Optical and Quantum Electronics (2005) 37:1397–1408
DOI 10.1007/s11082-005-4219-0© Springer 2006

Tomographic imaging of collagen ﬁber orientationin human tissue using depth-resolved polarimetryof second-harmonic-generation lightt a k e s h i y a s u i1,∗ , k u n i h i k o s a s a k i1 , y o s h i y u k i t o h n o2
a n d t s u t o m u a r a k i1
1 Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan
2 1st Department of Anatomy, Nara Medical University, Kashihara, Nara 634-8521, Japan(∗ author for correspondence: E-mail: t-yasui@me.es.osaka-u.ac.jp)
Received 2 February 2005; accepted 15 September 2005

Abstract. We propose a nonlinear optical probe method to image the distribution of collagen ﬁber orientation in human tissue by measuring the polarization of collagen-induced second-harmonic-generation(SHG) light (SHG polarimetry). Depth-resolved SHG polarimetry, with a depth resolution of 14 m, wasused to evaluate the cross-sectional proﬁle of collagen ﬁber orientation in Achilles tendon and dentin,
revealing a characteristic distribution of collagen orientation along the depth direction. We evaluated thetwo-dimensional (2D) lateral distribution of collagen ﬁber orientation in back reticular dermis and anklebone by polarization-resolved SHG imaging, and conﬁrmed an appreciable difference in the distribution
proﬁles of the two samples. We further extended the method to a depth-resolved measurement of thethree-dimensional (3D) distribution of collagen orientation in anklebone. The proposed system promises
to be a powerful tool for in vivo measurement of collagen ﬁber orientation in human tissue.
Key words: collagen, femtosecond laser, orientation, polarimetry, second-harmonic-generation, tomography

1. Introduction
Collagen molecules, which form three-dimensional (3D) structural aggregatesof successively different sizes (i.e., microﬁbrils, ﬁbrils, ﬁbers, and bundles),play an important role in determining the morphology and functional properties of tissues and organs as a structural protein. For example, transparency of the cornea is mainly due to polycrystalline lattice of the corneal
collagen ﬁbrils whereas inhomogeneous, tubelike structure of scleral ﬁbrils with thin hard shells results in maintaining of the high stiffness andelasticity of the sclera (Han et al. 2005). Therefore, there is a considerable need for an optical probe method that can reveal the 3D structure
and orientation of collagen ﬁbers and ﬁbrils in situ in biological studies and clinical medicine. Recent advances in ultrashort pulse lasers haveopened the door to new optical probe methods based on optical nonlinear
effects in biological tissue: e.g., two-photon ﬂuorescence (Denk et al. 1990),

1398

T. YASUI ET AL.

second-harmonic-generation (SHG) light (Fine and Hansen 1971), thirdharmonic-generation (THG) light (Yelin and Silberberg 1999), andcoherent anti-Stokes Raman scattering (CARS) light (Zumbusch et al. 1999;
Hashimoto et al. 2000). Such nonlinear optical probes based on multi-photon processes provide unique imaging modality: speciﬁc probing power, highspatial resolution, optical sectioning, non-invasion, and deep penetration.
Among these probes, the SHG method is an attractive tool for in vivo studiesof living biological tissues containing collagen molecules. When ultrashortpulse light is incident on collagen-rich tissues, light of half wavelength of
the incident light, known as collagen-induced SHG light, is often observed(Roth and Freund 1979). By using this naturally-endogenous SHG process
as a contrast mechanism, SHG imaging can clearly visualize structures ofcollagen ﬁbers and/or ﬁbrils in tissues without additional staining with ﬂuorochrome, and hence it is free from photodamage, photoxicity, or photobleaching. Furthermore, a combination of polarization methods can lead to
additional insights. The efﬁciency of SHG light is sensitive to collagen orientation when the incident light is polarized, and hence polarization measurements of SHG light are effective in probing the collagen orientation in tissues.
These SHG probing methods have been successfully applied to various tissue specimens: tendon (Roth and Freund 1979; Stoller et al. 2002), dermis(König and Reimann 2003), cornea (Stoller et al. 2002; Yeh et al. 2002; Hanet al. 2005), endometrium (Cox et al. 2003), and intervertebral disk (Stoller et al. 2002). However, few papers have been concerned with human tissue
subjects. We have previously proposed polarization-resolved measurements of
SHG light in a reﬂection optics conﬁguration (SHG polarimetry) as a method
to extract detailed information of collagen ﬁber orientation in human tissue(Yasui et al. 2004a). We extended the proposed method to two-dimensional(2D) lateral imaging (Yasui et al. 2003) and applied it to the characterizationof collagen orientation in human dermis (Yasui et al. 2004b). Since 3D structures of collagen ﬁbers and ﬁbrils perform various functions in determining
morphological and functional properties in tissues and organs, it is necessary
to further extend SHG polarimetry in the depth direction to investigate the
3D distribution of collagen ﬁber orientation in tissue.
In the present paper, we propose depth-resolved SHG polarimetry for the
tomography of collagen ﬁber orientation in human tissue. The penetration
power of the proposed method is ﬁrst investigated for human soft tissue
and hard tissue. Using the proposed method, the longitudinal distributionof collagen ﬁber orientation in human tissue is evaluated in the form ofdepth-resolved SHG radar graphs. Further, we demonstrate depth-resolved
imaging of the 2D lateral distribution of collagen orientation by extending
polarization-resolved SHG imaging in the depth direction, which enables
reconstructing of the 3D distribution of collagen ﬁber orientation in humantissue.

SHG TOMOGRAPHY OF COLLAGEN FIBER ORIENTATION IN HUMAN TISSUE

1399

2. Experimental setup
The experimental setup for depth-resolved SHG polarimetry is illustrated inFig. 1. We used an 80-MHz mode-locked Ti:Sapphire laser (Spectra Physics,
MaiTai) pumped by a 5-W frequency-doubled Nd:YVO4 laser running at
532 nm. The laser pulse had a duration of 100 fs and an average power of
800 mW at 800 nm. After passing through an optical chopper (OCP, chopping freq. = 3 kHz) and neutral density (ND) ﬁlter, ultrashort pulse lightfrom the laser is focused onto the sample with an objective lens [OL, magniﬁcation = 20, numerical aperture (NA) = 0.42, working distance = 20 mm],giving an analytical spot of 1.5 m in diameter and 10 m in depth in air.
Such a relatively low NA value prevents the OL from distorting the polarization of the focus spot of the incident laser light. Although the SHG
light induced in the sample mainly propagates in the same direction as theincident light, a portion of the SHG light is backscattered into the tissue, and then collected via the OL. After eliminating the unnecessary laser
light with a combination of two harmonic separators (HS, reﬂected wavelength = 400 nm) and a blue-pass ﬁlter (F, pass wavelength = 300–500 nm),the analytical SHG light is detected with a photomultiplier (PMT) and alock-in-ampliﬁer.
In general, SHG imaging techniques limit the probing volume to a localized focal volume, resulting in confocal-like imaging quality without using
a confocal pinhole. However, when applying it to optically thick, turbid
medium such as usual human tissue, we have to consider two effects that

Fig. 1. Experimental setup. OCP: optical chopper; ND: neutral density ﬁlter; PL: polarizer; HWP:
half-wave plate (rotatory); HS: harmonic separator; OL: objective lens; L: lens; PH: pinhole; F:
blue-pass ﬁlter; AN: analyzer; PMT: photomultiplier.

1400

T. YASUI ET AL.

may degrade the imaging characteristics: scattering and refractive index
mismatching. The imaging characteristics of two-photon microscopy ina turbid medium have been investigated comprehensively (Centonze andWhite 1998; Dunn et al. 2000; Dong et al. 2003), and it is reported that the
scattering does not signiﬁcantly affect the two-photon point-spread-function
down to a depth of 200 m (Dong et al. 2003). In the present SHG setup
with a probing depth of a few 100s m, it can be consider that the imaging
characteristics are hardly affected by the scattering in the same manner as
the two-photon microscopy although we have to consider that the SHG is acoherent nonlinear optical process. On the other hand, the refractive index
mismatching induces the spherical aberration depending on the imaging
depth, which degrades the point-spread-function (Dong et al. 2003). This
results in a blurred analytical spot when probing deep portions in a thicktissue sample. Hence, deep optical sectioning of the tissue requires a confocal conﬁguration with a detection pinhole, even if the pinhole decreases
the SHG signal level. In the present setup, to improve the spatial resolution for depth-resolved measurements, a confocal conﬁguration, comprising a pinhole (PH) and two lenses (L, f = 50 mm), is introduced in the
SHG optical path. Although a diameter of Airy disk is about 10 m at
the PH position, a PH of 50 m in diameter is selected by considering a
relationship between the achieved spatial resolution and the detected SHG
signal level. The resulting depth resolution was 14 m. Simultaneously, theconfocal conﬁguration decreases depolarization effects in the incident laser
light and analytical SHG light caused by multiple-scattering in the depthresolved measurement of a thick tissue sample.
For SHG polarimetry, a polarizer (PL), two half-wave plates (HWP, rotatory), and an analyzer (AN) are inserted in the light path. The polarization of the incident laser light and of the detected SHG light are rotatedsynchronously, keeping them parallel to each other, by the two mechanically-rotated HWP. Although this rotation procedure is equivalent to therotation of the sample around the optical axis under ﬁxed polarization
optics, it is difﬁcult to coincide the sample rotating axis and the opticalaxis within the analytical spot, and hence we chose to use a polarizationrotating setup. The distribution of the SHG light intensity is measured withrespect to the polarization angle. For the imaging measurement of the SHG
light, the sample is scanned by a 3D moving stage.

3. Sample
In this experiment, we used four kinds of human tissue samples rich in collagen. Back reticular dermis (from a cadaver) and Achilles tendon (froma cadaver) were prepared as soft tissue samples. The cadavers were treatedSHG TOMOGRAPHY OF COLLAGEN FIBER ORIENTATION IN HUMAN TISSUE

1401

by injecting a mixture of 36% ethanol, 13% glycerin, 6% phenol, and 6%
formalin through the femoral artery. After a typical dissection by medical
students in an anatomy class at Nara Medical University was carried out,
the tendon and skin were resected. The tissue samples were washed with
distilled water and then dried in air. We have conﬁrmed that these sample
preparation procedures (formalin ﬁxation and air-drying) do not affect theresults of SHG polarimetry (Yasui et al. 2004b). Two kinds of tendon specimens with different thicknesses (2 mm and 0.3 mm) were prepared by slicing
them along the axial direction. The skin was stripped off the cadaver’s backand subcutaneous tissue was removed with a scalpel to expose the reticulardermis layer. The exposed reticular dermis was cut into a 44 mm × 55 mm
square sheet of 1 mm thickness. As hard tissue samples, we prepared dentin (extracted by a dentist) and anklebone (from a cadaver). The dentin andanklebone were sliced to 1 mm thickness along the tooth axis and 2 mm
thickness across the section, respectively, with a tooth/bone cutter. We havepreviously conﬁrmed using SHG polarimetry that the Achilles tendon, theback reticular dermis, the dentin, and the anklebone possess uniaxial, randomly entangled, crossed biaxial, and parallel-to-crest orientated collagenﬁbers, respectively (Yasui et al. 2004a, b).

4. Results
4.1. penetration power of depth-resolved shg measurement
We ﬁrst evaluated the penetration power of the depth-resolved SHG measurements for an incident laser power of 25 mW. The analytical laser spot
was scanned in the depth direction from the sample surface. The sample
specimens examined were the Achilles tendon and the back reticular dermis, as human soft tissue samples, and the dentin as a human hard tissue
sample. Figure 2 shows decay curves of SHG light intensity with respect
to depth position for the three sample specimens. The depth resolution in
the present system was 14 m, determined from another experiment using a
sufﬁciently thin slice (thickness < 5 m) of a mouse tendon (not shown). At
a depth of 0 m, the analytical spot was focused on the sample surface. The
resulting maximum penetration depths were 300, 150, and 100 m for thetendon, dermis, and dentin, respectively. These penetration powers can be
improved by matching the refractive indices of the sample and the objective
lens by using an oil-immersion objective lens. An alternative method is touse a deep-penetrative femtosecond laser with a longer wavelength, suchas a mode-locked Cr:Forsterite laser with a center wavelength of 1250 nm.
Efforts to improve the penetration power by these two methods are in
progress.

1402

T. YASUI ET AL.

Fig. 2. Decay curves of SHG light intensity with respect to depth position for three different human tissues. The incident laser power and depth resolution are 25 mW and 14 m, respectively. A depth of 0 m
indicates the analytical spot focused onto the sample surface.

4.2. depth-resolved shg polarimetry
We next performed depth-resolved SHG polarimetry of a tissue sample
with a different collagen orientation along the depth direction, to evaluate the basic performance of this method. We stacked two sliced Achilles
tendons (thickness = 0.3 mm) with their collagen orientations perpendicular to each other, as shown in Fig. 3(a). Figure 3(b) shows the resulting
SHG radar graph with respect to polarization angle, scanning the analytical spot along the depth direction. Here we refer to 0 (or 180) and 90 (or270) degrees as horizontal and vertical polarization, respectively. The polarization angle of the major axis and the proﬁle of the SHG radar graphreveal, respectively, the direction of the absolute orientation and the degreeof organization of the collagen ﬁbers in the area of the light spot. The grayarrows in each of the plots of Fig. 3(b) indicate the expected direction ofcollagen orientation. In general, collagen SHG light is output strongly withthe same polarization as the incident light when the polarization directionof the incident light is parallel to the longitudinal direction of the collagenﬁber (Yasui et al. 2004a). However, the SHG light almost disappears if thelaser polarization is perpendicular to the collagen orientation. Such sharp
polarization sensitivity is due to differences in the χ (2) tensor components.
Hence, the SHG radar graph at 0-m depth (the surface of the ﬁrst tendon
layer) shows that the collagen ﬁber was well aligned along the horizontal
direction. The proﬁle was rotated about 90 degrees at 350-m depth, indicating vertical orientation of the collagen ﬁber in the second layer. Furthermore, a mixed proﬁle of horizontal and vertical orientation was observed

SHG TOMOGRAPHY OF COLLAGEN FIBER ORIENTATION IN HUMAN TISSUE

1403

Fig. 3. Basic characteristics of depth-resolved SHG polarimetry. (a) Sample. Two sliced Achilles tendons
stacked so that their collagen orientations are perpendicular to each other. (b) Depth-resolved SHG
radar graph. The 0, 300, and 350-m depths indicate that the analytical spot is located on the surfaceof the ﬁrst tendon layer, the boundary between the ﬁrst and the second tendon layers, and the second
tendon layer, respectively.

at a depth of 300 m, which locates the boundary between the ﬁrst andsecond tendon layers. These results clearly indicate that the proposed system is effective for the tomographic measurement of collagen orientation intissue. However, there is an outstanding issue to be addressed in the present system. The proﬁle of the SHG radar graph at 0-m depth exhibits anode at the origin, whereas no node is observed at 350-m depth, although
the ﬁgure-eight proﬁle is characteristic for SHG radar graphs of Achilles
tendon with uniaxial collagen orientation (Yasui et al. 2004a). This result
might imply some depolarization of the incident laser light and the analytical SHG light in the tissue when probing a deep portion of a sample.
Such a depolarization effect can be counteracted by using a sharper confocal conﬁguration at the expense of signal efﬁciency.
We applied depth-resolved SHG polarimetry to the human tissue samples.
Figure 4(a) and (b) show the resulting depth-resolved SHG radar graphsof the Achilles tendon (thickness = 2 mm) and the dentin. In these graphs,
the horizontal and vertical axes indicate the depth position and polarization
angle, respectively. Five SHG radar graphs are also illustrated from the vertical line proﬁles at ﬁve depth positions in the depth-resolved SHG radargraphs. The SHG radar graph in Fig. 4(a) of the Achilles tendon indicatesuniaxial collagen orientation in the vertical direction at each depth position.
In a previous study, we reported that human Achilles tendon possesses a1404

T. YASUI ET AL.

Fig. 4. Depth-resolved SHG radar graphs of human tissues. (a) Human Achilles tendon and (b) humandentin. The 0-m depth indicates that the analytical spot is focused onto the sample surface. Five SHG
radar graphs illustrate a vertical line proﬁle at each depth position in the depth-resolved SHG radargraph.

characteristic 2D lateral distribution of collagen orientation in which the collagen ﬁber is arranged with a high organization (Yasui et al. 2003). From thepresent results for the longitudinal direction and the previous results for the
2D lateral direction, we conclude that the collagen orientation is arranged
with a highly organized three-dimensional distribution. On the other hand,in Fig. 4(b) we observed that a crossed, biaxial orientation of collagen ﬁber
is uniformly distributed along the depth direction in the dentin. We previously conﬁrmed that the 2D lateral distribution of collagen orientation in
dentin gradually becomes disordered, from biaxial at the root to random at
the crown, depending on the growth lines (Yasui et al. 2004a). The present
tomographic measurement of the collagen orientation was performed along
the depth direction on the same growth line. Differences in the collagen orientation distribution in the longitudinal and 2D lateral directions are interesting from the viewpoint of both the size effect of the measurement region andmetabolic effects such as tooth aging. To conﬁrm these differences, further
examination of different types of teeth is essential.

4.3. 2d lateral distribution of collagen orientation at sample
surface
We performed polarization-resolved SHG imaging of the 2D lateral distribution of collagen orientation at the sample surface. Figure 5(a) and (b)show, respectively, vertically- and horizontally-polarized SHG images of theanklebone (image size = 3 mm × 3 mm). Although we can clearly observe

SHG TOMOGRAPHY OF COLLAGEN FIBER ORIENTATION IN HUMAN TISSUE

1405

Fig. 5. 2D lateral distribution of collagen ﬁber orientation at the surface of human anklebone. (a) Vertically-polarized SHG image, (b) horizontally-polarized SHG image, (c) ODCO image calculated from thetwo polarization-resolved SHG images. Image size is 3 mm by 3 mm. The color scale of the ODCO value
indicates the direction of collagen orientation: red for vertical, blue for horizontal.

the 2D distribution of SHG light along the crest of the anklebone, it
is difﬁcult to recognize the detailed distribution of the collagen orientation from a direct comparison between the two polarization-resolved SHG
images. Hence, we deﬁne a quantity for the polarization anisotropy of SHG
light, the organization degree of the collagen orientation (ODCO), using
the following equation:
ODCO = (IV − IH )/(IV + IH ),(1)

where IV and IH are the vertically- and horizontally-polarized SHG intensities, respectively. The collagen orientation is uniaxial for ODCO = ±1 andrandom or biaxial for ODCO = 0. The sign of ODCO gives the dominantdirection of the collagen orientation: positive for vertical orientation andnegative for horizontal orientation. Figure 5(c) shows the resultant ODCO
image of the anklebone. The color scale of the ODCO value indicates the
direction of the collagen orientation, with vertical indicated by red and horizontal by blue. Here, focusing on the relationship between the structureof the bone crest and the color in the image, we observe that the vertically-running crests are largely red while the horizontally-running crests arelargely blue. In other words, the anklebone possesses a characteristic 2D
lateral distribution of collagen ﬁber orientation at the surface in which thedirection of the collagen orientation is approximately consistent with thebone crest direction. This result is in good agreement with histological ﬁndings of bone structure (Langton and Njeh 2004).
In the next experiment, the 2D distribution of the collagen orientation
angle in the reticular dermis was investigated. For this purpose, we measured six polarization-resolved SHG images at every 30 degrees. Figure 6(a)1406

T. YASUI ET AL.

shows six polarization-resolved SHG images of 1 mm by 1 mm at 0, 30,
60, 90, 120, and 150 degrees. By comparing these six images, we can determine the polarization angle for each pixel by identifying the image that displays the maximum intensity for that pixel, hence indicating the orientation
direction. Figure 6(b) shows the resulting 2D vector map of the orientation
angle for the reticular dermis. Even in the 2D distribution, we can conﬁrm
the winding of the orientation angle, as indicated by the gray stripe, similar to the 1D distribution of the collagen ﬁber orientation of the reticulardermis in the previous study (Yasui et al. 2004b). The present result is consistent with the histological ﬁndings for reticular dermis, in which thicklygrowing collagen ﬁbers and bundles run entangled in the tissue.
4.4. depth-resoled imaging of 2d lateral distribution of collagenorientation
Finally, we performed depth-resolved imaging of the 2D lateral distribution of collagen orientation in the anklebone. For this purpose, we extended

Fig. 6. 2D lateral distribution of collagen ﬁber orientation at the surface of human back reticular dermis. (a) Polarization-resolved SHG images at 0, 30, 60, 90, 120, and 150 degrees (image
size = 1 mm × 1 mm). (b) 2D vector map of orientation angle. The orientation angle for any pixel
was determined to be the polarization angle showing the maximum SHG intensity among the six
polarization-resolved SHG images.

SHG TOMOGRAPHY OF COLLAGEN FIBER ORIENTATION IN HUMAN TISSUE

1407

Fig. 7. Depth-resolved ODCO images of 2D lateral distribution of collagen ﬁber orientation for humananklebone at depths of (a) 0 m (sample surface), (b) 50 m, (c) 100 m, and (d) 150 m (image
size = 2.5 mm × 2.5 mm). The color scale of the ODCO value indicates the direction of collagen orientation: red for vertical, blue for horizontal, respectively.

the 2D lateral ODCO imaging along the depth direction. Figure 7 shows
2D lateral ODCO images of the anklebone at depths of 0 m (sample surface), 50, 100, and 150 m (image size = 2.5 mm × 2.5 mm). Although there
is a lack of image signal at 150-m depth due to weak SHG light, the 3D
structure of the bone crest can be clearly visualized from the four successive
ODCO images at each depth. Furthermore, the collagen orientation parallel to the crest direction continuously develops along the depth direction in
the same manner as in the 2D lateral direction, the mechanical propertyof which is adapted to bear a mechanical load in any direction. A combination of such measurements and 3D control of laser polarization angle(Yoshiki et al. 2005) could reconstruct the 3D distribution of collagen ﬁberorientation in human tissue.

5. Conclusions
Depth-resolved SHG polarimetry with a confocal pinhole was proposed as
an effective method to probe the tomographic distribution of the collagen ﬁber orientation in human tissue. A maximum penetration depth of
100 m for human hard tissue and 150–300 m for two samples of humansoft tissue was achieved, for which the incident laser power and the depth
resolution were 25 mW and 14 m, respectively. We examined the efﬁcacyof the proposed method for the tomography of collagen orientation usingﬁrst a stacked sample, each of which had a known collagen orientation
along the depth direction, and then applied it to human tissue samples.
The resulting depth-resolved SHG radar graphs indicate a uniform distribution of the uniaxial orientation and the biaxial orientation along the depth
direction for Achilles tendon and dentin, respectively. We also investigated1408

T. YASUI ET AL.

the 2D lateral distribution of collagen orientation and demonstrated depthresolved imaging of this distribution based on 2D lateral ODCO imaging
calculated from two perpendicularly-polarized SHG images. Back reticulardermis exhibited an entangled structure of collagen ﬁber at the 2D lateralsurface, whereas anklebone possessed a 3D collagen orientation parallel tothe bone crest. The proposed system promises to be a powerful tool for invivo monitoring of human tissue collagen, such as dermis or cornea.

Acknowledgements
Portions of this work were supported by Grant-in-Aid for Scientiﬁc Research
Nos. 16300155 and 1720032, from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. We would like to thank Dr. M. Hashimotofor many insightful discussions.

References
Centonze, V.E. and J.G. White. Biophys. J. 75 2015, 1998.
Cox, G., E. Kable, A. Jones, I. Fraser, F. Manconi and M.D. Gorrell. J. Struct. Biol. 141 53, 2003.
Denk, W., J.H. Strickler and W.W. Webb. Science 248 73, 1990.
Dong, C.-Y., K. Koenig and P. So. J. Biomed. Opt. 8 450, 2003.
Dunn, A.K., V.P. Wallace, M. Coleno, M.W. Berns and B.J. Tromberg. Appl. Opt. 39 1194, 2000.
Fine, S. and W.P. Hansen. Appl. Opt. 10 2350, 1971.
Han, M., G. Giese and J.F. Bille. Opt. Express 13 5791, 2005. http://www.opticsexpress.org/abstract.cfm?
URI=OPEX-13-15-5791.
Hashimoto, M., T. Araki and S. Kawata. Opt. Lett. 25 1768, 2000.
König, K. and I. Riemann. J. Biomed. Opt. 8 432, 2003.
Langton, C.M. and C.F. Njeh. In: The Physical Measurement of Bone, Institute of Physics Publishing,London, 11, 2004.
Roth, S. and I. Freund. J. Chem. Phys. 70 1637, 1979.
Stoller, P., K.M. Reiser, P.M. Celliers and A.M. Rubenchik. Biophys. J. 82 3330, 2002.
Yasui, T., K. Shimabayashi, Y. Tohno and T. Araki. In: The 5th Paciﬁc Rim Conference on Lasers andElectro-Optics CLEO, Proc. The 5th Paciﬁc Rim Conference on Lasers and Electro-Optics, TaiPei, 15–
19 December 2003, 361, 2003.
Yasui, T., Y. Tohno and T. Araki. Appl. Opt. 43 2861, 2004a.
Yasui, T., Y. Tohno and T. Araki. J. Biomed. Opt. 9 259, 2004b.
Yeh, A.T., N. Nassif, A. Zoumi and B.J. Tromberg. Opt. Lett. 27 2082, 2002.
Yelin, D. and Y. Silberberg. Opt. Express 5 169, 1999. http://www.opticsexpress.org/abstract.cfm?
URI=OPEX-5-8-169.
Yoshiki, K., M. Hashimoto and T. Araki. Jpn. J. Appl. Phys. 44 L1066, 2005.
Zumbusch, A., G.R. Holtom and X.S. Xie. Phys. Rev. Lett. 82 4142, 1999.