Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis.
ABSTRACT Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring the mobility of fluorescently tagged proteins in living cells. Although FRAP presumes that high-intensity illumination causes only irreversible photobleaching, reversible photoswitching of many fluorescent molecules, including GFP, can also occur. Here, we show that this photoswitching is likely to contaminate many FRAPs of GFP, and worse, the size of its contribution can be up to 60% under different experimental conditions, making it difficult to compare FRAPs from different studies. We develop a procedure to correct FRAPs for photoswitching and apply it to FRAPs of the GFP-tagged histone H2B, which, depending on the precise photobleaching conditions exhibits apparent fast components ranging from 9-36% before correction and ∼1% after correction. We demonstrate how this ∼1% fast component of H2B-GFP can be used as a benchmark both to estimate the role of photoswitching in previous FRAP studies of TATA binding proteins (TBP) and also as a tool to minimize the contribution of photoswitching to tolerable levels in future FRAP experiments. In sum, we show how the impact of photoswitching on FRAP can be identified, minimized, and corrected.
- SourceAvailable from: Kateri J Spinelli[Show abstract] [Hide abstract]
ABSTRACT: Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of α-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study α-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human α-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic α-synuclein existed primarily in an unbound, soluble pool. In contrast, α-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing α-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for α-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, α-synuclein aggregates are selectively found in presynaptic terminals.Journal of Neuroscience 02/2014; 34(6):2037-50. · 6.75 Impact Factor
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ABSTRACT: Since the pioneering work of Hirschfeld, it is known that time-integrated emission (TiEm) of a fluorophore is independent of fluorescence quantum yield and illumination intensity. Practical implementation of this important result for determining exact probe distribution in living cells is often hampered by the presence of autofluorescence. Using kinetic modelling of photobleaching combined with pixel-wise bleach rate fitting of decay models with an updated plugin to the ImageJ program, it is shown that the TiEm of a fluorophore in living cells can be determined exactly from the product of bleaching amplitude and time constant. This applies to mono-exponential bleaching from the first excited singlet and/or triplet state and to multi-exponential combinations of such processes. The TiEm can be used to correct for illumination shading and background autofluorescence without the need for fluorescent test layers or separate imaging of non-stained cells. We apply the method to simulated images and to images of cells, whose membranes were labelled with fluorescent sterols and sphingolipids. Our bleaching model can be extended to include a probability density function (PDF) of intrinsic bleach rate constants with a memory kernel. This approach results in a time-dependent bleach rate coefficient and is exemplified for fluorescent sterols in restricted intracellular environments, like lipid droplets. We show that for small deviations from the classical exponential bleaching, the TiEm of decay functions with rate coefficients remains largely independent of fluorescence lifetime and illumination, and thereby represents a faithful measure of probe distribution.Molecules 08/2014; 19(8):11096-130. · 2.10 Impact Factor
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ABSTRACT: Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring protein dynamics in live cells that has provided many important biological insights. Although FRAP presumes that the conversion of a fluorophore from a bright to a dark state is irreversible, GFP as well as other genetically encoded fluorescent proteins now in common use can also exhibit a reversible conversion known as photoswitching. Various studies have shown how photoswitching can cause at least four different artifacts in FRAP, leading to false conclusions about various biological phenomena, including the erroneous identification of anomalous diffusion or the overestimation of the freely diffusible fraction of a cellular protein. Unfortunately, identifying and then correcting these artifacts is difficult. Here we report a new characteristic of an organic fluorophore tetramethylrhodamine bound to the HaloTag protein (TMR-HaloTag), which like GFP can be genetically encoded, but which directly and simply overcomes the artifacts caused by photoswitching in FRAP. We show that TMR exhibits virtually no photoswitching in live cells under typical imaging conditions for FRAP. We also demonstrate that TMR eliminates all of the four reported photoswitching artifacts in FRAP. Finally, we apply this photoswitching-free FRAP with TMR to show that the chromatin decondensation following UV irradiation does not involve loss of nucleosomes from the damaged DNA. In sum, we demonstrate that the TMR Halo label provides a genetically encoded fluorescent tag very well suited for accurate FRAP experiments.PLoS ONE 09/2014; 9(9):e107730. · 3.53 Impact Factor
Minimizing the Impact of Photoswitching of Fluorescent Proteins on FRAP
Florian Mueller,6Tatsuya Morisaki,6Davide Mazza, and James G. McNally*
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, Maryland
mobility of fluorescently tagged proteins in living cells. Although FRAP presumes that high-intensity illumination causes only irre-
versible photobleaching, reversible photoswitching of many fluorescent molecules, including GFP, can also occur. Here, we
show that this photoswitching is likely to contaminate many FRAPs of GFP, and worse, the size of its contribution can be up
to 60% under different experimental conditions, making it difficult to compare FRAPs from different studies. We develop a proce-
dure to correct FRAPs for photoswitching and apply it to FRAPs of the GFP-tagged histone H2B, which, depending on the
precise photobleaching conditions exhibits apparent fast components ranging from 9–36% before correction and ~1% after
correction. We demonstrate how this ~1% fast component of H2B-GFP can be used as a benchmark both to estimate the
role of photoswitching in previous FRAP studies of TATA binding proteins (TBP) and also as a tool to minimize the contribution
of photoswitching to tolerable levels in future FRAP experiments. In sum, we show how the impact of photoswitching on FRAP
can be identified, minimized, and corrected.
Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring the
Over the past decade, fluorescence recovery after photo-
bleaching (FRAP) has been used extensively to measure
the mobility of fluorescently tagged proteins in living cells
(1,2). These measurements have provided many insights
into the dynamics of cellular processes, but there remain
a number of assumptions in FRAP that have yet to be thor-
oughly evaluated (3).
One fundamental presumption is that the conversion from
a bright state to a dark state is irreversible (4). This means
that the measured fluorescence recovery is due exclusively
to the influx of fluorescent molecules into the bleach spot,
therefore providing a direct assay of molecular mobility.
However, light exposure can convert fluorescent molecules
to a dark state either irreversibly (photobleaching) or revers-
ibly (photoswitching). Photoswitched molecules can later
revert to the bright state, yielding a reversible fraction,
a process also known as reversible photobleaching (5–9).
An undetected reversible fraction will cause overestimates
of protein mobility in FRAP. Many fluorescent molecules,
including GFP and its derivatives, exhibit some degree of
photoswitching. The photophysical properties of this
process have been well characterized (6,7,10,11), but the
impact of photoswitching on FRAP, namely howmuch over-
estimation of mobility occurs, has only been investigated in
a handful of studies (5,9).
By photobleaching fixed cells, Dayel et al. showed that
GFP can undergo rapid photoswitching with a reversible
fraction of 5–15% (5). Sinnecker et al. (9) also investigated
photoswitching, but of the GFP derivatives CFP and YFP in
live cells. They performed FRAP on the same membrane
protein tagged either with YFP or CFP (9). They found
that the YFP fusion was largely immobile, whereas the
CFP fusion had a 60% fast component, which they attrib-
uted to photoswitching of CFP.
The preceding studies indicate that photoswitching of
GFP and its derivatives can influence FRAPs, but the esti-
mated size of the reversible fraction differed markedly. As
a result, it remains unclear whether photoswitching has
a significant impact on most FRAPs. The primary reason
for this is that there is no established procedure to evaluate
how much photoswitching occurs in a FRAP experiment. As
a consequence, most FRAP studies to date have ignored the
possibility of photoswitching.
Here, using typical FRAP conditions, we show that pho-
toswitching occurs with five different fluorescent proteins:
GFP, YFP, mCherry, TagRFP, and mTFP1. We find that
for GFP, the size of the reversible fraction depends very
sensitively on the strength of the intentional photobleach,
and we show that this can give rise to very different
FRAP recoveries for the histone H2B-GFP and the TATA-
binding protein (TBP)-GFP under FRAP conditions
commonly used by different laboratories.
We develop a mathematical method to correct for revers-
ible behavior in FRAP and apply it to H2B-GFP FRAPs, ob-
taining a fast component of 0.8 5 0.8%. With this
knowledge, we show that FRAP of H2B-GFP can be used
by estimating the contributions of reversible fractions in
these different studies. We also show that FRAP of H2B in
Submitted September 15, 2011, and accepted for publication February 13,
6Florian Mueller and Tatsuya Morisaki contributed equally to this work.
Florian Mueller’s present address is Institut Pasteur, Imaging and Modeling
Group CNRS, Paris, France
Editor: Anne Kenworthy.
? 2012 by the Biophysical Society
1656 Biophysical Journal Volume 102April 20121656–1665
minimize the reversible effect. Once these optimal photo-
bleaching conditions are selected, we find errors of no
parameters and fast components before and after applying
our mathematical method to correct for reversible fractions.
This suggests that selection of optimal photobleaching
conditions by performing FRAP of H2B in live cells may
be sufficient to minimize the effects of photoswitching in
many FRAP studies. If the reversible fraction cannot be
reduced to tolerable levels, or if more accurate estimation
is needed, our correction procedure can be applied.
MATERIALS AND METHODS
Cells and constructs
We used a previously described H2B-GFP construct (12). To produce TBP-
GFP, the open reading frame encoding human TBP was amplified using
appropriate oligonucleotides flanked with XhoI and EcoRI, and then in-
serted into pEGFP-N1 (Clontech, Mountain View, CA). For FRAPs,
HeLa cells were transiently transfected with pEGFP-N1, pEYFP-N1,
pmCherry-C1, pTagRFP-N (Axxora, San Diego, CA), pmTFP1-N (Allele
Biotech, San Diego, CA), H2B-GFP, or TBP-GFP DNA (12) using Lipo-
fectamine LTX reagent (Invitrogen, Carlsbad, CA) according to the manu-
facturer’s instructions. At 24 h after transfection, cells were also prepared
for live-cell FRAP experiments as previously described (13), whereas for
fixed-cell FRAPs, cells were first incubated in 3.5% paraformaldehyde
for 60 min at room temperature on a shaker at 50 rpm, and then washed
three times with fresh medium.
Data collection and processing
The FRAP experiments with unconjugated fluorescent proteins were per-
formed on a Zeiss LSM 5 LIVE DuoScan confocal microscope using
a 63X/1.4 NA oil immersion objective. The temperature of the incubated
stage was set to 37?C, and the CO2content was held at 5%. For each fluo-
rescent protein, imaging sizewas 500 ? 500 pixels (50 ? 50 mm2), imaging
time was 50 ms, and the optical slice was 2.0 mm. We used different lasers,
dichroics, and filter sets depending on the spectral characteristics of each
protein (Table S1 in the Supporting Material). For photobleaching, we
used the same 100-mW 488-nm laser line, because this laser line yielded
the strongest bleach for each of the proteins (better than a 50-mW 405 laser
line for mTFP1 or a 40-mW 561 laser line for TagRFP and mCherry—see
Table S1 for details of the optical configurations).
The H2B-GFP whole-nucleus FRAP experiments were performed under
the same conditions for GFP as described above except that imaging size
was set to 250 ? 250 pixels (25 ? 25 mm2), and imaging time was
33 ms. The intentional photobleaching was performed with the 488-nm
line under the different conditions described in the Results. Control
measurements for observational photobleaching and subsequent data pro-
cessing were carried out as described earlier (14). FRAPs on fixed cells
were performed in the same medium and under incubation conditions iden-
tical to those for live cells. The strip FRAPs for H2B-GFP were performed
on a Zeiss LSM510.Thecircle FRAPsfor H2B-GFPwereperformedunder
the same conditions as for the H2B-GFP whole-nucleus bleaches, except
that the intentional photobleach was performed with a small circle of
2.0 mm in diameter. See Table S1 for details.
In an attempt to match the conditions used in the previous TBP-GFP
FRAP experiments, we performed FRAP on a Zeiss LSM 510, which
was the same instrument used in the preceding two TBP FRAP studies in
mammalian cells. See Section 5 and Table S2 in the Supporting Material
for details of both the published protocols and our protocols designed
to approximate them. To correct the reversible fraction of TBP, we per-
formed FRAPs on a Zeiss LSM 5 LIVE with the same conditions used
for H2B-GFP, except that the acousto-optic tunable filter was set to 20%
to observe dimmer cells, since TBP overexpression had been reported to
yield slower FRAPs and lead to underestimation of the fast component (15).
Fitting of the FRAP data
The FRAP model equations were programmed in MATLAB (The Math-
Works, Natick, MA), and the routine lsqcurvefit was used to fit the models
to experimental data. Details of the fitting procedure can be found in
Mueller et al. (14). The MATLAB source code for the newly developed
FRAP procedureto correctfor photoswitching(Eq. 5in Resultsand Discus-
sion) and the modeling protocol are available upon request.
RESULTS AND DISCUSSION
GFP exhibits photoswitching after intentional
Our objective is to characterize and ultimately mitigate the
effects of photoswitching in FRAP experiments. This photo-
switching behavior is likely to be sensitive to a variety of
environmental conditions, including pH (9), so our experi-
ments have been designed to determine these effects within
livecells, or as close as possible to livecells, namely in fixed
Thus, to test for photoswitching of GFP we transfected
live cells with unconjugated GFP, photobleached the entire
cell, and then measured the recovery over 20 s. Although
this procedure should bleach virtually all of the fluorescence
to yield no recovery, we consistently measured a fluores-
cence recovery. We quantified the recovery by renormaliz-
ing it such that the intensity before the bleach was 1 and
the intensity immediately after the bleach was 0 (Fig. 1 A).
One possible explanation for this renormalized recovery
(~9%) was that it reflects the percentage of the fluorescence
that returns to the fluorescent state from the dark state.
An alternative explanation for this 9% recovery was
detector photoblinding (14), which is a transient loss in
sensitivity that can occur on a photomultiplier tube after it
has been exposed to the intense fluorescence produced
by a photobleach. When present, detector photoblinding
produces an artifactual increase in measured fluorescence
as the detector regains sensitivity. However, we found that
the CCD camera used for the measurement in Fig. 1 A
did not exhibit detector photoblinding (Fig. S1 A), thereby
excluding this possibility.
A second alternative explanation for the 9% recovery was
that the photobleach was nonuniform, particularly along the
z axis, where a conical photobleach profile is expected (16).
If so, then small spatial gradients in fluorescence could exist
and their relaxation by diffusion could conceivably produce
some influx of fluorescence into the focal plane. To exclude
this, we performed the same whole-cell photobleach on
Biophysical Journal 102(7) 1656–1665
Minimizing Photoswitching in FRAP1657
fixed cells, measuring a fluorescence recovery (~10%)
comparable to that in live cells (Fig. S1 B). This suggests
that diffusion in the live cells did not contribute to the
However, in principle, these very similar recoveries in
live and fixed cells could have arisen by chance if the
60 min paraformaldehyde fixation procedure failed to
cross-link a 10% fraction of the GFP. To test for this possi-
bility, we also fixed cells containing GFP-H2B, most of
which is thought to be tightly associated to chromatin inside
of cells with only a small diffusible component (17,18).
Despite this small free fraction, the fluorescence recovery
in the fixed H2B cells was very similar to the recovery in
the fixed GFP cells (Fig. S1 C), indicating that these recov-
eries were not due to diffusion of unfixed molecules.
We conclude that the 9% recovery seen in a whole-cell
photobleach primarily reflects the photoswitching of GFP
molecules within live cells.
Four other fluorescent proteins also exhibit
Any sort of reversible behavior can complicate FRAP exper-
iments, so we tested four alternative tags, namely YFP,
mCherry, TagRFP, and mTFP1 to see if any would provide
a better label for FRAP. We estimated the reversible fraction
for each tag by using the same whole-live-cell photobleach
procedure described above. An optimal tag for FRAP would
show a very small reversible fraction that recovered very
mTFP1 yielded a much larger reversible fraction than
GFP (20% vs. 9%) but a somewhat faster reversion rate
(2–3 s vs. 3–4 s) (Fig. 1 B). YFP yielded a similar reversible
fraction to GFP (~9%) but a slower reversion rate (~20 s)
(Fig. 1 B). mCherry also displayed a slower reversion
rate (~20 s), but with a smaller reversible fraction (5%)
(Fig. 1 B).
TagRFP was unique in that it showed very slow conver-
sion to the dark state, such that measurements of the
recovery phase still contained a fluorescence loss over the
first few seconds (Fig. 1 B). Only by 4–5 s after the photo-
bleach had TagRFP finally decayed to a steady-state value,
which then increased only slightly, suggesting a small
reversible fraction. Despite the negligible reversible fraction
for TagRFP, like GFP, it required ~4 s to achieve an equili-
brated state (Fig. 1 B), so it is also unsuited for most FRAP
experiments. Thus, none of these fluorescent proteins
yielded satisfactory behavior for FRAP.
The reversible fraction of GFP varies
considerably depending on the bleaching
The preceding data indicate that complicated photoswitch-
ing behaviors are a common property of fluorescent
proteins. GFP has been the most widely used fluorescent
protein for FRAP, and our tests of other fluorescent proteins
suggest that there is not a simple alternative. Thus, we pro-
ceeded to further investigate the consequences of GFP’s
reversible behavior for FRAP. To do so, we again used
H2B-GFP, since it is mostly bound to chromatin and is ex-
pected to have at best a small free component (17,18). We
subjected live cells transfected with H2B-GFP to a whole-
nucleus photobleach, and then to varied photobleaching
parameters typically used in FRAP experiments. First, we
gradually decreased the laser power during the photobleach
to replicate the effects of weaker photobleaches that can
arise in real FRAPs due to older or misaligned lasers.
Second, we also changed the number of iterations of the
fractions. (A) Photoswitching behaviors of GFP, with a zoomed-in view
(inset). To eliminate gradients in fluorescence, whole cells were photo-
bleached, and then whole-cell intensity was measured over time. The result-
ing recovery curves were renormalized such that prebleach intensity was 1
and postbleach intensity was 0. Thus, the renormalized curve shows the
relative contribution of photoswitching to FRAP. (B) Photoswitching
behaviors of four other fluorescent proteins. In this assay, an ideal probe
for FRAP would show rapid photobleaching and then no recovery. (C)
Live cells expressing H2B-GFP were subjected to whole-nuclear bleaches
using a single bleach iteration at different laser intensities. Stronger laser
intensities reduced the reversible fraction. (D) In a real FRAP experiment
(here using a strip photobleach), the fast component of H2B can reach
60% even with a reasonable bleach depth (inset; the bleach depth was
not normalized to zero). The asterisk next to the curve indicates the point
on the measured curve where the change in slope is minimized, which is
how we estimated fast components.
Fluorescent proteins exhibit markedly different reversible
Biophysical Journal 102(7) 1656–1665
1658Mueller et al.
We found that the laser intensity of the intentional photo-
bleach had a striking effect on the reversible fraction, which
increased from 5% to 30% as the laser intensity decreased
(Fig. 1 C). Consistent with this, the number of bleach itera-
tions also had a marked effect on the reversible fraction,
which could be reduced to as low as 1% when using five
bleach iterations (data not shown). These effects in whole
nuclear FRAPs directly translated to real FRAPs. Bleaching
of a 0.5-mm-wide strip across the nucleus produced H2B
recoveries with fast components ranging from 5% to 60%
that depended on the strength of the photobleach (the worst
case (60%) is shown in Fig. 1 D). It is important to note that
even this weakest photobleach (with a 60% H2B fast
component) still produced FRAP curves with a substantial
bleach depth (Fig. 1 D, inset) demonstrating that large
reversible fractions can arise under standard FRAP
A mathematical model to correct for
photoswitching in FRAP
The preceding results show that FRAPs of GFP-H2B in live
cells under different photobleaching conditions can give rise
to a wide range of recoveries. Our data strongly suggest that
the differences are due to different photoswitchable frac-
tions induced by different photobleaching conditions, since
we could obtain very different fast components simply by
changing the intensity or duration of the photobleach. Our
smallest fast component for H2B was 5%, which provides
an upper bound on the size of the true H2B fast component,
but it is not clear how much of this 5% fast component is
still due to a reversible fraction. This will be a general
problem in any FRAP experiment.
To address this, we developed a correction procedure for
photoswitching in FRAP. A first approach might be to esti-
mate the size of the photoswitchable fraction for the photo-
bleaching conditions in use and then subtract this from the
initial part of the FRAP. However, this neglects the fact
that a substantial fraction of the photoswitchable molecules
may diffuse out of the bleach spot if the protein under study
has a large fast component. The subtraction procedure will
then lead to underestimation of the fast component. Alterna-
tively, the time required for the photoswitchable fraction to
recover could be measured, and then this part of the initial
FRAP curve could be ignored. This, however, will also
lead to underestimation of the true fast component, since
some of the discarded recovery can reflect the true recovery
of the protein under study. Thus, proper correction of photo-
switching in FRAP requires a more rigorous mathematical
model that accounts for the movements of fluorescent mole-
cules into the bleach spot, as well as the appearance of mole-
cules that revert to the bright state, some of which may
diffuse out of the bleach spot.
Let IMðr;tÞ be the measured FRAP recovery that should
already be corrected for observational photobleaching, if it
occurs. Note that in some cases, the observational photo-
bleaching correction is itself also complicated by photo-
switching (see Section 2 in the Supporting Material for
This measured recovery, IMðr;tÞ, reflects two compo-
nents: the entry of fluorescent molecules into the bleach
spot described by the conventional FRAP equations,
FRAPðr;tÞ, plus a new component arising from a fraction
of the molecules, Irevðr;tÞ, that revert to the bright state
from the dark state. Thus,
IMðr;tÞ ¼ FRAPðr;tÞ þ Irevðr;tÞ:
To find equations that describe the reversible fraction Irev,
we note that this contribution to the measured recovery
curve is effectively a time-dependent reactivation of the
photoswitched GFP. This is mathematically equivalent to
a photoactivation or FLAP (fluorescence loss after photoac-
tivation) experiment, except that in these experiments all of
the molecules are instantly photoactivated, whereas in pho-
toswitching, molecules revert to the bright state over a time
course. Thus, to describe the reactivation occurring in pho-
toswitching, we must modulate the equations to describe
a FLAP by a function accounting for the fraction of mole-
cules that have reverted to the bright state, RðtÞ. Note in
fact that Irev depends on the history of the FLAP up to
time t rather than just the current FLAP at time t; however,
the following equation is assumed for theoretical and
Irevðr;tÞ ¼ RðtÞ ? FLAPðr;tÞ:
Like FRAP, FLAP depends on space and time. FLAP has
been quantified before (19), but to our knowledge the
connection between FRAP and FLAP has not been directly
demonstrated. In Section 3 of the Supporting Material, we
FLAPðr;tÞ ¼ 1 ? FRAPðr;tÞ:
To implement this equation, the initial condition for the
FLAP model, namely, the photoactivation profile ðPactÞ,
must be converted to an equivalent photobleaching profile
ðP00ble00Þ, where the quotes on the subscript ‘‘ble’’ indicate
that this is not a true photobleach, but instead the
photobleach that would occur by transforming the FLAP
experiment into the corresponding FRAP experiment.
We show in the Section 3 of the Supporting Material
that the equivalent photobleaching profile is given by
P00ble00ðrÞ ¼ 1 ? PactðrÞ.
Substitution of Eqs. 2 and 3 in Eq. 1 yields the full equa-
tion to describe the recovery profile after the photobleach:
IMðr;tÞ ¼ FRAPðr;t;PbleÞ þ Irevðr;tÞ
¼ FRAPðr;t;PbleÞ þ RðtÞ
? ½1 ? FRAPðr;t;P00ble00Þ?;
Biophysical Journal 102(7) 1656–1665
Minimizing Photoswitching in FRAP1659
where the terms Pbleand P00ble00 have been added to the func-
tional dependence of the FRAP terms to indicate that they
have fundamentally different initial conditions. This is
because the number of molecules that undergo reactivation
is only a fraction of the number of molecules in the dark
To use Eq. 4 to fit FRAP data, Pble, P00ble00, and R(t) must be
measured. Pblecan be obtained directly from the live-cell
FRAP data, but to isolate the reversible behavior character-
ized by P00ble00 and R(t), measurements must be made in fixed
cells expressing H2B-GFP. For these measurements, we
estimated that the fraction of freely diffusible H2B-GFP
after fixation is at most 0.08% (Section 1 in the Supporting
Material), and so the observed recovery can be attributed
solely to photoswitching of GFP in the fixed cells. As noted
above, photoswitching in fixed cells is similar to photo-
switching in live cells, but they are not exactly the same.
As described in the next section, we show that fixed-cell
reversible fractions can be converted into live-cell reversible
fractions by use of a rescaling factor, a. Thus, the final equa-
tion to describe the recovery profile after the photobleach is
IMðr;tÞ ¼FRAPðr;t;PbleÞ þ aRðtÞ
? ½1 ? FRAPðr;t;P00ble00Þ?:
Measurements required for the photoswitching
To determine the equivalent photobleaching profile for the
reactivation P00ble00, we performed a circular photobleach in
fixed cells with the same conditions used for live cells and
then measured the radial-intensity profile as a function of
time (Fig. 2 A). After a few seconds, the reversible fraction
equilibrated, as indicated by no further change in the profile.
This stationary profile reflects only the irreversibly photo-
bleached molecules that remain. The difference between
the first profile after the photobleach and this stationary
profile therefore reflects the total profile of reactivated mole-
cules arising from photoswitching. We found that this
profile could be fit with a piecewise constant function
with Gaussian flanks (Fig. 2 B), and so we used one minus
this function as the initial condition for the second FRAP
term in Eq. 5.
To determine the function accounting for the fraction of
molecules that have reverted to the bright state, R(t), we
measured the average intensity across the central part of
the reversible recovery profiles in Fig. 2 A as a function
of time. After renormalizing from 0 to 1, this yielded a
curve that could be fit with a double exponential (Eq. 6,
Fig. 2 C).
experiment. (A) FRAP was performed in a small circular
region (diameter 2.0 mm) in fixed cells expressing H2B-
GFP. The radial-intensity profile, Pt, was measured at
different time points, as described earlier (14). The equil-
ibrated profile was measured by averaging the profiles
after no measurable increase of fluorescence was detected
(Pequil). (B) The reactivation profile was measured by sub-
tracting the profile at t ¼ 0 s (P0) from the equilibrium
profile (Pequil). These data were fit (equation shown in
the plot) to yield the initial conditions for the reactivation.
(C) The rate of reversal was measured in the center of the
bleach region (dashed line in A) by computing the area
under the curve for Pt– P0at all time points t. After re-
normalization, the resultant curve was fit with a double
exponential. (D) The reversible behaviors in fixed and
live cells were different, as demonstrated by whole-
nuclear FRAPs of H2B-GFP. However, for these bleach
conditions, the entire fixed-cell recovery can be rescaled
by a factor of 0.75 to closely (but not perfectly) match
the live-cell recovery.
Quantification of photoswitching in a FRAP
Biophysical Journal 102(7) 1656–1665
1660 Mueller et al.
RðtÞ ¼ 1 ? A1? exp
? ð1 ? A1Þ ? exp
To obtain the rescaling factor, a, between fixed and live
cells, we performed whole-nuclear bleaches in fixed and
live cells, and compared reversible behaviors. We found
that the fixed reversible recovery could be converted to the
live reversible recovery by a one-parameter rescaling of
the fixedcurve(Fig. 2 D). By measuring this rescaling factor
at a series of photobleaches with increasing intensity, we
generated a calibration curve relating the scale factor to
the size of the reversible fraction in fixed cells (Fig. S4).
We then measured the reversible fraction produced by our
FRAP conditions in fixed cells and used the calibration
curve to find the appropriate rescaling factor, a.
With measurements of Pble, P00ble00, R(t), and a, Eq. 5 can
be used to fit the measured FRAP recovery. This requires
a FRAP model that describes the mobility of the protein
under study. In the next section for H2B correction, we
used our reaction diffusion model (13) that presumes diffu-
sion and binding of the protein to immobile chromatin sites.
This model has three unknown parameters, the molecule’s
diffusion constant and its association and dissociation rates
with chromatin. Once the parameters are estimated, the true
FRAP recovery is obtained by substituting these estimated
parameters into the reaction-diffusion term for FRAP in
Eq. 5, namely FRAPðr;t;PbleÞ.
Note that the preceding general approach is suitable for
any FRAP bleach geometry. In Section 3 of the Supporting
Material, we develop the procedure for a circular FRAP.
Also note that the new model is an extension of the basic
FRAP model described in Mueller et al. (14) and is subject
to all of the assumptions described for that model. This
includes the assumption of no variation in the photobleach
profilewith z, which means in practice that bleach diameters
should be on the order of 1 mm or larger. It also includes the
assumption that the first measurement is made immediately
after the photobleach, which in practice means the delay
between the bleach and first measurement should be mini-
mized (our delay is 45 ms).
Tests of the photobleaching correction procedure
We performed two tests of the effectiveness and accuracy of
the correction procedure for photoswitching. First, we asked
how reproducible our measurement was of the reversible
fraction in fixed cells, since this is the primary component
in the correction procedure. We measured this fraction
under seven different photobleaching conditions and per-
formed each measurement five times. From this, we calcu-
lated the standard deviation of the estimated reversible
fraction under each condition. These standard deviations
ranged from 0.4% to 3.7%, indicating that the reversible
fraction can be accurately estimated. (Table S3).
Second, we asked how much variation there would be in
estimating the true fast component of H2B. Using Eq. 5 as
implemented for a circle FRAP, we corrected the seven
FRAP curves of H2B, which showed fast components that
ranged from 8.5% to 35.8% (Fig. 3, A and C). After correc-
tion, the fast components ranged from 0.0% to 2.2% (Fig. 3,
B and C), with a mean value of 0.8 5 0.8%. These results
also suggest that the correction procedure is not subject to
large variations, since we could obtain a very similar fast
component for H2B regardless of the size of the initial
fast component. Our estimate of an ~1% fast component
of H2B is also consistent with the expectation that virtually
all of H2B is tightly bound to chromatin (17,18).
Based on these results, we propose that FRAP of H2B-
GFP in live cells can be used as a standard for evaluating
the presence of photoswitching under a certain set of photo-
bleaching conditions. The cell line and the plasmid encod-
ing H2B-GFP in use here are readily available and so
could be obtained by any laboratory performing FRAP
nuclei of live cells expressing H2B-GFP bleaching a small circular spot
(diameter 2.0 mm) with different laser intensities (see legend below A
and B). (B) After application of our correction procedure for photoswitch-
ing, the fast components of all H2B curves were reduced to 0–2% with
a mean fast component of 1%. Note that the corrected curves are smooth,
since they reflect a fit of the data using the photoswitching model. (C)
We estimated the fast component of H2B under each photobleaching condi-
tion before and after the correction. The fast components ranged from 8.5–
35.8% before the photoswitching correction and were reduced to a mean
value of 0.8 5 0.8% after the photoswitching correction.
Photoswitching correction. (A) We performed FRAP on
Biophysical Journal 102(7) 1656–1665
Minimizing Photoswitching in FRAP 1661
experiments. The photobleaching conditions selected for an
experiment can then be applied to live cells transfected with
H2B-GFP and the fast component estimated. If this fast
component exceeds a few percent, then the photobleaching
conditions give rise to photoswitching, and so the conditions
should be changed until the H2B-GFP fast component is
reduced to tolerable levels. Better bleaching conditions
can be determined empirically by increasing the bleaching
laser power, number of bleach iterations, or pixel dwell
time. If the reversible fraction cannot be reduced to tolerable
levels, or more accurate quantitative estimation is needed,
then the correction procedure described above can be
applied. We apply this general strategy in the next section
to FRAP analysis of TBP, which is a key component of
the polymerase complex.
A potential contribution of photoswitching to
discrepant TBP FRAP curves
A large body of in vitro data suggests that TBP is stably
bound at transcriptionally active promoters. However, in
mammalian cells, two different studies have produced
very different TBP FRAP curves, leading to potentially
different conclusions about TBP mobility (15,20). An espe-
cially puzzling question raised by these two studies has been
how FRAP data could be so different between two groups
studying the same protein.
The differences between these two FRAP curves are clear
when the data are extracted from each study and plotted on
the same graph (Fig. 4 A). de Graaf et al. (15) argued that
this difference could have been due to overexpression of
TBP in Chen et al. (20). This is plausible, since de Graaf
et al. showed that the protein BTAF1 reduces TBP’s resi-
dence time on DNA. Thus, higher TBP expression levels
could eventually titrate out the protein BTAF1, leading to
slower FRAPs. Consistent with this, de Graaf et al. showed
that brighter cells yielded slower TBP FRAPs, and we
confirmed this finding when we performed FRAPs of TBP
in dim and bright cells (Fig. S5 C). However, under our
conditions, the change in the FRAP curves due to TBP
concentration was ~10%, which was not large enough to
explain the ~30% difference between the published curves.
To investigate other possible explanations for the differ-
ence between the published TBP FRAP curves, we attemp-
ted to reproduce the published data by approximating the
imaging and photobleaching conditions reported (see
Table S2). We could not do this exactly, because not all
the parameter settings were reported, and furthermore,
a key parameter, the laser power, will depend on the age
and alignment of the laser in use. Nevertheless, we were
able to find conditions (referred to as conditions 1 and 2)
that allowed us to nearly replicate the published curves,
but now, it is important to note, using the same cells with
the same expression levels of TBP (Fig. 4 A and Table 1
for the estimated fast components). The similarities between
our curves and the published curves include a good match of
the actual bleach depths (see Fig. S5, A and B, in which
bleach depths were not normalized to zero).
Although it is impossible to exactly replicate the original
experiments, we can use the two conditions we identified to
investigate how different FRAPs can arise purely due to
changes in imaging and bleaching conditions. As we show
below, two factors account for the differences in our exper-
iments: the time to acquire the first image of the bleached
region and the contribution of photoswitching.
The time to acquire the first image of the bleached region
was 482 ms longer for condition 2 compared to condition 1.
This difference arose because condition 2 used a larger
image that introduced a longer interval between the end of
the bleach and the first image of the bleached region. The
(A) We were able to roughly match two published FRAP curves by approx-
imating the different imaging conditions used in the two studies (15,20).
Our two FRAP curves were obtained on the same cells expressing similar
levels of TBP. The difference in fast component between conditions 1
and 2 is ~30%. (See Fig. 1 legend for the procedure to estimate fast compo-
nents). (B) We renormalized the curve from condition 2 with the true bleach
depth by measuring the intensity in the bleach region as quickly as possible
after the condition 2 photobleach. This reduced the difference in fast
component between conditions 1 and 2 to ~20%. (C) We applied the photo-
bleaches of conditions 1 and 2 to H2B-GFP in live cells and found an ~20%
difference in reversible fractions, roughly accounting for the remaining
difference from B. (D) We performed FRAP using a circular photobleach
on cellsexpressinglow levelsof TBPand thenappliedourcorrectionproce-
dure for photoswitching to the TBP curve, yielding an estimated fast
component of 26%. The uncorrected H2B curve shows the level of photo-
switching present under these conditions.
Reconciling discrepant predictions of TBP fast components.
Biophysical Journal 102(7) 1656–1665
1662 Mueller et al.
reason for this is that the bleached square is in the middle of
the image in condition 2, so the top half of the image must
first be scanned, requiring 482 ms before the bleached
region is reached. This does not occur in condition 1, which
bleaches and then immediately images the same strip. Due
to this poor temporal sampling in condition 2, some of the
fast component of TBP is not detected. To correct for this,
we estimated the full extent of the bleach depth in condition
2 by performing a photobleach with the same laser intensity
and bleach pattern but then measuring the fluorescent inten-
sity in the bleached region as quickly as possible (with no
delay rather than 482 ms). We then used this value to
normalize the bleach depth to zero for the FRAP data
from condition 2. This correction reduced the difference
between the fast components observed in conditions 1 and
2 from ~30% to ~20% (Fig. 4 B).
We then asked whether photoswitching could explain the
remaining difference between the two curves. We cannot
directly correct the FRAPs from either condition 1 or condi-
tion 2, since our current implementation of the correction
method is not designed for FRAPs where only the bleached
region is imaged (condition 1) or for FRAPs with square
bleach profiles (condition 2). Instead, we used H2B-GFP
in live cells as a benchmark to estimate how much photo-
switching occurred under conditions 1 and 2. The difference
between the resultant H2B-GFP fast components was ~20%
(Fig. 4 C), which roughly accounts for the remaining differ-
ence in the fast components estimated from the delay-time-
corrected TBP FRAP curves (Fig. 4 B). Note that these
fast-component estimates are only approximate, particularly
since the temporal sampling of condition 2 is poor. Note also
that this simple approach of subtracting out reversible frac-
tions is crude, since the actual contribution to the FRAP will
also be influenced by the rate of reversion balanced by the
rate at which these molecules leave the bleached region.
Nevertheless, our analysis suggests that by accounting for
the temporal sampling rate and the amount of photoswitch-
ing, the FRAP curves from the two previous studies are in
fact very similar. This is reassuring, because it demonstrates
that there are not fundamental differences in the behavior of
TBP in these two published studies.
Finally, we tested our suggested procedure of using FRAP
of H2B to select photobleaching conditions that would mini-
mize the reversible fraction. We used a 45-ms circular pho-
tobleach and thenvaried the laser intensity, pixel dwell time,
and number of iterations during the photobleach. The small-
est fast component of H2B that we could obtain was 6%,
suggesting a residual 5% reversible fraction under these
optimal conditions. We then used these optimized condi-
tions to perform FRAP in TBP, and subsequently applied
our mathematical procedure to correct for the residual
reversible fraction. We obtained estimates of TBP free frac-
tions that showed ~5% change before and after correction
(35 5 2.7% vs. 30 5 3.0%), indicating that the correction
procedure had a minimal effect once optimal photobleach-
ing conditions were used.
To test the effects on quantification when optimal condi-
tions were not used for the photobleach, we reexamined
TBP FRAPs under conditions 1 and 2, used to mimic
previous FRAP studies of TBP. As noted above, our imple-
mentation of the correction method cannot estimate free
fractions from these FRAPs, so instead we estimated the
size of the fast component for conditions 1 and 2 and
compared that to the size of the fast component using our
optimized condition before and after correction. Here, we
found large differences in the size of the fast component
between our optimized condition and either conditions 1
or 2 (Table 1), indicating that optimizing the photobleach
condition is critical. Again, however, we found a small
difference in the size of the corrected fast component once
the optimized photobleach condition was selected.
Our principal finding is that the reversible fraction arising in
a GFP photobleach is highly variable, ranging up to ~60%
under typical conditions used in a FRAP experiment. This
has potentially serious consequences, since most FRAP
studies with GFP presume that the reversible fraction is
negligible. We found that the reversible fraction grows
with weaker photobleaches, contributing substantially to
the first seconds of the FRAP recovery. In many cases, the
first few seconds of the recovery must be recorded to capture
the true fast component of the protein under study. Conse-
quently, some FRAP data may inadvertently incorporate
significant reversible fractions that can lead to overestima-
tion of mobility.
as large as 60% even though H2B is known to be tightly
bound to chromatin. Therefore we developed a correction
procedure for this photoswitching that accounted for the
behavior of the reversible molecules during a FRAP. By
TABLE 1Estimated TBP fast components
Condition 1 Condition 2
Fast component (%) 5 SD 49.1 5 8.4 14.6 5 9.1 30.6 5 2.4 26.4 5 2.6
Fast components of TBP were estimated from FRAP curves of condition 1 (our replication of de Graaf et al. (15)), condition 2 (our replication of Chen et al.
(20)), and our optimal condition before and after the photoswitching correction.
Biophysical Journal 102(7) 1656–1665
Minimizing Photoswitching in FRAP 1663
applying this procedure to H2B-GFP FRAPs performed
under different photobleaching conditions, we obtained
a consistent fast component of 0.8 5 0.8%. This suggests
that our correction procedure is effective over a large range
of reversible fractions, but we recommend first optimizing
the photobleach conditions to minimize the reversible effect
rather than relying exclusively on the laborious and time
consuming correction procedure.
One potential concern with this H2B calibration proce-
dure is that the reversible fraction of GFP likely depends
on pH (9), which might vary from one cell type to the
next. Nevertheless, we found very similar reversible behav-
iors for H2B-GFP in HeLa and mouse adenocarcinoma cells
(F. Mueller and T. Morisaki, unpublished observations),
suggesting that reversible fractions of GFP do not vary
widely at least in common tissue culture cells. If there is
uncertainty about whether the reversible fraction may
change significantly in another cell type, then the correction
procedure that we have devised here can be applied.
Our data suggest that the H2B test to minimize reversible
behavior will suffice for the vast majority of FRAP experi-
ments. We found that under conditions suitable for FRAP
of TBP, we could minimize the reversible fraction to ~5%.
Then our correction procedure produced only small changes
from the TBP FRAP.
Another option for the future to reduce the impact of
reversible behaviors may be the identification or creation of
other fluorescent proteins more suitable for FRAP. Although
none of the fluorescent proteins that we tested were optimal,
other fluorescent proteins might be identified or engineered
that would have better reversible behaviors, namely very
small reversible fractions that recover very quickly. An
alternate possibility might be to use organic fluorophores,
which may be less subject to reversible behavior than the
fluorescent proteins. Such fluorophores can be bound by
certain fusion proteins, for example, the SNAP tag (21).
At the moment, given the prevalence of GFP FRAPs, the
H2B live-cell calibration procedure should be a useful assay
to choose photobleaching conditions that minimize revers-
ible behavior. We used the H2B test to investigate the very
different GFP-TBP FRAP curves reported in two published
studies (15,20). We were able to closely match these
discrepant published FRAP curves using photobleach and
imaging conditions based on the published experimental
protocols. We showed that the difference in the resultant
TBP FRAP curves in our experiments was due partly to
the different time intervals between the photobleach and
first image of the bleached region and partly to different
amounts of photoswitching.
These effects are probably responsible for at least some of
the difference reported in the two published studies, but
other factors, such as the expression levels of TBP and the
details of the TBP-GFP constructs, could also contribute
to differences. For example, it appears that Chen et al.
(20) and de Graaf et al. used N-terminal GFP fusions while
we used a C-terminal fusion. The expression level of TBP is
certainly a key variable as both we and de Graaf et al. (15)
found that overexpression of TBP could reduce its fast
component. One piece of evidence that favors some role
for photoswitching in the published difference is that
de Graaf et al. (15), who reported the larger TBP fast
component also performed FRAP of H2B obtaining a 20%
fast component. This might reflect a significant reversible
fraction in their experiments, since our correction procedure
suggests that the H2B fast component is ~1%. We should
point out however that de Graaf et al. (15) fit their TBP
data with a diffusion and binding model that included
a procedure to account for photoswitching (A. Houtsmuller,
Josephine Nefkens Institute, personal communication,
2011), so they did not ignore this effect in their published
quantitative estimates for the binding rates of TBP.
With respect to the previous TBP studies, it is important
to point out that our analysis in no way alters the conclu-
sions that have been reported (15) about the role of other
cellular factors in mobilizing TBP. By measuring altered
FRAP curves after perturbing the levels of TBP interacting
factors, BTAF1 and NC2, de Graaf et al. (15) demonstrated
that both factors regulate TBP residence times on chro-
matin, with BTAF1 reducing TBP residence times and
NC2 increasing TBP residence times. The conclusions
from these and other comparative studies are in general
not affected by photoswitching, since as long as the compar-
isons are done under similar conditions, the reversible frac-
tion will be relatively constant, and so differences in the
FRAP curves reflect the underlying biological mechanisms.
comparing FRAP curves reported by different laboratories,
as we suggest here, or whenever any form of quantitative
information is extracted from an early phase of a FRAP
curve that is not optimized to minimal reversible behavior.
In these circumstances, it is vital to perform the photo-
switching test as described above for H2B to minimize the
In conclusion, we have shown that photoswitching can be
a serious problem in GFP FRAP, but it should now be easy
to identify, minimize, and, if necessary, correct using the
procedures we describe.
Five sections, references, and tables are available at http://www.biophysj.
We thank Tom Misteli for plasmids and Claudio Fenizia for cDNA. We are
grateful to Carolyn Smith for her comments on the manuscript. We thank
Tim Stasevich for suggesting the intuitive approach used to modify the
FRAP equations in the presence of photoswitching.
This research was supported in part by the intramural program of the
National Institutes of Health, National Cancer Institute, Center for Cancer
Biophysical Journal 102(7) 1656–1665
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