CORRESPONDENCE
We apply the system to directed evolution,
FISH-quant: automatic counting of
transcripts
in 3D FISH images
Ultrahigh-throughput
them at rates of thousands per second.
Counts
No. Alexa 488
Colocalization (%)
Normalized frequency
Normalized frequency
To the Editor: Transcription is inherently stochastic even in
clonal cell populations1. Studies at the single-cell, single-molecule level enable a quantitative understanding of the underlying regulatory mechanisms 2,3. A widely used technique is single-molecule RNA fluorescence in situ hybridization (FISH),
in which fluorescent probes target the
1-million-fold reduction in cost.
mRNA of interest, and individual mol0.4
Mature mRNA detection
Mature mRNA
ecules appear as bright, diffraction-lim- a
per cell (n = 150)
3
Data (xy)
Fit (xy)
explosive
growth
experimental
itedThe
spots (Fig.
1a) . Recent
250
0.2
progress
has made
FISH easy toof
use4, but
150
in our
knowledge
0.5 μm
a dedicated image analysis tool is cur50
0
cakes,
cookies,
and
0
100
200
rently lacking. Available methods allow
No. of mature mRNA
counting
of
isolated
mature
mRNAs
but
candies is driving everNascent mRNA quantification
Nascent mRNA
0.3
cannot reliably quantify the dense mRNA
per cell (n = 150)
increasing
fundamental
PSF superposition
aggregates
at transcription
sites in three
0.2
Data (xy)
47 transcripts
dimensions,
particularly
those
of
highly
0.1
2,500
understanding of the
transcribed genes. We developed FISH1,500
0
gastronomy,
enabling
0
50
100
0.5 μm
quant to close this gap (Supplementary
5 μm
500
No. of nascent mRNA
Note
q 1).
ualitatively new
d 100 Cy3 Alexa 488
FISH-quant first detects and then b Both colors Only Alexa 488 Only Cy3 c
studies
complex
localizes
matureof
mRNA
in three dimen350
80
sions
by fitting
Gaussians
fluorescent
food
mixtures
andtotheir
60
5
5 μm
then undergoes
spots ; each mRNA
evolution1.
250
40
a quality test based on the measured
point-spread function (PSF) (Fig. 1a Figure
and
20
1.
Visualization
of
the
microfluidics
system
for
the directed
This knowledge also
150
Supplementary Note 2). This provides
Alexa (A). Section view (B).
evolution of candies
0
150
250
350
Cy3
1
3
5
7
9 11
488
the drives
three-dimensional
modern (3D) position of
No. is
Cy3
Imagewebsite.
Note: Supplementary information
available on the DeNature Methods
mRNAs inside cells and the distribution
biotechnologies,
such
as
molecular
f
Simulated FISH-quant:
FISH-quant:
of mRNA counts across the cell popula- e
AUTHOR
CONTRIBUTIONS
Max. int.
Integrated
intensity
Integrated intensity: 23 ± 16
Amplitude: 11 ± 7
Amplitude
PSF superposition
and food
chemistry1002, which
MB produced the device,
run the experiments,
analyzed the
tiongastronomy
(Fig. 1a). We validated
this method
Max. int.: 14 ± 7
PSF superposition: 26 ± 18
100
TS radius
200out system design and
TS radius
data
and wrote the paper. RD carried
in simulations
and
experimentally
by
300
nm
have enormous potential to address
urgent
300
0
nm
80
80
complained about the candies’ size.
dual-color FISH against mRNA of RPB1,
60
60
200
problems,
developing
potent
new
which
encodes theincluding
largest subunit
of RNA
100
40
40 ACKNOWLEDGMENTS
polymerase
excellent
100
candiesII, and
andwe obtained
providing
environmentally
This
work
was
supported
by MB personal funding. We thank
20
20
agreement in the number of estimated
and A. Sautel-Caillé
0
0 A. Griffiths, E. Brient-Litzler, A. Trouchet
0
friendly
foods.
0
spots
and a high
degree of colocalization
0
50
100
0
50
100
0 10 20 30 40 50 60 70 80
0 10 suggestions.
20 30 40
for
useful
discussions
and
Simulated no. of nascent mRNA
Estimated no. of nascent mRNA
(Fig.
1b–d,
Supplementary
Note
3
and
Here, for the first time, we present a general
Supplementary Methods).
COMPETING FINANCIAL INTERESTS
Figure 1 | Counting mature and nascent mRNA in FISH-quant. (a) FISH image of Hygro-MS2-x96ultrahigh-throughput
screening
The authors declare no competing financial interests.
Counting
nascent mRNAs is more
chal- bGH platform
reporter (Supplementary Note 6). Shown is a cell (green outline) with detected mRNA (red
lenging
because
their high localmicrofluidics
density
circles) andthat
transcription site (TS; blue box). Red box indicates individual mRNA (“Data”) fit with a
using
drop-based
Matteo
Raphael
Doineau
prevents the resolution of individual mol3D Gaussian (“Fit”), yielding
localizationBellucci
(blue dot) with&
subpixel
accuracy; blue
box indicates the
o v FISH-quant
e r c o m e soffers
t h etwo
s esolutions:
l i m i t a tTSi owith
n stranscripts
a n d (plus signs)
quantified
using
superposition.
Histograms
show distributions
ecules.
Ecole
Superieure
dePSF
Physique
et de Chimie
Industrielles,
of nascent
andof
mature mRNA
per cell, obtained
by batch
processing 10
of 150
(b) Validation
ParisTech,
Laboratoire
de Biochimie,
Ruecells.
Vauquelin,
Paris, of
(i) comparison
of the integrated
revolutionizes
both theintensity
scale and
speed
dual-color
FISH against RPB1. Detection with 15 Alexa 488 probes (left)
France
of the transcription site to that of mature mature mRNA detection by 75231,
and 15
Cy3 probes
Spot [email protected]
counts from two colors in 11 analyzed images. (d) Colocalization
candies’
screening (Fig.
1). We
use
flat (right). (c)
e-mail:
mRNA
and (ii) reconstruction
of the tranin each image. (e) Validation of TS quantification in simulations: resolution-limited TS (left) and
scription
site
signal
by
iterative
superposicandies dispersed in dark chocolate
as TS (right).
1. Artusi,
P. ,maximum
Murtha Baca
& Luigi
Science
in the Kitchen
spatially extended
Max. int.,
intensity.
(f) TSBallerini
quantification
of Hygro-MS2x96tion of weighted PSFs5 (Supplementary bGH with simple methods (left)
and the
of Eating
Well. Values
Lorenzolisted
Da Ponte
Ed. (2003).
and Art
FISH-quant
(right).
are mean
± s.d. of nascent
picoliter-volume
reaction
vessels
and
screen
2. McGee H. On Food and Cooking: The Science and Lore of the
Note 4). Whereas simple methods such mRNA counts.
Estimated no. of nascent mRNA
npg
© 2013
2013
De Nature America, Inc. All rights reserved.
screening in drop-based
microfluidics for directed
evolution of candies
as comparing maximum intensities 6 ignore the 3D extent of
theidentifying
transcriptionnew
site, variants
FISH-quant
for it.
of explicitly
candies accounts
exhibiting
Also,
method
(ii)
uses
the
experimentally
measured
PSF
tastes more than 10 times sweeter than theirand
restricts the intensity of the weighted PSFs to the range previparent,
which
is already
a very
ously
determined
for mature
transcripts,
therebysugared
taking into
consideration
aberration
effects
and
variable
labeling
candy. Compared to state-of-the-art efficiency.
food
Note that FISH-quant estimates an equivalent number of fully
screening
systems,
wepositioning
perform of
the
with on
elongated
transcripts;
different
theassay
FISH probes
theamRNA
affects the results
and canin
be used
to inferand
important
1,000-fold
increase
speed
a
Kitchen. Scribner; Rev Upd edition (2004).
277
DE NATURE METHODS | VOL.10 NO.4 | APRIL 2013 |111