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Novel Fluorescent Probes for the Oxidative Milieu

where b and ttrip are the fraction and relaxation time of the triplet GðtÞ~ 1 (1z b e{ttD )gx(t)gy (t)gz (t) ð10Þ
state, respectively, N is the mean molecule number in the confocal vN 1{b
volume, s and tD are the structural parameter and diffusional w
relaxation time. The equation for the multi-component analysis
was descrbed previously [32]. The best fit numbers were obtained Since estimation of the best-fit numbers requires calculation of
with Levenberg-Marquardt algorithm using OriginPro version 8.0
(OriginLab Corp). error functions, we used FFS Data Processor ver. 2.3 (SSTC,

To estimate the time dependency of diffusion coefficient, Belarus).
autocorrelation function including the anomalous exponent a, PCH analysis. An observation volume of PCH is character-

GðtÞ~ 1 (1z b {t )( ar1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiaffi ) ð4Þ ized by the point-spread function (PSF) of the instrument. For
vN 1{b convenience, a scaled PSF, PSF , was considered, such that the
etD
volume defined for FCS (fluorescence cÐorrelation spectroscopy), is
w (1z t 1z 1 t equal to the volume of PSF , VPSF ~ PSF ðrÞdr: According to
tD s2 tD this model, the probability (p(1)(k;V0,e)) of detecting k photons from
a single fluorescent molecule in a volume V0 large enough to
was used. Autocorrelation function measurements were normal- contain the illuminated volume is,
ized and the averaged decay profiles were fitted with (4) (Figure
S1). 1 ð ÂÃ
V0 Poisson k,ePSF ðrÞ dr
Alternatively, if ER cargo motion is confined in a smaller space p(1) ðk; V0,eÞ~ ð11Þ
than the optical confocal volume, it may be appropriate to
consider a model of diffusion limited by boundaries [31]. In this V0
model, a free diffusing 3D autocorrelation model ignoring the
triplet state population, where e is the molecular brightness, which is a product of the
average number of photon counts ,k. and a ratio V0/VPSF. For n
GðtÞ~ 1 1 t qffiffiffiffiffi1ffiffiffiffiffiffiffiffiffiffiffi ð5Þ independent molecules, total PCH is given by a convolution of the
vN w 1z tD 1z t single molecule PCH. A 3D Gaussian approximation was
S2 tD originally proposed to describe the observation volume profile
for one-photon excitation. However, different from FCS, an actual
is written as a product of three autocorrelation function gi(t), brightness profile by PCH is often disturbed by several optical
factors such as photons in the out-of-focus region [51,52]. Also,
1 ð6Þ since the original assumption of PCH was that fluorescence
GðtÞ~ vNw gx(t)gy(t)gz(t) intensity emitted by a molecule is constant, there is a clear effect of
bin time for diffusing molecules [53]. Hence, we used a global
We consider rz and rxy, distances in axial and lateral directions at analysis procedure that simultaneously obtains time-dependent
which the intensity of the laser beam is dropped by e22, and di, decay of correlation function and PCH to recover relevant and
distance in i-direction at which boundary planes localize. The common parameters [28].

degree of confinement is expressed as di/ri. If diffusion is confined Information on fluorescence intensity fluctuation was acquired
in the optical axis and in x- and y-directions, gx(t) is given as. using a confocal laser scanning microscope A1R (NIKON)
equipped with a time-correlated single photon counting module
gx ðtÞ~ qffiffiffi1ffiffiffiffiffiffiffiffiffi ð7Þ (LSM Upgrade Kit, Picoquant). Expressions of fluorescent
1z t proteins were carried out in COS7 cells plated on 4-well glass
tD bottom dish (Matsunami Glass Ind., Osaka, Japan) kept in a closed
stage chamber (95% air and 5% CO2; Tokai Hit CO.,
A closed form expression of gi(t) is unknown, however, if di/ Fujinomiya, Japan). The cells were excited using a 488 nm diode
ri#8, gi(t) can be approximated as. laser (85BCD020, Mellies Griot) or a 561 nm diode laser
(85YCA010, Mellies Griot). The applied irradiance at the top of
pffiffiffi ½1zfpYffiffiffi erf ðY Þ exp½{qKffiffiðffiYffiffiffiffiÞffiffiÀffiffiYffip Á2 t Š an objective lens was 5.3 mW (488 nm laser line) or 1.1 mW
p p erf 2 pYffiffi tD Š (561 nm laser line). The light signal was collected through a water-
gyðtÞ~ {1g ð8Þ immersion objective (x60/NA1.27, Nikon) and split into two
Y 2 1z t signals by a half-beam splitter. The two signals were fed to two
tD separate single photon counting avalanche photodiodes (PDM
Seires, MPD) through a bandpass filter (BP520/35, Semrock) for
gz ðtÞ~ pffiffiffi ½1zfpZffiffiffi erf ðZÞ {1g exp½{qKffiffiffiðffiffiZffiffiffiÞffiffiÀffiffisffipffiZffiffiÁ2ttDŠŠ ð9Þ 488 nm excitation or a longpass filter (BA590, Nikon) for 561 nm
p p erf 2 pZffiffi 1z t excitation. The cross-correlation function of the two signals
s2 tD detected in the two photodiodes through the identical filter sets
Z 2 was used for accurate estimation of dynamic processes. The
molecular brightness of each fluorescent protein was determined
where Y and Z are dy/rxy and dZ/rxy, respectively, and by finding a best-fit from globally linked datasets of a cross-
K(i) = 0.689+0.34exp(20.37(i20.5)2) [31]. Taking the triplet frac- correlation function and four PCH that were generated with bin
tion into consideration, the FCS model becomes time of 0.03, 0.06, 0.12 and 0.24 ms from one measuremt by using
FFS data processor. A first-order correction for out-of-focus
emission [51] and corrections for triplet and 3D diffusion were
applied in the fitting as described [28].

PLoS ONE | www.plosone.org 13 May 2012 | Volume 7 | Issue 5 | e37551