific excitation. It is possible to
excite fluorescent proteins in a very small area of tissue or cells using a laser beam. Confocal and twophoton laser scanning microscopy (Zipfel et al., 2003; Conchello and Lichtman, 2005) are high-end
fluorescent microscopy techniques that “erase” out-of-focus fluorescence from the background by
exciting a point of a sample with a focused laser beam and generating 2-D or 3-D images by scanning.
Especially, two-photon laser scanning microscopy enables the visualization of fluorescent signals in
thicker specimens, such as brain tissue (>500 µm) in a living mouse by using a laser with a longer
wavelength (Peters et al., 2014). In the circadian rhythm field, daily changes in neuronal activity have
been monitored in the zebrafish brain (Appelbaum et al., 2010), the fly brain (Bushey et al., 2015), and
in a marine annelid (Tosches et al., 2014) using calcium sensors based on fluorescent proteins that we
will introduce later in this article.
Disadvantages of fluorescent proteins are: (1) Photobleaching. Fluorophores are damaged by photons
and lose the ability to fluoresce. Photobleaching is problematic in real-time imaging, in which reporters
are repeatedly exposed to excitation light to observe the pattern of expression over time (Giloh and
Sedat, 1982; White and Stelzer, 1999). (2) Phototoxicity. When fluorophores are exposed to light,
especially of higher energy, the process tends to generate reactive oxygen species (ROS), extremely
reactive molecules that can damage DNA, RNA, and proteins by oxidization (Dixit and Cyr, 2003; Godley
et al., 2005). This phototoxicity also complicates applications of fluorescent protein reporters to live-cell
imaging. (3) High background. Because cells and organisms contain compounds that naturally fluoresce
(exhibit autofluorescence), signals from fluorescent proteins have to be high enough compared to
autofluorescence to rise above the background (Niswender et al., 1995; Shaner et al., 2005) or have to
be different enough in wavelength from autofluorescence to be separated by filtration (Fig. 6).
Fluorescence imaging in plant cells or other photosynthetic organisms, such as cyanobacteria, is
especially challenging because photosynthetic pigments, such as chlorophyll, exhibit high levels of
autofluorescence (Zhang et al., 2010). But fluorescence imaging can be performed by choosing filters
specifically designed to block the transmission of autofluorescence wavelengths (Cohen and Golden,
2015). (4) High stability. GFP is a very stable protein (Sacchetti et al., 2001; Kitsera et al., 2007). Stability
of proteins is advantageous to visualize localization of proteins, but problematic for measuring dynamics
of expression level because pre-existing proteins continue to accumulate. Thus, you can’t tell how much
“new” protein was made at a particular time. Quantification of gene expression is possible with
fluorescent proteins by including degradation sequences into the reporter protein that recruit the
cellular proteolysis machinery, shortening half-life.
7
Applications of bioluminescent and fluorescent reporters in circadian
rhythm studies
Luciferase and fluorescent protein reporters are used widely in circadian rhythm studies for various
purposes. The selection and design of reporters are different depending on the purpose of each
experiment. To understand the experimental results in a paper or presentation better, it will be helpful
for students to consider if the reporter is representing activity of a promoter or expression of a protein,
spatial and/or temporal information, and activity in live or fixed cells. In this section, we summarize
representative use of luciferase and fluorescent protein reporters in experiments and introduce
examples of applications of each case in circadian rhythm research.
Application flow chart
The first check point (Fig. 7A) to evaluate the experiment is to determine if the reporter is luciferase
or a fluorescent protein. Luciferase reporters are mostly used to quantify gene or protein expression
level. On the other hand, fluorescent proteins are mostly used to locate where the gene or protein is
expressed rather than quantifying the expression level.
Figure 7A. The first check point to evaluate the use
of reporters in experiments. Is the reporter luciferase
or a fluorescent protein?
[Attribution and copyright information: This
diagram was generated by Takako Noguchi. CC BY
4.0.]
The second check point (Fig. 7B) is whether the reporter gene is directly connected to a promoter of a
gene or designed to create a fusion protein with a protein of interest. Both promoter fusions and protein
fusions may be used with both kinds of reporters (Fig. 8). If the coding region of the luciferase reporter
gene alone is directly connected to the promoter of the gene of interest, bioluminescence level
represents promoter activity (Fig. 8A). In other words, it reports how frequently the promoter turns on
and causes an mRNA to be transcribed. But the mRNA that is translated into a protein is just the
luciferase, not the protein that is usually controlled by that promoter. Thus, this type of reporter
predicts mRNA levels of the gene of interest, but does not provide information about quantity or
functions of the protein of interest that the gene normally controls.
Figure 7B. The second check point to evaluate the use of reporters in
experiments. Is it the reporter promoter fusion or protein fusion?
[Attribution and copyright information: This diagram was generated by
Takako Noguchi. CC BY 4.0.]
8
Alternatively, a fusion protein can be created by attaching the codons of a luciferase gene seamlessly to
the codons from a gene of interest, such that when a ribosome reads the resulting mRNA, it will insert
all of the amino acids for one protein and start putting in the amino acids for the next, all in one
polypeptide. For example, in Fig. 8A the promoter and codons for per2 are fused seamlessly to codons of
the luciferase gene. The resulting protein is a fused product, in which the protein of interest and the
luciferase reporter are both present and attached to one another – in this case, PER2::LUC. When
luciferase is physically fused to a protein of interest, the bioluminescence level represents the level of
the protein of interest as well as of the reporter, because they are part of the same molecule (Fig. 8A).
The luciferase reporter will be degraded together with the fused protein of interest.
The same sorts of fusions can be made with genes for fluorescent proteins (Fig. 8B), and the light
emitted from a fluorescent protein is usually bright enough to make a distinct spot. With a promoter
fusion, the fluorescent protein is usually expressed in the cytosol and visualized as the shape of an entire
cell in which the promoter is activated, such as both the cell body and neural processes of a subset of
neurons in a fruit fly brain. However, sometimes it is important to also know where in the cell a protein
of interest goes (subcellular localization information), which cannot be provided by the promoter for a
gene. When subcellular localization information is desirable, the reporter protein needs to be physically
attached to the protein of interest, so that they move together. Subcellular localization of a protein is
controlled by short amino acid sequences (localization signals) that guide a protein to a certain cellular
compartment or to be secreted out of the cell. Commonly used reporter proteins have no localization
signal of their own, and just stay in the cytoplasm after it being translated by the ribosomes. However,
when the codons for a fluorescent reporter gene are fused to those for a protein of interest (Fig. 8D),
localization signals in the protein of interest bring the attached reporter protein along for the ride to a
specific subcellular location (Fig. 6, 8D).
Advanced concept: Both luciferase and fluorescent protein reporters can be engineered to be
expressed in only specific cells of a multicellular organism using methods in which the reporter gene is
silent unless a cell-type-specific signal turns it on, such as the GAL4-UAS system used commonly in fruit
fly (Brand and Perrimon, 1993), and the Cre-Lox recombination system used in mice and other
eukaryotes (Sauer, 1998). In these cases, the expression of reporters can be indirectly controlled by a
particular promoter, and it is necessary to look carefully at the details to find out what is controlling the
expression of the reporter.
The third check point (Fig. 7C) is to determine whether the reporter signal was collected from fixed
(immobilized) cells, in which the live activity of cells is arrested, such as by cross-linking (fixing) protein
structures with formalin (Nakagawa et al., 2015), or from living cells, tissues, or organisms where
transcription and translation are still occurring. When scientists use fixed cells, the results of
Figure 7C. The third check point to evaluate the use of reporters in
experiments. Are samples fixed cells or live-cells?
[Attribution and copyright information: This diagram was generated by
Takako Noguchi. CC BY 4.0.]
9
experiments do not convey temporal information. However, scientists can take samples in a time series
and fix cells from each time point to convey temporal information. When reporter expression is
monitored continuously from the same living cells, tissues, or organisms, the results of experiments
convey temporal information directly, showing dynamic changes in expression of the gene or protein of
interest in real time.
The 4th check point is to determine whether the data were collected as a summed output (added up
total of signal) from a whole population of cells or tissues, or imaged at the single-cell level (Fig. 7D).
Luciferase reporters can be analyzed both ways. Fluorescent protein reporters are analyzed from
images. Summed output from a population conveys temporal and quantity information, but not spatial
information. But with images, scientists can analyze detailed cellular or spatial events. Using fluorescent
reporters fused to proteins of interest, scientists can analyze subcellular- or organelle-level localization
of the proteins. (Keep in mind that localization signals are parts of proteins, and a promoter can’t tell a
reporter where to go in the cell after it is made.)
The last check point to consider is whether the reporter is destabilized or not (Fig. 7E). Luciferases are
naturally unstable compared to fluorescent proteins, and they are often destabilized further to increase
Figure 7D. The 4th check point to evaluate the use of reporters in
experiments. Are data summed outputs or images?
[Attribution and copyright information: This diagram was generated by
Takako Noguchi. CC BY 4.0.]
temporal resolution. Fluorescent proteins are generally not destabilized, because accumulation
improves ability to visualize localization of proteins. However, it is possible to quantify promoter activity
using fluorescent proteins if they are destabilized.
Once you have evaluated check points discussed above, you can understand the use of reporters in
experiments by following the entire flowchart (Fig. 7). Determine whether the reporter is representing
quantity and/or location, activity of a promoter or level of a protein, and activity in live or fixed cells.
Figure 7E. The 5th check point to evaluate the use of reporters in
experiments. Is the reporter destabilized or not?
[Attribution and copyright information: This diagram was generated by
Takako Noguchi. CC BY 4.0.]
10
Figure 7. Flowchart to tell the use of reporters in experiments from the design of genetically encoded reporters
and key features of experiments.
[Attribution and copyright information: This diagram was generated by Takako Noguchi. CC BY 4.0.]
Examples of applications in circadian rhythm research
One-timepoint gene expression quantification
The best example of one-timepoint gene expression quantification (Fig. 7i) is a promoter-fusion assay
that tests the functions of specific sequences within promoters or the interaction of transcription factors
with promoters. Luciferase reporters are widely used in promoter assays in many types of biological
science as well as circadian rhythm research. In the circadian rhythm field, for example, the E-box, a
transcription factor-binding site, was shown to be important for rhythmic Period gene expression in
Drosophila (Darlington et al., 1998) and in mouse using luciferase promoter assays (Gekakis et al., 1998).
Temporal gene expression or protein quantification
In the circadian rhythm field, scientists often use luciferase to quantify temporal changes of clock
gene expression. Clock genes encode molecular components that generate circadian rhythms, and most
of these genes increase and decrease in expression level over the course of the day (Mohawk et al.,
2012). Luciferase was first applied as a reporter to monitor circadian oscillation in expression of a gene
in living Arabidopsis plants (Millar et al., 1995). In cyanobacteria, a luciferase reporter was used to find
the core clock genes (Kondo et al., 1993; Kondo et al., 1994). In mice, timing of accumulation of some
clock proteins is delayed several hours relative to mRNA expression (Morse and Sassone-Corsi, 2002)
(Fig. 8A-B). This difference can be studied by measuring transcription (promoter fusion) and protein
levels (protein fusion) over time using luciferase (Fig. 7ii, iv). For example, to compare transcription and
11
translation levels of Per2, scientists generated two mouse lines: one expressing a luciferase coding
sequence under control of Per2 promoter elements, and the other expressing a luciferase fused to PER2
protein (PER2::LUC mouse) by fusing their coding sequences together in the correct reading frame (Yoo
et al., 2005) (Fig. 8A-B). They cultured the SCN, which is a master circadian pacemaker of mammals
located in the hypothalamus, monitored luciferase activity in real time over a week (Yoo et al., 2005),
and demonstrated a delay of several hours in luciferase peaks from the promoter fusion (transcriptional
fusion reports Per2 mRNA transcription) and the protein fusion (translational fusion reports PER2
protein levels).
Figure 8. (A-B) Examples of luciferase gene fusion constructs to measure transcriptional (promoter) activity
(parallel to mRNA expression) (A) and translational activity (parallel to protein expression) of a clock gene, Per2 (B).
Degradation of luciferase is important to measure temporal changes of transcription or translation level. Without
active degradation, the circadian trough would not be detectable. A schematic graph shows bioluminescence level
obtained from transcriptional and translational fusion constructs of Per2. Note that promoter activity peaks hours
before protein expression. (C-D) Examples of fluorescent reporter gene fusion constructs to visualize localization of
control YFP (C), and KaiC, a clock protein in cyanobacteria (D). Fluorescent proteins are generally stably expressed
in cytosol without large amount of degradation. When YFP is directly fused to promoter and not fused to
localization signals (transcriptional fusion), it is expressed in cytosol (C). When YFP is fused to KaiC (translational
fusion), it localize together with KaiC. Note that fluorescence intensity reports where YFP located, but do not
necessary quantify the activity level of promoter (C) or fused protein level because YFP is stably expressed.
[Attribution and copyright information: These diagrams were generated by Takako Noguchi. CC BY 4.0.]
Spatiotemporal gene and protein quantification
Bioluminescence is very dim, but it is possible to capture single-cell level images (Welsh et al., 2004).
Scientists perform spatiotemporal (“where and when”) gene expression or protein quantification (Fig. 7
iii, v) by imaging to study activities that are not evident from a summed output of a whole population.
For example, synchronization among the SCN neurons was observed by imaging the SCN of a mouse
expressing a luciferase under control of Per1 promoter (Yamaguchi et al., 2003). Images of the SCN of
PER2::LUC mouse are shown in Fig. 5. Single fibroblasts were found to have independent clocks that can
keep oscillating in constant conditions for over a month using PER2::LUC mouse fibroblasts (Welsh et al.,
2004; Leise et al., 2012). Bioluminescence can also be measured in living rodents. For example, circadian
rhythms in peripheral tissues in anesthetized PER2::LUC mice are monitored by in vivo imaging (Tahara
12
et al., 2012), and even in free-moving mice (Saini et al., 2013). Per1-luc expression in the SCN is detected
by an optic fiber in free-moving mice and rats (Yamaguchi et al., 2001; Yamaguchi et al., 2016).
One-timepoint gene and protein location
Fluorescent proteins are most commonly used for one-timepoint gene or protein location (Fig. 7vi,
viii). This means that a sample is imaged once, but in some cases multiple samples are imaged over a
time course to reconstruct dynamic changes. By introducing a construct that connects a promoter and
the gene that encodes a fluorescent protein, scientists can locate the tissues or cells in the organism in
which the gene is expressed. For example, neurons expressing clock genes were identified by Enhanced
Green Fluorescent Protein (EGFP) using the GAL4-UAS system in fruit fly brains (Zhao et al., 2003).
Nuclear entry of clock proteins or their complexes are studied by creating a fusion protein that consists
of a clock protein and a fluorescent protein in mammalian cells (Vielhaber et al., 2000; Lee et al., 2015).
Time-dependent localization of the cyanobacterial clock protein KaiC is studied by sampling cells from a
population at different time points and imaging fluorescent fusion proteins (Cohen et al., 2014) (Fig. 6).
One-timepoint gene expression or protein localization using fluorescent proteins produces similar
results to immunohistochemical (IHC) staining, which locates proteins by antibodies often tagged with
synthetic fluorescent molecules. Scientists often show combined fluorescent images of IHC and
genetically encoded fluorescent reporters. In these cases, students have to carefully identify what the
fluorescence represents.
Spatiotemporal gene quantification by fluorescent proteins
Spatiotemporal gene expression (Fig. 7vii) can be measured by destabilizing fluorescent proteins so
that they do not accumulate and their expression levels better reflect recent synthesis. The genes that
encode fluorescent proteins are modified to encode destabilization signal to the polypeptide that are
recognized by protein degradation machinery, such as by fusing 2 PEST sequences (Kuhlman et al.,
2000). For example, circadian Rev-Erbα promoter activity was imaged by destabilized Venus, a variant of
YFP, in NIH3T3 cells, a mouse cell line (Nagoshi et al., 2004). Circadian Per1 promoter activity in the SCN
was reported by destabilized EGFP (LeSauter et al., 2003) (Fig. 5A, C).
Variations of luciferase reporters
Luciferases have various characteristics depending on the purposes for which they evolved in the
original organisms. Luciferases with different characteristics are commercially available as gene
expression or protein reporters. The most commonly used luciferase reporter is North American firefly
luciferase (FLuc). Modified luciferases with different properties are constantly being invented through
genetic engineering to make experiments more efficient and easier. In this section, we introduce various
types of luciferases and technologies using them.
Flash- and glow-type luciferases
There are luciferase reporters with different reaction kinetics. For example, “flash” type luciferase, such
as the sea pansy Renilla luciferase (RLuc) and Gaussia luciferase (GLuc), generate high intensity
bioluminescence in short time (within 10 s) (Bhaumik and Gambhir, 2002; Tannous, 2009). On the other
hand, “glow” type luciferases, such as Fluc or bacterial luciferase (Lux) generate stable long-lasting
bioluminescence (more than 30 min) (Andreu et al., 2010). Flash-type luciferase is a useful tool for onetimepoint quantification, but glow type is more suitable for the long-term recording required in
circadian rhythm applications.
Calcium-sensitive luciferase
Aequorin, a calcium-sensitive luciferase, was isolated from the jellyfish Aequorea together with GFP
(Shimomura et al., 1962, 1963). Aequorin uses the substrate coelenterazine (Granatiero et al., 2014a)
13
and can measure wide range of calcium concentrations (0.1 -10 µM in natural form, up to 100 µM in
mutated form) (Granatiero et al., 2014b). Aequorin was successfully applied to measure circadian
calcium rhythms in living plants continuously (Johnson et al., 1995) and neuronal activity in free-moving
zebrafish (Naumann et al., 2010). Aequorin has advantages in (1) wide dynamic range, (2) low
background noise, (3) low calcium buffering, and (4) low toxicity. However, the disadvantages include (1)
some properties of coelenterazine, such as less membrane permeability, less stability, and higher price
than other luciferins, (2) the requirement of specialized detectors, and (3) a long reconstitution time of
aequorin and coelenterazine complex. Because of these downsides, calcium measurements in circadian
rhythm and other biological fields are mostly performed by calcium-sensitive dyes, such as Fra-2 and
fluo-4, or GFP-based genetically encoded calcium indicators (GECIs) (Chen et al., 2013; Sun et al., 2013).
Calcium-sensitive dyes are effective for short-term (within 1 h) recording (Colwell, 2000). GECIs are
effective for long-term (more than a week or months) recording (Ikeda et al., 2003; Brancaccio et al.,
2013).
Bioluminescence resonance energy transfer (BRET)
Purified aequorin emits blue light; however, the result is green light emitted by the jellyfish body. This
difference happens because GFP naturally forms a complex with aequorin and energy generated by
aequorin is transferred to GFP, which then emits fluorescence. This process is called Förster resonance
energy transfer (FRET). Especially when a donor of energy is a luciferase, it is called bioluminescence
resonance energy transfer (BRET) (Wilson and Hastings, 1998; Pfleger et al., 2006). BRET can occur when
the donor and acceptor are very close (10-100 Å apart). During BRET, the energy from an excited
electron in a donor molecule jumps to an electron in the acceptor molecule, exciting it without turning
energy into photons in between; only the acceptor electron gives off light when it falls back to ground
state. BRET happens in some luciferase and florescent protein pairs, such as RLuc and GFP (Wilson and
Hastings, 1998). Changes in emission color by BRET can be used to test protein-protein interactions. In
this assay, scientists can fuse one protein to a luciferase and the other protein to a fluorescent protein.
When the two proteins interact (physically associate closely), the spectrum changes from shorter to
longer wavelength (e.g., blue to green). For example, a protein interaction assay by BRET was first
applied in the circadian rhythm field to study interaction between the cyanobacterial oscillator proteins
KaiA and KaiB, and interacting subunits of KaiB itself (Xu et al., 1999).
Brighter luciferases
Dimness of signal is the major weakness of bioluminescent reporters. Bioengineers are making efforts to
generate brighter luciferases. Recently introduced NanoLuc (Promega Madison, WI), derived from the
deep sea shrimp Oplophorus gracilirostris, is ~150-fold brighter than FLuc and has glow-type kinetics.
NanoLuc has been applied successfully to image nuclear entry of proteins (Hall et al., 2012). Application
to circadian rhythm research has not yet been achieved. The other brighter luciferase ELuc (10-fold
brighter than FLuc), derived from the click beetle Pyrearinus termitilluminans, was applied to image
promoter activity of the clock gene, Bmal1, in the SCN as well as protein nuclear entry (Nakajima et al.,
2010).
Color variations
Color spectra of luciferases are different depending on the organisms they are derived from or various
chemical conditions. Fireflies exhibit green-yellow bioluminescence in nature, but purified FLuc exhibits
green at pH 8, and red at pH 6. Higher temperature and the presence of heavy metal cations also red
shift the spectrum of FLuc (Nakatsu et al., 2006; Viviani et al., 2008). Luciferases derived from other
luminescent beetles use the same substrates as FLuc, D-luciferin, but are insensitive to pH, temperature,
and heavy metal cations, and stably exhibit one color spectrum (Viviani et al., 2008). For example, stable
red- or green-emitting luciferase derived from railroad worms and click beetles are available. In the
14
circadian rhythm field, the expression patterns of two different clock genes that peak at different times
of the day (Bmal1 and Per2) were monitored simultaneously using mice expressing red- and greenemitting luciferases (Noguchi et al., 2012). Luciferases derived from marine organisms often exhibit blue
bioluminescence, as is true for Renilla and Gaussia, (Zhao et al., 2005; Viviani, 2007; Maguire et al.,
2009).
Other luciferase reporters
Luciferases can be engineered to localize to the cytosol, nucleus, or other organelles by the addition of
localization signals. Some luciferases such as Ostracod Cypridina noctiluca (CLuc), and Gaussia (GLuc) are
naturally secreted proteins. In the circadian rhythm field, promoter activity of Per2 was monitored by
secreted CLuc in the blood of rats (Yamada et al., 2013).
In the bacterial Vibrio harveyi luciferase (Lux) assay system, the substrate is a long-chain aldehyde,
unrelated to invertebrate luciferins, which can be readily engineered to be expressed in the host
organism along with the luciferase (Meighen, 1991; Shultzaberger et al., 2015). The co-expression of the
luciferase LuxAB and its substrate enables bioluminescence monitoring without adding luciferin, and is
often favored for circadian monitoring in cyanobacteria.
Variations of fluorescent protein reporters
A wide variety of reporters or sensors based on fluorescent proteins has been developed (Shaner et al.,
2005). In this section, we introduce various fluorescent reporters used in circadian rhythms research.
Color variations
Expression of multiple genes can be analyzed simultaneously by using florescent proteins of different
colors. Color variants of fluorescent proteins are either generated by mutating existing fluorescent
proteins, such as GFP, or newly cloned from organisms exhibiting different fluorescent colors. There are
many color variants, but common fluorescent proteins that exhibit red, yellow, green, cyan (blue), are
generally called RFP, YFP, GFP, and CFP, respectively. Each color variant has its own name, such as
mPlum emitting far-red, mCherry and DsRed emitting red, mOrange, mCitrine and Venus emitting
yellow-green, Emerald and EGFP emitting green, and mCFPm and Cerulean emitting cyan (Shaner et al.,
2005). For example, in circadian rhythm research, a transgenic mouse line expressing two fusion
proteins, PER1-Venus and PER2-DsRED, was developed to locate and quantify the expression of PER1
and PER2 proteins in mouse brain (Cheng et al., 2009).
FRET
FRET (Förster resonance energy transfer, described above) happens between fluorescent protein pairs
with different colors when they are in close distance. In this case, the word FRET could stand for
fluorescent resonance energy transfer. When a donor of energy is a fluorescent molecule, an external
light source must excite the donor, whose excited state is transferred to the acceptor, which then
fluoresces. FRET can be used to study protein interactions similarly to BRET. For example, to study KaiC
and KaiB interaction, scientists made KaiC-Cerulean and KaiB-Venus fusion proteins (Ma and
Ranganathan, 2012). Cerulean itself is excited by 433 nm and emits at 475 nm. Venus itself is excited
maximally by 515 nm (but can also absorb slightly shorter or longer wavelengths) and emits at 528 nm.
But when KaiB and KaiC interact and bring Cerulean and Venus close, energy from 433 nm light is
absorbed by Cerulean, transferred to Venus, and emitted as 528 nm light. Thus, scientists can tell that
KaiB and KaiC interact by analyzing the emission color. If there were no interaction, the 433 nm
excitation would result in only 475 nm emission (Fig. 9).
15
Figure 9. Schematic diagram of assays based on FRET. When two light-responsive proteins are very close, energy of
the donor is transferred to excite the acceptor by FRET. (A) Protein interaction assay by FRET. CFP and YFP are
tagged to protein A and B, respectively. Interaction between protein A and B can be detected by the emission color
change. (B) FRET based Ca2+ sensor. FRET occurs only when a Ca2+ sensitive linker changes conformation by Ca2+
binding and bring two light-responsive proteins very close.
[Attribution and copyright information: These diagrams were generated by Takako Noguchi. CC BY 4.0.]
FRET-based small molecule sensors
The concentration of small molecules, such as Ca2+ or cyclic adenosine monophosphate (cAMP), can be
measured by FRET-based sensors. Epac1-camps, a genetically encoded cAMP FRET sensor, is a chimeric
protein of Epac (exchange proteins directly activated by cAMP) sandwiched between CFP and YFP
(DiPilato et al., 2004; Ponsioen et al., 2004). FRET occurrence in Epac1-camps is reduced in the presence
of cAMP because the two fluorescent protein components are pushed too far away from each other for
energy transfer. For example, the cAMP response to a neurotransmitter in fly clock neurons was
measured using Epac1-camps (Shafer et al., 2008). The Yellow Cameleons (YCs), genetically encoded
calcium FRET sensors, are chimeric proteins of CFP and YFP linked by calcium-sensitive proteins,
calmodulin (CaM), a glycylglycine linker, and CaM-binding domain of myosin light chain (M13) (Miyawaki
et al., 1999; Russell, 2011; Horikawa, 2015). FRET occurrence increases in the presence of calcium
because two fluorescent protein components are brought close when the CaM and M13 bind Ca2+. For
example, circadian calcium rhythms can be imaged in cultured mouse SCNs (Ikeda et al., 2003; Enoki et
al., 2012). These FRET-based sensors can respond to fast cellular events in millisecond timescales, such
as the intracellular calcium concentration change triggered by a single action potential (Lutcke et al.,
2010; Yamada et al., 2011). This is because they detect targeted molecules by conformation changes in
pre-existing proteins, whereas the gene and protein expression reporters described above require
transcription, translation, and degradation to respond to changes.
Single fluorescent protein-based sensors
Changes in calcium concentration can be measured by single fluorescent protein-based genetically
encoded calcium sensors (GECIs), such as GCaMPs and Pericams (Horikawa, 2015). In this type of GECI,
intensity of fluorescent protein emissions are modulated by calcium-binding modules (Tian et al., 2009;
16
Chen et al., 2013). Fluorescent proteins are split into N and C terminal parts, then linked by CAM and
M13 (N-CAM-M13-C), but the order of protein segments is circularly permuted (M13-C-N-CaM) so that
the proximity of the N- and C-terminal protein domains is modulated by CaM (Baird et al., 1999). These
GECIs are able to detect intracellular calcium concentration changes triggered by a single action
potential (Lutcke et al., 2010; Yamada et al., 2011) as well as slow calcium changes as a summed effect
of multiple action potentials or intracellular calcium stores. In the circadian rhythm field, slow cytosolic
calcium changes over days are measured in mouse SCN or in fly clock neurons (Brancaccio et al., 2013;
Liang et al., 2016).
Other fluorescent protein sensors
Single fluorescent protein-based voltage sensors have been developed to directly detect action
potentials (Jin et al., 2012). In the circadian rhythm field, a single action potential was detected in
Drosophila clock neurons using ArcLight (Cao et al., 2013).
Many other fluorescent protein-based genetically encoded sensors are available. For example, there are
heme sensors (Hanna et al., 2016), redox sensors (Sugiura et al., 2015; Gibhardt et al., 2016), and
extracellular glutamate sensors (Xie et al., 2016). These sensors may open new aspects of circadian
biology to future investigation.
Conclusion
Bioluminescence reporters are highly quantitative and nontoxic tools that enable noninvasive long-term
recording. In the circadian rhythm field, luciferases are frequently used to monitor temporal gene and
protein quantification. Fluorescent reporters have high spatial resolution and are often used to visualize
the sites of gene or protein expression and are less frequently used to quantify gene or protein
expression. Fluorescent protein-based sensors are also useful tools to monitor circadian rhythms of
small molecules, such as calcium.
References
Adams JU (2010) How Do Cells Decode Genetic Information into Functional Proteins? In: Essential of Cell
Biology (O'Connor C, ed). Cambridge, MA: NPG Education.
http://www.nature.com/scitable/ebooks/cell-biology-for-seminars-14760004/how-do-cellsdecode-genetic-information-into-14760047
Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U,
Robertson BD, Wiles S (2010) Optimisation of bioluminescent reporters for use with
mycobacteria. PLoS One 5:e10777. http://www.ncbi.nlm.nih.gov/pubmed/20520722
Appelbaum L, Wang G, Yokogawa T, Skariah GM, Smith SJ, Mourrain P, Mignot E (2010) Circadian and
homeostatic regulation of structural synaptic plasticity in hypocretin neurons. Neuron 68:87-98.
http://www.ncbi.nlm.nih.gov/pubmed/20920793
Arnold KE, Owens IP, Marshall NJ (2002) Fluorescent signaling in parrots. Science 295:92.
http://www.ncbi.nlm.nih.gov/pubmed/11778040
Arpino JA, Rizkallah PJ, Jones DD (2014) Structural and dynamic changes associated with beneficial
engineered single-amino-acid deletion mutations in enhanced green fluorescent protein. Acta
Crystallogr D Biol Crystallogr 70:2152-2162. http://www.ncbi.nlm.nih.gov/pubmed/25084334
Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green
fluorescent proteins. Proc Natl Acad Sci U S A 96:11241-11246.
http://www.ncbi.nlm.nih.gov/pubmed/10500161
Bhaumik S, Gambhir SS (2002) Optical imaging of Renilla luciferase reporter gene expression in living
mice. Proc Natl Acad Sci U S A 99:377-382. http://www.ncbi.nlm.nih.gov/pubmed/11752410
17
Brancaccio M, Maywood ES, Chesham JE, Loudon AS, Hastings MH (2013) A Gq-Ca2+ axis controls
circuit-level encoding of circadian time in the suprachiasmatic nucleus. Neuron 78:714-728.
http://www.ncbi.nlm.nih.gov/pubmed/23623697
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating
dominant phenotypes. Development 118:401-415.
http://www.ncbi.nlm.nih.gov/pubmed/8223268
Bushey D, Tononi G, Cirelli C (2015) Sleep- and wake-dependent changes in neuronal activity and
reactivity demonstrated in fly neurons using in vivo calcium imaging. Proc Natl Acad Sci U S A
112:4785-4790. http://www.ncbi.nlm.nih.gov/pubmed/25825756
Cao G, Platisa J, Pieribone VA, Raccuglia D, Kunst M, Nitabach MN (2013) Genetically targeted optical
electrophysiology in intact neural circuits. Cell 154:904-913.
http://www.ncbi.nlm.nih.gov/pubmed/23932121
Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB,
Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for
imaging neuronal activity. Nature 499:295-300.
http://www.ncbi.nlm.nih.gov/pubmed/23868258
Cheng HY, Alvarez-Saavedra M, Dziema H, Choi YS, Li A, Obrietan K (2009) Segregation of expression of
mPeriod gene homologs in neurons and glia: possible divergent roles of mPeriod1 and mPeriod2
in the brain. Hum Mol Genet 18:3110-3124. http://www.ncbi.nlm.nih.gov/pubmed/19477955
Choy G, O'Connor S, Diehn FE, Costouros N, Alexander HR, Choyke P, Libutti SK (2003) Comparison of
noninvasive fluorescent and bioluminescent small animal optical imaging. Biotechniques
35:1022-1026, 1028-1030. http://www.ncbi.nlm.nih.gov/pubmed/14628676
Cohen SE, Golden SS (2015) Circadian Rhythms in Cyanobacteria. Microbiol Mol Biol Rev 79:373-385.
http://www.ncbi.nlm.nih.gov/pubmed/26335718
Cohen SE, Erb ML, Selimkhanov J, Dong G, Hasty J, Pogliano J, Golden SS (2014) Dynamic localization of
the cyanobacterial circadian clock proteins. Curr Biol 24:1836-1844.
http://www.ncbi.nlm.nih.gov/pubmed/25127213
Colwell CS (2000) Circadian modulation of calcium levels in cells in the suprachiasmatic nucleus. Eur J
Neurosci 12:571-576. http://www.ncbi.nlm.nih.gov/pubmed/10712636
Conchello JA, Lichtman JW (2005) Optical sectioning microscopy. Nat Methods 2:920-931.
http://www.ncbi.nlm.nih.gov/pubmed/16299477
Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS, Kay
SA (1998) Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and
tim. Science 280:1599-1603. http://www.ncbi.nlm.nih.gov/pubmed/9616122
DiPilato LM, Cheng X, Zhang J (2004) Fluorescent indicators of cAMP and Epac activation reveal
differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl
Acad Sci U S A 101:16513-16518. http://www.ncbi.nlm.nih.gov/pubmed/15545605
Dixit R, Cyr R (2003) Cell damage and reactive oxygen species production induced by fluorescence
microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J
36:280-290. http://www.ncbi.nlm.nih.gov/pubmed/14535891
Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gartner W, Jaeger KE
(2007) Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 25:443-445.
http://www.ncbi.nlm.nih.gov/pubmed/17351616
Enoki R, Kuroda S, Ono D, Hasan MT, Ueda T, Honma S, Honma K (2012) Topological specificity and
hierarchical network of the circadian calcium rhythm in the suprachiasmatic nucleus.
Proceedings of the National Academy of Sciences of the United States of America 109:2149821503. http://www.ncbi.nlm.nih.gov/pubmed/23213253
18
Gandia-Herrero F, Garcia-Carmona F, Escribano J (2005) Botany: floral fluorescence effect. Nature
437:334. http://www.ncbi.nlm.nih.gov/pubmed/16163341
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ (1998) Role of
the CLOCK protein in the mammalian circadian mechanism. Science 280:1564-1569.
http://www.ncbi.nlm.nih.gov/pubmed/9616112
Gibhardt CS, Zimmermann KM, Zhang X, Belousov VV, Bogeski I (2016) Imaging calcium and redox
signals using genetically encoded fluorescent indicators. Cell Calcium
10.1016/j.ceca.2016.04.008. http://www.ncbi.nlm.nih.gov/pubmed/27142890
Giloh H, Sedat JW (1982) Fluorescence microscopy: reduced photobleaching of rhodamine and
fluorescein protein conjugates by n-propyl gallate. Science 217:1252-1255.
http://www.ncbi.nlm.nih.gov/pubmed/7112126
Godley BF, Shamsi FA, Liang FQ, Jarrett SG, Davies S, Boulton M (2005) Blue light induces mitochondrial
DNA damage and free radical production in epithelial cells. J Biol Chem 280:21061-21066.
http://www.ncbi.nlm.nih.gov/pubmed/15797866
Granatiero V, Patron M, Tosatto A, Merli G, Rizzuto R (2014a) The use of aequorin and its variants for
Ca2+ measurements. Cold Spring Harb Protoc 2014:9-16.
http://www.ncbi.nlm.nih.gov/pubmed/24371311
Granatiero V, Patron M, Tosatto A, Merli G, Rizzuto R (2014b) Using targeted variants of aequorin to
measure Ca2+ levels in intracellular organelles. Cold Spring Harb Protoc 2014:86-93.
http://www.ncbi.nlm.nih.gov/pubmed/24371314
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G,
Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F,
Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a
novel imidazopyrazinone substrate. ACS Chem Biol 7:1848-1857.
http://www.ncbi.nlm.nih.gov/pubmed/22894855
Hamamatsu Photonics KK (2006) Photomultiplier Tubes: Basics and Applications, 3a Edition: Hamamatsu
Photonics KK. https://www.hamamatsu.com/resources/pdf/etd/PMT_handbook_v3aE.pdf
Hanna DA, Harvey RM, Martinez-Guzman O, Yuan X, Chandrasekharan B, Raju G, Outten FW, Hamza I,
Reddi AR (2016) Heme dynamics and trafficking factors revealed by genetically encoded
fluorescent heme sensors. Proc Natl Acad Sci U S A 10.1073/pnas.1523802113.
http://www.ncbi.nlm.nih.gov/pubmed/27247412
Horikawa K (2015) Recent progress in the development of genetically encoded Ca2+ indicators. J Med
Invest 62:24-28. http://www.ncbi.nlm.nih.gov/pubmed/25817279
Ikeda M, Sugiyama T, Wallace CS, Gompf HS, Yoshioka T, Miyawaki A, Allen CN (2003) Circadian
dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons. Neuron
38:253-263. http://www.ncbi.nlm.nih.gov/pubmed/12718859
Jin L, Han Z, Platisa J, Wooltorton JR, Cohen LB, Pieribone VA (2012) Single action potentials and
subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe.
Neuron 75:779-785. http://www.ncbi.nlm.nih.gov/pubmed/22958819
Johnson CH, Knight MR, Kondo T, Masson P, Sedbrook J, Haley A, Trewavas A (1995) Circadian
oscillations of cytosolic and chloroplastic free calcium in plants. Science 269:1863-1865.
http://www.ncbi.nlm.nih.gov/pubmed/7569925
Kitsera N, Khobta A, Epe B (2007) Destabilized green fluorescent protein detects rapid removal of
transcription blocks after genotoxic exposure. Biotechniques 43:222-227.
http://www.ncbi.nlm.nih.gov/pubmed/17824390
Kondo T, Tsinoremas NF, Golden SS, Johnson CH, Kutsuna S, Ishiura M (1994) Circadian clock mutants of
cyanobacteria. Science 266:1233-1236. http://www.ncbi.nlm.nih.gov/pubmed/7973706
19
Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M, Golden SS, Johnson CH (1993) Circadian rhythms
in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc Natl
Acad Sci U S A 90:5672-5676. http://www.ncbi.nlm.nih.gov/pubmed/8516317
Kuhlman SJ, Quintero JE, McMahon DG (2000) GFP fluorescence reports Period 1 circadian gene
regulation in the mammalian biological clock. Neuroreport 11:1479-1482.
http://www.ncbi.nlm.nih.gov/pubmed/10841361
Leclerc GM, Boockfor FR, Faught WJ, Frawley LS (2000) Development of a destabilized firefly luciferase
enzyme for measurement of gene expression. Biotechniques 29:590-591, 594-596, 598 passim.
http://www.ncbi.nlm.nih.gov/pubmed/10997273
Lee Y, Jang AR, Francey LJ, Sehgal A, Hogenesch JB (2015) KPNB1 mediates PER/CRY nuclear
translocation and circadian clock function. Elife 4.
http://www.ncbi.nlm.nih.gov/pubmed/26319354
Leise TL, Wang CW, Gitis PJ, Welsh DK (2012) Persistent cell-autonomous circadian oscillations in
fibroblasts revealed by six-week single-cell imaging of PER2::LUC bioluminescence. PLoS One
7:e33334. http://www.ncbi.nlm.nih.gov/pubmed/22479387
LeSauter J, Yan L, Vishnubhotla B, Quintero JE, Kuhlman SJ, McMahon DG, Silver R (2003) A short halflife GFP mouse model for analysis of suprachiasmatic nucleus organization. Brain Research
964:279-287. https://www.ncbi.nlm.nih.gov/pubmed/12576188
Liang X, Holy TE, Taghert PH (2016) Synchronous Drosophila circadian pacemakers display
nonsynchronous Ca(2)(+) rhythms in vivo. Science 351:976-981.
http://www.ncbi.nlm.nih.gov/pubmed/26917772
Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2:910-919.
http://www.ncbi.nlm.nih.gov/pubmed/16299476
Lutcke H, Murayama M, Hahn T, Margolis DJ, Astori S, Zum Alten Borgloh SM, Gobel W, Yang Y, Tang W,
Kugler S, Sprengel R, Nagai T, Miyawaki A, Larkum ME, Helmchen F, Hasan MT (2010) Optical
recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and
freely moving mice. Front Neural Circuits 4:9. http://www.ncbi.nlm.nih.gov/pubmed/20461230
Ma L, Ranganathan R (2012) Quantifying the rhythm of KaiB-C interaction for in vitro cyanobacterial
circadian clock. PLoS One 7:e42581. http://www.ncbi.nlm.nih.gov/pubmed/22900029
Maguire CA, Deliolanis NC, Pike L, Niers JM, Tjon-Kon-Fat LA, Sena-Esteves M, Tannous BA (2009)
Gaussia luciferase variant for high-throughput functional screening applications. Anal Chem
81:7102-7106. http://www.ncbi.nlm.nih.gov/pubmed/19601604
Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent
proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969-973.
http://www.ncbi.nlm.nih.gov/pubmed/10504696
Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev 55:123-142.
http://www.ncbi.nlm.nih.gov/pubmed/2030669
Millar AJ, Carre IA, Strayer CA, Chua NH, Kay SA (1995) Circadian clock mutants in Arabidopsis identified
by luciferase imaging. Science 267:1161-1163. http://www.ncbi.nlm.nih.gov/pubmed/7855595
Miyawaki A, Griesbeck O, Heim R, Tsien RY (1999) Dynamic and quantitative Ca2+ measurements using
improved cameleons. Proc Natl Acad Sci U S A 96:2135-2140.
http://www.ncbi.nlm.nih.gov/pubmed/10051607
Mohawk JA, Green CB, Takahashi JS (2012) Central and peripheral circadian clocks in mammals. Annu
Rev Neurosci 35:445-462. http://www.ncbi.nlm.nih.gov/pubmed/22483041
Morse D, Sassone-Corsi P (2002) Time after time: inputs to and outputs from the mammalian circadian
oscillators. Trends Neurosci 25:632-637. http://www.ncbi.nlm.nih.gov/pubmed/12446131
20
Nagoshi E, Saini C, Bauer C, Laroche T, Naef F, Schibler U (2004) Circadian gene expression in individual
fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell
119:693-705. http://www.ncbi.nlm.nih.gov/pubmed/15550250
Nakagawa A, Alt KV, Lillemoe KD, Castillo CF, Warshaw AL, Liss AS (2015) A method for fixing and
paraffin embedding tissue to retain the natural fluorescence of reporter proteins. Biotechniques
59:153-155. http://www.ncbi.nlm.nih.gov/pubmed/26345508
Nakajima Y, Yamazaki T, Nishii S, Noguchi T, Hoshino H, Niwa K, Viviani VR, Ohmiya Y (2010) Enhanced
beetle luciferase for high-resolution bioluminescence imaging. PLoS One 5:e10011.
http://www.ncbi.nlm.nih.gov/pubmed/20368807
Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H (2006) Structural basis for the
spectral difference in luciferase bioluminescence. Nature 440:372-376.
http://www.ncbi.nlm.nih.gov/pubmed/16541080
Naumann EA, Kampff AR, Prober DA, Schier AF, Engert F (2010) Monitoring neural activity with
bioluminescence during natural behavior. Nat Neurosci 13:513-520.
http://www.ncbi.nlm.nih.gov/pubmed/20305645
Niswender KD, Blackman SM, Rohde L, Magnuson MA, Piston DW (1995) Quantitative imaging of green
fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion
proteins and detection limits. J Microsc 180:109-116.
http://www.ncbi.nlm.nih.gov/pubmed/8537958
Noguchi T, Ikeda M, Ohmiya Y, Nakajima Y (2012) A dual-color luciferase assay system reveals circadian
resetting of cultured fibroblasts by co-cultured adrenal glands. PLoS One 7:e37093.
http://www.ncbi.nlm.nih.gov/pubmed/22615906
Oba Y, Schultz DT (2014) Eco-evo bioluminescence on land and in the sea. Adv Biochem Eng Biotechnol
144:3-36. http://www.ncbi.nlm.nih.gov/pubmed/25084993
Oliveira AG, Desjardin DE, Perry BA, Stevani CV (2012) Evidence that a single bioluminescent system is
shared by all known bioluminescent fungal lineages. Photochem Photobiol Sci 11:848-852.
http://www.ncbi.nlm.nih.gov/pubmed/22495263
Peters AJ, Chen SX, Komiyama T (2014) Emergence of reproducible spatiotemporal activity during motor
learning. Nature 510:263-267. http://www.ncbi.nlm.nih.gov/pubmed/24805237
Pfleger KD, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the realtime detection of protein-protein interactions. Nat Protoc 1:337-345.
http://www.ncbi.nlm.nih.gov/pubmed/17406254
Ponsioen B, Zhao J, Riedl J, Zwartkruis F, van der Krogt G, Zaccolo M, Moolenaar WH, Bos JL, Jalink K
(2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer:
Epac as a novel cAMP indicator. EMBO Rep 5:1176-1180.
http://www.ncbi.nlm.nih.gov/pubmed/15550931
Promega Corporation (2000) Technical Bulletin: Luciferase assay system Promega Corporation.
https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/0/luciferaseassay-system-protocol.pdf
Purtov KV, Petushkov VN, Baranov MS, Mineev KS, Rodionova NS, Kaskova ZM, Tsarkova AS, Petunin AI,
Bondar VS, Rodicheva EK, Medvedeva SE, Oba Y, Oba Y, Arseniev AS, Lukyanov S, Gitelson JI,
Yampolsky IV (2015) The Chemical Basis of Fungal Bioluminescence. Angew Chem Int Ed Engl
54:8124-8128. http://www.ncbi.nlm.nih.gov/pubmed/26094784
Russell JT (2011) Imaging calcium signals in vivo: a powerful tool in physiology and pharmacology. Br J
Pharmacol 163:1605-1625. http://www.ncbi.nlm.nih.gov/pubmed/20718728
Sacchetti A, Cappetti V, Marra P, Dell'Arciprete R, El Sewedy T, Crescenzi C, Alberti S (2001) Green
fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells. J Cell Biochem
Suppl Suppl 36:117-128. http://www.ncbi.nlm.nih.gov/pubmed/11455577
21
Saini C, Liani A, Curie T, Gos P, Kreppel F, Emmenegger Y, Bonacina L, Wolf JP, Poget YA, Franken P,
Schibler U (2013) Real-time recording of circadian liver gene expression in freely moving mice
reveals the phase-setting behavior of hepatocyte clocks. Genes Dev 27:1526-1536.
http://www.ncbi.nlm.nih.gov/pubmed/23824542
Salih A, Larkum A, Cox G, Kuhl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are
photoprotective. Nature 408:850-853. http://www.ncbi.nlm.nih.gov/pubmed/11130722
Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14:381-392.
http://www.ncbi.nlm.nih.gov/pubmed/9608509
Shafer OT, Kim DJ, Dunbar-Yaffe R, Nikolaev VO, Lohse MJ, Taghert PH (2008) Widespread receptivity to
neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by
real-time cyclic AMP imaging. Neuron 58:223-237.
http://www.ncbi.nlm.nih.gov/pubmed/18439407
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905909. http://www.ncbi.nlm.nih.gov/pubmed/16299475
Shimomura O (2006) Bioluminescence Chemical Principles and Methods Preface. Bioluminescence:
Chemical Principles and Methods http://www.worldscientific.com/worldscibooks/10.1142/6102
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a
bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol
59:223-239. http://www.ncbi.nlm.nih.gov/pubmed/13911999
Shimomura O, Johnson FH, Saiga Y (1963) Further Data on the Bioluminescent Protein, Aequorin. J Cell
Physiol 62:1-8. http://www.ncbi.nlm.nih.gov/pubmed/14051831
Shultzaberger RK, Paddock ML, Katsuki T, Greenspan RJ, Golden SS (2015) High-throughput and
quantitative approaches for measuring circadian rhythms in cyanobacteria using
bioluminescence. Methods Enzymol 551:53-72.
http://www.ncbi.nlm.nih.gov/pubmed/25662451
Sparks JS, Schelly RC, Smith WL, Davis MP, Tchernov D, Pieribone VA, Gruber DF (2014) The covert world
of fish biofluorescence: a phylogenetically widespread and phenotypically variable
phenomenon. PLoS One 9:e83259. http://www.ncbi.nlm.nih.gov/pubmed/24421880
Sugiura K, Nagai T, Nakano M, Ichinose H, Nakabayashi T, Ohta N, Hisabori T (2015) Redox sensor
proteins for highly sensitive direct imaging of intracellular redox state. Biochem Biophys Res
Commun 457:242-248. http://www.ncbi.nlm.nih.gov/pubmed/25592971
Sun XR, Badura A, Pacheco DA, Lynch LA, Schneider ER, Taylor MP, Hogue IB, Enquist LW, Murthy M,
Wang SS (2013) Fast GCaMPs for improved tracking of neuronal activity. Nat Commun 4:2170.
http://www.ncbi.nlm.nih.gov/pubmed/23863808
Sundlov JA, Fontaine DM, Southworth TL, Branchini BR, Gulick AM (2012) Crystal structure of firefly
luciferase in a second catalytic conformation supports a domain alternation mechanism.
Biochemistry 51:6493-6495. http://www.ncbi.nlm.nih.gov/pubmed/22852753
Tahara Y, Kuroda H, Saito K, Nakajima Y, Kubo Y, Ohnishi N, Seo Y, Otsuka M, Fuse Y, Ohura Y, Komatsu
T, Moriya Y, Okada S, Furutani N, Hirao A, Horikawa K, Kudo T, Shibata S (2012) In vivo
monitoring of peripheral circadian clocks in the mouse. Curr Biol 22:1029-1034.
http://www.ncbi.nlm.nih.gov/pubmed/22578421
Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in
vivo. Nat Protoc 4:582-591. http://www.ncbi.nlm.nih.gov/pubmed/19373229
Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA,
Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL (2009) Imaging neural activity in
worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6:875-881.
http://www.ncbi.nlm.nih.gov/pubmed/19898485
22
Tosches MA, Bucher D, Vopalensky P, Arendt D (2014) Melatonin signaling controls circadian swimming
behavior in marine zooplankton. Cell 159:46-57.
http://www.ncbi.nlm.nih.gov/pubmed/25259919
Vielhaber E, Eide E, Rivers A, Gao ZH, Virshup DM (2000) Nuclear entry of the circadian regulator mPER1
is controlled by mammalian casein kinase I epsilon. Mol Cell Biol 20:4888-4899.
http://www.ncbi.nlm.nih.gov/pubmed/10848614
Viviani V (2007) TERRESTRIAL BIOLUMINESCENCE. In: Photobiological sciences online (American Society
for Photobiology., ed), p 1 online resource. McLean, VA: American Society for Photobiology,
Viviani VR, Arnoldi FG, Neto AJ, Oehlmeyer TL, Bechara EJ, Ohmiya Y (2008) The structural origin and
biological function of pH-sensitivity in firefly luciferases. Photochem Photobiol Sci 7:159-169.
http://www.ncbi.nlm.nih.gov/pubmed/18264583
Vukusic P, Hooper I (2005) Directionally controlled fluorescence emission in butterflies. Science
310:1151. http://www.ncbi.nlm.nih.gov/pubmed/16293753
Welsh DK, Noguchi T (2012) Cellular bioluminescence imaging. Cold Spring Harb Protoc 2012.
http://www.ncbi.nlm.nih.gov/pubmed/22854570
Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA (2004) Bioluminescence imaging of individual fibroblasts
reveals persistent, independently phased circadian rhythms of clock gene expression. Curr Biol
14:2289-2295. http://www.ncbi.nlm.nih.gov/pubmed/15620658
White J, Stelzer E (1999) Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol
9:61-65. http://www.ncbi.nlm.nih.gov/pubmed/10087620
Wilson T, Hastings JW (1998) Bioluminescence. Annu Rev Cell Dev Biol 14:197-230.
http://www.ncbi.nlm.nih.gov/pubmed/9891783
Xie Y, Chan AW, McGirr A, Xue S, Xiao D, Zeng H, Murphy TH (2016) Resolution of High-Frequency
Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR. J
Neurosci 36:1261-1272. http://www.ncbi.nlm.nih.gov/pubmed/26818514
Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system:
application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 96:151-156.
http://www.ncbi.nlm.nih.gov/pubmed/9874787
Yamada Y, Nishide SY, Nakajima Y, Watanabe T, Ohmiya Y, Honma K, Honma S (2013) Monitoring
circadian time in rat plasma using a secreted Cypridina luciferase reporter. Anal Biochem
439:80-87. http://www.ncbi.nlm.nih.gov/pubmed/23624321
Yamada Y, Michikawa T, Hashimoto M, Horikawa K, Nagai T, Miyawaki A, Hausser M, Mikoshiba K (2011)
Quantitative comparison of genetically encoded Ca indicators in cortical pyramidal cells and
cerebellar Purkinje cells. Front Cell Neurosci 5:18.
http://www.ncbi.nlm.nih.gov/pubmed/21994490
Yamaguchi S, Isejima H, Matsuo T, Okura R, Yagita K, Kobayashi M, Okamura H (2003) Synchronization of
cellular clocks in the suprachiasmatic nucleus. Science 302:1408-1412.
http://www.ncbi.nlm.nih.gov/pubmed/14631044
Yamaguchi S, Kobayashi M, Mitsui S, Ishida Y, van der Horst GT, Suzuki M, Shibata S, Okamura H (2001)
View of a mouse clock gene ticking. Nature 409:684.
http://www.ncbi.nlm.nih.gov/pubmed/11217850
Yamaguchi Y, Okada K, Mizuno T, Ota T, Yamada H, Doi M, Kobayashi M, Tei H, Shigeyoshi Y, Okamura H
(2016) Real-Time Recording of Circadian Per1 and Per2 Expression in the Suprachiasmatic
Nucleus of Freely Moving Rats. J Biol Rhythms 31:108-111.
http://www.ncbi.nlm.nih.gov/pubmed/26656624
Yoo SH, Ko CH, Lowrey PL, Buhr ED, Song EJ, Chang S, Yoo OJ, Yamazaki S, Lee C, Takahashi JS (2005) A
noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo. Proc Natl Acad
Sci U S A 102:2608-2613. http://www.ncbi.nlm.nih.gov/pubmed/15699353
23
Zhang L, Gase K, Baldwin I, Galis I (2010) Enhanced fluorescence imaging in chlorophyll-suppressed
tobacco tissues using virus-induced gene silencing of the phytoene desaturase gene.
Biotechniques 48:125-133. http://www.ncbi.nlm.nih.gov/pubmed/20359296
Zhao H, Doyle TC, Coquoz O, Kalish F, Rice BW, Contag CH (2005) Emission spectra of bioluminescent
reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J
Biomed Opt 10:41210. http://www.ncbi.nlm.nih.gov/pubmed/16178634
Zhao J, Kilman VL, Keegan KP, Peng Y, Emery P, Rosbash M, Allada R (2003) Drosophila clock can
generate ectopic circadian clocks. Cell 113:755-766.
http://www.ncbi.nlm.nih.gov/pubmed/12809606
Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences.
Nat Biotechnol 21:1369-1377. http://www.ncbi.nlm.nih.gov/pubmed/14595365
https://www.hamamatsu.com/resources/pdf/etd/PMT_handbook_v3aE.pdf
24