COL 10(7), 071701(2012) CHINESE OPTICS LETTERS July 10, 2012
Optimization of FRET algorithms for sensitized-emission
FRET detection
Sheng Wang (
ǑǑǑ
), Qiong Wu (
ÇÇÇ
), and Zichun Hua (
uuu
fff
SSS
)
∗
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences and
School of Stomatology, Af f iliated Stomatological Hospital, Nanjing University, Nanjing 210093, China
∗
Corresponding author: zchua@nju.edu.cn
Received October 26, 2011; accepted February 12, 2012; posted online April 12, 2012
Sensitized-emission fluorescence resonance energy transfer (FRET) detection method based on three-
channel fluorescence microscopy is widely used. Several FRET algorithms, such as N
FRET
, FR ET
N
, FR ,
FRET
R
, and F
C
/Df, are developed recently to quantitatively gauge and compare FR ET signals between
different experimental groups. However, the algorithms are difficult to choose and interpret. In this letter,
we optimize the suitable yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) concentration
ratio range for the above FRET algorithms. We also test the effect of YFP-to-CFP concentration ratio
on the calculated energy transfer efficiency E and use t he optimized FRET algorithms in the analysis of
fas-associated protein with death domain (FADD) self-association directly in living cells.
OCIS codes: 170.2655, 170.2520, 170.1530.
doi: 10.3788/COL201210.071701.
Fluorescence resonance energy transfer (FRET) is a
technique used to measure the interaction between two
molecules labeled with two different fluorophores by the
transfer of energy from the excited donor to the acceptor.
FRET involves the transfer of energy from a fluorescence
donor in its excited state to another excitable acceptor.
In biological application, this technique can be used to
gauge protein–protein interaction in living cells
[1]
. Cyan
fluorescence protein (CFP) and yellow fluorescence pro-
tein (YFP) are a commonly used fluores c e nce donor a nd
acceptor pair for tagging interested protein for live cell
FRET imaging
[2]
. When FRET occurs, emission of the
donor is decr e ased, whereas that of the acceptor is in-
creased (sensitized emission). Many methods have been
applied recently in measuring FRET in vivo, such as sen-
sitized emission measurement
[3]
, acceptor photobleach-
ing measurement
[4]
, spectral FRET measurement
[5]
, and
fluorescence lifetime imaging microscopy (FLIM)-FRET
measurement
[6]
. Sensitized emission FRET measurement
based on three- channel fluor escence microscopy, with its
easy conduction and high spatiotemporal resolution for
live cell FRET imaging, is commonly a nd widely us e d.
Spectr al cross-talk and spectral bleed-through, and vari-
able CFP- to-YFP expre ssion ratio may complicate the
detection of FRE T signals
[7]
. Therefore, many algo-
rithms have been developed recently for the correction
and determination of the FRET signals, such as N
FRET
,
FRET
N
, FR, FRET
R
, and F
C
/Df
[8−12]
. In this letter,
we characterize and optimize the YFP-to-CFP concen-
tration r atio for each o f the algorithms mentioned above,
and then test the effect of YFP-to-CFP concentration
ratio on the calculated energy transfer efficiency E. Fi-
nally, we analyze the fas-associa ted death domain pro-
tein (FADD) self-associatio n in living cells using the op-
timized FRET algorithms.
The expression vectors for CFP- or YFP-tagged
FADDs were constructed by inserting full length FADD
in-frame with CFP-C1 and YFP-C1 (Clontech) vectors.
The coding o f vectors pECFP- YFP for the CFP-YFP fu-
sion protein was generated by inserting YFP cDNA into
CFP-C1 vectors. All the constructs were s e quenced to
ensure correct reading frame, orientation, and sequences.
Spectr oscopic measurements using a fluoresce nce spec-
trophotometer (F-7 000, Hitachi) revealed no spectral
change for the CFP and YFP fluorescence in the tagged
protein.
Hela cell line was grown in Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum and antibi-
otics in a 5% CO
2
incubator. Exponentially growing cells
were dispersed with trypsin, seeded at 2×10
5
cells per 35
mm glass bottom dish in 1.5 ml of culture medium. The
transfection of CFP and YFP fusion protein constructs
was carried out using the calcium phosphate precipita-
tion method.
Hela cells were plated onto 0.17-mm-thick bottom glas s
dishes a nd were transient transfected using the ca lcium
phosphate precipitation method 24 h later. The cell
were washed twice with PBS (PH 7.4), then covered
with 1 ml fresh medium. Images were taken with an
inverted microscope (IX81, Olympus) equipped with a
60×NA=1.45 oil immersion objective lenses and cooled
charge-coupled devices. Excitation light was delivered
by a n X-cite light source. For imaging, Image-pro Plus
software version 6.0 (Media Cybernetics) was used. In
most experiments, the excitation intensity was attenu-
ated down to 25% of the maximum power of the light
source. Images were acquired using 1×1 binning mode
and 400 integration time. For quantitative FRET mea-
surements, the methods of sensitized FRET have been
in detail described in Refs. [13–15]. Images were ac-
quired sequentially through YFP, FRET, and CFP filter
channels. Here, the filter sets used were YFP (S500/20
nm; Q5 15lp; S 535/30 nm, Chroma); CFP (S436/20
nm; Q4 55lp; S480/40 nm, Chroma); FRET(S43 6/20 nm;
Q4 55lp; S535/30 nm, Chroma). The background images
were removed from the raw images before carrying out
FRET calculation.
Many algorithms have been developed recently based
on three-channel FRET micr oscopy to gauge FRET
signals
[16]
. In this letter, we used the two-letter sym-
1671-7694/2012/071701(4) 071701-1
c
2012 Chinese Optics Letters
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