-- Supplemental material
Confined Detection Volume of Fluorescence
Correlation Spectroscopy by Bare Fiber Probes
Guowei Lu,1,2* Franck H. Lei,1 Jean-François Angiboust, 1 and Michel Manfait1
1
Unité MéDIAN, UFR Pharmacie, Université de Reims Champagne-Ardenne, UMR
CNRS 6237, 51 rue Cognacq-Jay, 51096 Reims cedex, France
2
State key Laboratory for Mesoscopic Physics, Department of Physics, Peking
University, Beijing 100871, China
*
Corresponding author: guowei.lu@univ-reims.fr
1. Molecular binding on surface
Recently, the influence of molecule adsorption onto the coverslip surface on FCS
measurements has been studied in detail by C. Boutin et al. (Boutin et al. 2007). The
surface hydrophobicity influences on FCS measurements strongly when the sampling
zone locates within a few microns above the coverslip solution interface. This
influence increases with hydrophobicity degree. Usually in the conventional confocal
FCS, this influence can be avoided by adjusting the focal plane of objective lens far
away from the coverslip solution interface. Since the working distance of objective
lens is at least 200 μm, being large enough to avoid the influence of the dye
adsorption onto the surface. However, in fiber tip based near-field illumination FCS
measurements, the sampling zone is very close to the fiber tip within several microns,
1
hence, the dye absorption is one of the major problems influencing FCS
measurements. In order to avoid surface adsorption, special sample or surface
treatments are often necessary. For example, the surfactant Nonidet P-40 was added in
the experiments of Ref. (Foquet et al. 2004) to suppress dye adsorption to the
capillary wall, where microfluidic channels were applied to confine the focal volume.
A blocking reagent (N101, NOF, Tokyo, Japan) was used in the TIR-FCS in vitro
experiments (Ohsugi et al. 2006) to prevent nonspecific adsorption of fluorescent
molecules. And oxygen plasma technique with more efficient surface treatment was
reported in the study of TIR-FCS by Ruckstuhl et al. and Hassler et al. respectively
(Ruckstuhl and Seeger 2004; Hassler et al. 2005).
Fig. S1. Intensity spatial trace recorded when objective focal moves across the
interface and return: a) R6G water solution on coverslip treated only by ultrasonic, b)
R6G Tris buffer solution on coverslip treated only by ultrasonic, c) R6G Tris buffer
solution on coverslip treated by oxygen plasma.
In our previous study (Lu et al. 2008), the near-field FCS measurements were
performed with Rhodamine 6G (R6G) in Tris buffer solution. Although FCS
2
auto-correlation signal can be observed by adding buffer solution to the R6G sample,
it was believed that the buffer solution cannot avoid completely dye adsorption onto
the surface. The surface dye binding effect made it impossible to observe directly
shorter diffusion time because of the sub-diffraction limit illumination as expected.
Here, the oxygen plasma technique is used to treat the surface both of the coverslip
and the fiber tip to obtain a super-hydrophilic surface. In general, the intensity time
trace on the coverslip treated with oxygen plasma is more homogenous and no
obvious intensity burst like as that cleaned only by sonication. Similar experimental
results of time tracks of the fluorescence intensity were presented already (Ruckstuhl
et al. 2004). Our results show that dye binding onto the surface can also be observed
simply by adjusting the objective lens focal volume to pass through the solution glass
interface, and recording fluorescence signal intensity at different positions in
real-time.
As shown in Fig. S1 curve (a) shows the fluorescence intensity trace of 10 nM R6G
water solution on the coverslip treated only by ultrasonic. The spatial fluorescence
intensity was recorded when the objective focal plane was moving across the solution
glass interface (which is indicated by the black dotted line) with a constant velocity.
The fluorescence intensity increased greatly at the glass solution interface, which
means a lot of dye molecules adsorbed onto the coverslip surface. This is the major
reason why near-field FCS measurements failed in R6G water solution. Because a
large number of molecules adsorbed onto the surface of fiber tip leading a strong
noises, which overwhelmed the signal fluctuation in sampling volume and made it
3
impossible to observe auto-correlation curves easily. When R6G molecules were
diluted in Tris buffer solution, as shown by Fig.S1 curve (b) presents less intensity
burst at the interface than the curve (a), because the surfactant Tween and salt in the
solution can prevent dye binding mostly. Thus, the near-field FCS auto-correlation
signals can be observed in this situation (Lu et al. 2008). However, the dye binding
effect still exists and results in high fluorescence intensity and long diffusion time of
FCS measurements at the solution glass interface. If the coverslip was treated with
oxygen plasma technique for 5 minutes, the intensity burst at the solution glass
interface disappeared completely as shown by the green curve (c) in FigS1.
Fig. S2. Typical original auto-correlation data excited by the objective lens for R6G
molecules (10 nM) in Tris buffer on a coverslip treated only by ultrasonic. The curves
were measured at the positions indicated by two black spots in Fig. S1 curve (b), far
away from the coverslip surface and near the surface separately. The insert shows the
raw total fluorescence signal for these.
4
Here, FCS measurements were performed near the glass solution interface by
positioning the objective focal volume completely (far away from the coverslip
surface) or partly in the sample solution (near the coverslip surface). The precise
position of the objective lens focal plane, although difficult to determine, can be
roughly estimated by the spot size of light focus and the pattern of light interference
rings at the interface. The focal plane position near the interface can be further
adjusted according to the fluorescence intensity. For the coverslip treated only by
ultrasonic, the fluorescence intensity is almost the same when the focal volume is
completely within the solution, but it will increase greatly if the focal volume is
adjusted close to the coverslip surface because of dye molecules adsorption onto the
surface. As shown in FigS2, the diffusion time near the coverslip surface becomes
longer than that when the sampling volume is far away from the surface, and presents
larger signal intensity. A super-hydrophilic surface treated with oxygen plasma
technique can avoid the influence of surface adsorption on FCS measurements.
For the coverslip treated by oxygen plasma technique, typical FCS results near the
surface are shown in Fig. S3 for three points where the intensity are i) ~55%, ii)
~70%, and iii) 100% respectively, compared to that of the focal volume completely
within the solution. After fitting the FCS curves with Eq. 2, the diffusion time of FCS
measurements is about 50±5µs, almost the same for free solution and the solution
glass interface. Because of super-hydrophilic surface, the diffusion time becomes
some orders of magnitude smaller compared to FCS measurements on untreated
5
coverslip. Moreover, it is easy to understand that the value of G(0) at the interface
increases
Fig. S3. Typical original auto-correlation data (rough curves) excited by the objective
lens for R6G molecules (10 nM) in Tris buffer on an oxygen plasma treated coverslip:
i) the objective focal plane posited at solution-glass interface with ~55% fluorescence
intensity, ii) the objective lens focal volume is partly posited within the solution with
~70% fluorescence intensity, iii) the objective lens focal volume is completely posited
within the solution (100%). The insert shows the raw total fluorescence signal for i),
ii) and iii). And the solid smooth lines are fitted curves according to Eq.2.
because the objective focal volume is partly out of the sample solution due to the
coverslip mechanic isolation. This detection volume reduction is a mechanical
confinement of the detection volume similar to the confinement by waveguide
structures (Foquet et al. 2004). Anyway, the oxygen plasma treatments can almost
completely eliminate the surface effect influence on the FCS measurements. Hence,
6
it is reasonable to suppose that the surface effect influence would also disappear
for the fiber tip surface after the oxygen plasma treatments because the coverslip
and the fiber tip are both made of silica material.
2. FCS measurements by metal coated tapered fiber tips
Undoubtedly, a metal coated tapered fiber tip with sub-wavelength aperture can
provide smaller excitation volume than a bare fiber tip. Here, aluminum coated fiber
tips prepared by common heating and pulling method (Lovalite, France) were applied
to perform FCS measurements. At first, a fiber tip with 300 nm diameter aperture was
used to ensure enough light intensity emitted from such the probe. The probe was
glued onto the piezo-bimorph, and positioned about 100 µm above the coverslip
surface for FCS measurements. Typical FCS curves illuminated by the metal coated
fiber tip are shown in Fig. S4, in which curve (b) was treated by subtracting
afterpulsing contribution. In order to obtain reliable FCS curves, we averaged the FCS
data collected near the tip apex over 5 times. For comparison of diffusion time, the
FCS curve (a) excited by the objective lens (the same as Fig. S3 iii curves) is also
presented after normalizing its G(0) value to the curve (b). The results of Fig S4
illustrates that the FCS diffusion time near the metal coated fiber tip is obviously
shorter than that of the conventional confocal FCS, which means a smaller lateral
extension of the detection volume. The black solid smooth lines in Fig. S4 are fitted
curves obtained from Eq. 2. However, the FCS curves (b) cannot be fitted well with
the standard models containing only one diffusion component because of the large
7
divergence angle of optical field distribution from the tip aperture. Thus, a model
taking into account two diffusion components was utilized to provide a better fitting
line for curves (b) as indicated by the green solid line in Fig. S4 (Boutin et al. 2007).
Here, showing exact diffusion time performed by the meat coated probe is not
physical sense because the light excitation profile of the probe is obviously different
from the standard Gaussian beam excitation shape. The proper simulation for such
case has been shown already for the probe tip close to a surface in Ref. (Vobornik et
al. 2008). Even so, it is reasonable to draw a conclusion roughly (i.e. excitation
volume by such probe is sub-wavelength) from the approximating fitting by Eq.2.
Fig. S4. Typical original FCS auto-correlation data (rough curve b) excited by an
aluminum coated fiber tip with 300nm aperture diameter for R6G molecules (~ 10
nM) in Tris buffer, and collected by the objective lens near the tip apex. The curve a
excited by objective lens is also presented for comparison. Black solid smooth lines
are fitted curves obtained from Eq. 2. The green solid line is the fitting line using a
model with two diffusion time components.
8
However, the G(0) value of curves (b) in Fig. S4 near the tip apex, another important
indicator of the detection volume, is smaller than that taken with the objective lens,
not larger than the conventional confocal FCS as expected. This could attribute to
unexpected noise from the tip apex (e.g. broad emitting background of nanostructured
metal, containment of metal surface, and optical fiber fluorescent background). These
background signals would influence greatly the G(0) value of FCS measurements.
The signal collection of the SNON-FCS system is very dependent on the relative
position between the objective focus and the probe tip apex, this brings an uncertain
factor when the background signal is estimated. Moreover, various probe properties
and their surface states also introduce uncertain background. All of above factors
makes it nontrivial to calibrate background noises for an individual probe.
Fig. S5. Typical original FCS auto-correlation data excited by an aluminum coated
chemical etched fiber tip with 300nm aperture diameter for R6G molecules (~ 10 nM)
in Tris buffer: curve (a) collected near the tip apex and curve (b) collected at several
µm away from the tip apex.
9
Because of the above, the FCS signal with the metal coated fiber tip shows poorer
signal to noise ratio than the conventional confocal FCS, which could mainly be
attributed to low excitation light power emitting from the fiber tip aperture. Usually,
the light transmittance efficiency of metal coated fiber tips is very low (about
0.1-1%), and the fragility of the metal coated layer limits the light input power. As we
know, the metal coated chemically etched fiber tip usually possesses higher light
transmittance efficiency than the pulled fiber tip. More recently, a new type fiber tip
with high transmittance was reported (Chevalier et al. 2006), the efficiency reached
the order of 10% for a tip with 450 nm aperture. A dielectric material MgF2 were
coated onto chemically etched tip prior to metal coating. The chemically etched
optical tips were made from S460 single mode fiber with pure silicon core. Typical
auto-correlation curves excited by such fiber tip (Attocube, Germany) for the R6G
solution in Tris buffer are shown in Fig.S5 in which curves (a) and (b) were collected
very close to and several microns away from the tip apex, respectively. The
performance of FCS measurements improves evidently thanks to the high throughput
efficiency of such type fiber tip. Whereas the surface of dielectric material MgF2 layer
can not form a super-hydrophilic surface (like as dielectric material SiO2) after
common oxygen plasma treatments, which induces interaction between dye molecules
and solid surface (mainly dye adsorption and release process on the surface). The
existence of such surface effect as discussed above leads to a long diffusion time of
FCS measurements near the tip apex surface, and the diffusion time decreases when
the collection position is adjusted away from the tip apex. Similar results were
10
observed already using other type of tips without oxygen plasma treatments (bare
chemical etched tip or metal coated tip). This phenomenon has been reported in detail
recently by C. Boutin et al (Boutin et al. 2007). If the dielectric material MgF2 layer
of the fiber probe was replaced by SiO2 layer or focused ion beam technique was used
to mill fiber probe, those could resolve the problem of surface effect. Moreover, the
threshold of input light power is much lower in aqueous solutions than in air for the
metal coated fiber probes, which brings on a break of metal coated layer on fiber tip
apex easily in aqueous. Usually, the metal coated layer was found broken off slightly
even if the input power was kept within 100 µW. The near-field FCS measurements
under the control of SNOM system were not performed for both kinds of metal-coated
fiber tip because of these disadvantages. Much effort is still needed for better
metal-coated optical fiber probe with subwavelength aperture, which is in developing.
At the time this work was prepared, the authors just knew recently a same works was
being done concurrently by Vobornik (Vobornik et al. 2008). They successfully
utilized a chemically etched fiber tip and focused ion beam milled probe to perform
SNOM-FCS measurements with good signal to noise ratio. Whereas the detection
volume derived from the G(0) value was not yet discussed because of unexpected
noise probably.
3. FCS auto-correlation model equation
FCS measurement monitors the total fluorescent count rate I t  , which is proportional
to the number of molecules N t  present in the detection volume, as a function of
11
time t . The G( ) shape of the temporal autocorrelation curve of the time-dependent
signal I t  encodes diffusion (Brownian motion) of fluorescent particles through the
volume and emission characteristics like triplet state occupation, or changes of the
molecular conformation influencing the emission characteristics. In order to
determine the shape and to estimate the size of the detection volume, the molecule
detection efficiency (MDE) function needs to be calculated. The MDE is product of
the excitation field intensity distribution I  r  and the collection efficiency function
(CEF).
MDE r   CEF r  I r 
(1)
The CEF is defined as the fraction of the light emitted by a point source that passes
through the pinhole. In our experiments, the fluorescent signal illuminated by the fiber
tip or the objective lens is collected all through the objective lens and the pinhole. The
CEF of the fiber tip illumination is almost the same as that of the objective lens except
for some alteration considering variation of fluorescent dye behaviors near the
surface, such as emitter anisotropy and lifetime etc. The excitation intensity profile
I  r  of fiber tip illumination changes obviously compared to that of the objective lens.
For the cleaved end of a single mode fiber, the intensity distribution is well
approximated by a TEM00 Gaussian beam with a simple analytical expression
(Marcuse 1978). But I  r  of the bare chemical etched fiber tip and tapered metal
coated fiber tip are not easy to describe by simple analytical expressions for their near
field and far field (Obermuller et al. 1995; Obermuller and Karrai 1995). There are
many research reports about numerical simulation of the field distribution near fiber
12
tip apex (Novotny et al. 1995; Chavez-Pirson et al. 1999; Antosiewicz et al. 2007).
The complete description of fiber tip illumination MDE would require to calculate
both the CEF and the field distribution and that is beyond the scope of this paper.
As an approximation, the detection volume of 3D-Gaussian beam excitation shape
is often applicable. And it is also applied effectively in nano-holes FCS analysis
(Rigneault et al. 2005; Wenger et al. 2005; Leutenegger et al. 2006). The
auto-correlation function G( ) can be modeled approximately by a standard equation
for free diffusion of single fluorescent species through the sampling volume (Eq. 2).
Where N is the average number of molecules in the sampling volume,  d the lateral
diffusion times, s the ratio of axial over lateral extension of the sampling volume of
3D-diffusion, pt the probability of molecules in the triplet state,  t the correlation
time of this triplet state population, I b the background count rate, and I the mean
count rate. Moreover, the systematic bias of afterpulsing influence on short diffusion
times is corrected by subtracting the estimated afterpulsing contribution. The
afterpulsing contribution is estimated by averaging autocorrelation for an uncorrelated
daylight source (Bismuto et al. 2001).
G( )
2
1 
I b  

 1
1   1 
N
I  
d




1


1  2
s d

13




1
2
 
pt

exp  
1  pt
 t





(2)
References
Antosiewicz TJ, Szoplik T (2007) Description of near and far field light emitted from
metal coated tapered fiber tip. Opt. Express. 15:7845
Bismuto E, Gratton E, Lamb DC (2001) Dynamics of ANS binding to tuna
apomyoglobin measured with fluorescence correlation spectroscopy. Biophys. J.
81:3510
Boutin C, Jaffiol R, Plain J, and Royer P (2007) Influence of the surface
hydrophobicity on fluorescence correlation spectroscopy measurements. Proc. of
SPIE. 6444:64440J1
Chavez-Pirson A and Chu ST (1999) Polarization effects in near field
excitation-collection probe optical microscopy of a single quantum dot. J. Microscopy.
194: 421
Chevalier N, Sonnefraud Y, Motte JF, Huant S, and Karrai K (2006) Aperture size
controlled optical fiber tips for high resolution optical microscopy. Review of
Scientific instruments.77: 063704
Foquet M, Korlach J, Zipfel WR, Webb WW, and Craighead HG (2004) Focal volume
confinement by submicrometer size fluidic channels. Anal. Chem. 76:1618
Hassler K, Leutenegger M, Rigler P, Rao R, Rigler R, Gosch M, and Lasser T (2005)
Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with low
background and high count-rate per molecule. Opt. Express 13:7415
Leutenegger M, Gosch M, Perentes A, Hoffmann P, Martin OJF, and Lasser T (2006)
Confining the Sampling Volume for Fluorescence Correlation Spectroscopy Using a
Sub-wavelength Sized Aperture. Opt. Express. 14:956
14
Lu GW, Lei FH, Angiboust JF, Roche Y, Huang LY, and Manfait M (2008) Near-field
fluorescence correlation spectroscopy by using a tapered optical fiber tip as excitation
source. Research Signpost: Advanced technologies and applications on scanning
probe microscopy
Marcuse D. (1978) Gaussian approximation of the fundamental modes of graded
index fibers. J. Opt. Soc. Am. 68:103
Novotny L, Pohl DW, and Hecht B (1995) Scanning near field optical probe with
ultrasmall spot size. Opt. Lett. 20: 970
Obermuller C, Karrai K, Kolb G, and Abstreiter G (1995) Transmitted Radiation
through a Subwavelength Size Tapered Optical Fiber Tip. Ultramicroscopy. 61:171
Obermuller C, and Karrai K (1995) Far field characterization of diffraction circular
apertures. Appl. Phys. Lett. 67:3408
Ohsugi Y, Saito K, Tamura M, and Kinjo M (2006)Lateral mobility of
membrane-binding proteins in living cells measured by total internal reflection
fluorescence correlation spectroscopy. Biophys. J. 91:3456
Ruckstuhl T and Seeger S (2004) Attoliter detection volumes by confocal
total-internal-reflection fluorescence microscopy. Opt. Lett. 29:569
Vobornik D, Banks DS, Lu ZF, Fradin C, Taylor R and Johnston LJ (2008)
Fluorescence correlation spectroscopy with sub-diffusion-limited resolution using
near-field optical probes. App. Phys. Lett. 93:163904
Rigneault H, Capoulade J, Dintinger J, Wenger J, Bonod N, Popov E, Ebbesen TW
and Lenne P-F (2005) Enhancement of Single-Molecule Fluorescence Detection in
Subwavelength Apertures. Phys. Rev. Lett. 95:117401
Wenger J, Lenne P-F, Popov E, Rigneault H, Dintinger J, and Ebbesen TW (2005)
Single molecule fluorescence in rectangular nano-apertures. Opt. Express. 13:7035
15