Second harmonic microscopy of axonemes

Second harmonic microscopy ofaxonemes

Christophe Odin1, Claire Heichette2, Denis Chretien2,Yann Le Grand1

1 Institut of Physics of Rennes IPR/UMR CNRS 6251, Universityof Rennes I, Campus deBeaulieu, Bat 11A, 35042 Rennes Cedex, FRANCE

2Institut Federatif de Recherche 140, Genetique Fonctionnelle et Sante,UMR CNRS 6026,University of Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, FRANCE

[email protected]

Abstract: We performed Second Harmonic Microscopy of axonemesobtained from sea urchin sperm. Using polarization analysis and a trade-offbetween signal and photodamage, we were able to determine, for the firsttime to our knowledge, the nonlinear susceptibilityχzxx/χxzx = 1.1± 0.2andχzzz/χxzx= 4±0.5 of axonemes.

© 2009 Optical Society of America

OCIS codes: (180.4315) Nonlinear microscopy; (160.1435) Biomaterials ; (170.3880) Medicaland biological imaging

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#108928 - $15.00 USDReceived 18 Mar 2009; revised 13 May 2009; accepted 13 May 2009; published 18 May 2009

(C) 2009 OSA 25 May 2009 / Vol. 17, No. 11 / OPTICS EXPRESS 9235

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1. Introduction

Axonemes are complex organelles, about 250 nm wide and up to several micrometers in length,made up of nine microtubule doublets plus a central pair (9+2architecture), and involved inseveral important cell activities such as sperm motility orflows of mucus and cerebrospinalfluids. Defects in axoneme structure are associated with a broad range of diseases known as cil-iopathies (reviewed in [1]). Thus, a non invasive techniquethat would allow to detect axonemeanomalies in situ would be a valuable tool for clinical investigations of ciliary diseases.

Second harmonic microscopy (SHM) provides intrinsic optical sectioning and high in-depthpenetration due to the inherent localization of the nonlinear excitation at the objective focal vol-ume, while drastically reducing out-of-focus photobleaching and phototoxicity. In addition, avariety of biological macromolecules, such as collagen or myosin, give rise to endogenous SHGsignal, allowing visualization of organized biological assemblies in intact cells and tissues,invitro or in vivo [2, 3, 4, 5, 6]. SHM has also been used to visualize polar arrays of microtubulesin brain tissues, and may bring interesting insights into neurodegenerative diseases [7, 8, 9, 10].

In this paper, we have asked whether we could visualize and characterize axonemes purifiedfrom sea urchin sperm using SHM. We will show, that despite very small second harmonic (SH)signals, nonlinear optical properties of axonemes can be assessed.

2. Experimental methods

2.1. Axoneme sample preparation and Differencial Interference Contrast light Microscopy

Demembranated axonemes were purified from the sea urchinSphaerechinus granularisac-cording to published procedures [11]. The concentration ofaxonemes was chosen to avoidoverlappings and aggregation. Samples were prepared by injecting 10µ l of solution in a per-fusion chamber made of a slide and a coverslip separated by two strips of double-sided tape,and rinsed twice with 10µ l BRB80 to keep only adsorbed axonemes. Fresh samples were im-mediately imaged. Video-enhanced Differencial Interference Contrast (DIC) microscopy wereperformed as described in [12].

2.2. SHM experimental setup

Our imaging setup was based on a modified confocal microscopecomposed of an OlympusIX71 inverted stand and a FluoView 300 scanning head (Olympus, Hamburg, Germany). Afemtosecond Ti:Sapphire laser (Mira900-Verdi5, Coherent, Saclay, France) was coupled to themicroscope and was tuned at a wavelength of 830 nm for all experiments. Linearly polarized200-fs pulses at a repetition rate of 76 MHz were sent to a high-NA 60x water-immersion mi-croscope objective (UplanApo/IR 60xW NA1.2, Olympus). This latter was slightly underfilledby the input laser beam to match the NA of the water-immersioncondenser (IX2-TLW NA0.9,Olympus) collecting the SHG light in transmission. The average laser power in the focal planewas set to< P >= 30mW to limit photodamage (see section 3). The SH light was detectedthrough a 2-mm thick BG39 filter (Lambda Research Optics, CA)that blocks the excitationwavelength, and a 415-nm (10-nm FWHM) bandpass filter (Edmund Optics, York, UK) by a



photomultiplier tube (PMT) module with thermoelectric cooler (H7844, Hamamatsu Photon-ics, Massy, France). The PMT module was connected to a transimpedance amplifier (C7319,Hamamatsu) so as to match the SH signal to the full range of microscope hardware and soft-ware. The laser polarization was controlled by a zero-orderhalf-wave plate (Edmund Optics,UK), mounted in a motorized rotation stage (PR50CC, Newport) synchronized to the imageframe. The stage was inserted in the place of the fluorescent cube turret of the microscope.512x512 SH images with zoom 10X and 12-bit intensity resolution were acquired from Flu-oView microscope software, then recorded as TIFF files. The pixel dwell time was≈ 10µs.

2.3. Orientation Field-Second Harmonic Microscopy and imaging conditions

The principles of Orientation Field-Second Harmonic Microscopy (OF-SHM) were presentedin [13, 14]. The main assumptions of our method are : (i) the SHintensity is interpreted froma nonlinear susceptibility tensorχ (2) of Cn(n≥ 6) symmetry, with symmetry axis in the focalplane XZ at angleφ to X-axis ; this hypothesis is consistent with the axonemeC9 symmetry[15]; (ii) no polarization analysis is performed at detection ; the laser is linearly polarized atangleψ to X-axis. Then, only the three componentsχzzz, χzxxandχxzx (z‖Cn≥6 symmetry axis)contribute to the intensity [16]:

I2ω(φ ,ψ) ∝ [χxzx sin2θ ]2 +[

χzzzcos2 θ + χzxxsin2 θ]2

(1)

with θ = ψ −φ the angle between laser polarization and theC∞ symmetry axis. Only the ratiosξ = χzxx/χxzxandρ = χzzz/χxzxcan be measured, andφ . An isotropic imageU can be obtainedby averaging intensities acquired with N polarizationsnπ/N (n= 0..N−1) whenN ≥ 3. WhenKleinman symmetries are further valid,ξ = 1.

In the caseξ = 1, we showed that combining only 4 images acquired at laser polarizationsnπ/4 allows the determination of the unknownsU , φ andρ [13, 14]. However, ifξ is unknown,all the parameters can be determined from at least 6 images acquired at polarizationsnπ/6 usinga nonlinear least-square fit of pixel intensities with Eq. 1.Image analysis was performed withhomemade routines written inMatlab (the MathWorks, Natick, MA).

3. Results and discussion

Typical DIC and SHM images with the same field of view are presented in Figs. 1(a) and(b) respectively. The axonemes are well resolved, and most of them are straight. Note that, incontrast to DIC, the SHM is background free and that the axoneme profile can be resolved.

Figure 1(c) displays a zoom of a vertical axoneme, with transverse intensity profiles.The two-photon Point Spread Function (PSF2) of the microscope is well represented by a

gaussian∼ exp[−(r/wxy)2] wherewxy is the radius at 1/emaximum intensity [4]. As shown in

Fig. 1(c), axoneme profiles are well fitted by gaussians∼ exp[−(r/w)2], with w≈ 0.21µm. As-suming that the profile can be approximated by the convolution of thePSF2 with the axoneme

cylindrical profile of radiusR, we obtainedw ≈√

w2xy+ αR2 with α ≈ 1. WhenNA > 0.7,

wxy = 0.23λ/(NA0.91), and we obtained for our experimental set-upwxy ≈ 0.16− 0.19µm(NA∼1.2-1 when the objective entrance pupil is respectively overfilled or slightly underfilled).The diameter of the axonemes was measured by electron microscopy asR= 0.125µm. Thusfor NA∼ 1.2−1,w≈ 0.2−0.22µm. These values are consistent with our experimental results.

Moreover, the detection of the SHG signal from the axonemes pushed our confocal basedSHG microscope to its limits, indicating that the SHG signalemitted by the axonemes is verysmall. The numbern of photoelectrons delivered by the PMT photocathode can be roughlyestimated from the hypothesis that the signalS= g n, with g a gain.n obeys Poisson statis-



Fig. 1. (color online) :(a) DIC images of axonemes; (b) Isotropic SHG images of axonemes( both images 23.5×23.5 µm2) ; (c) SHG : 2.4×5.5µm2 zoom on a vertical axoneme .Right : two examples of horizontal profiles. Bottom : mean profile integrated along all theaxoneme. Continuous lines represent the best gaussian fits of the data.

tics (therefore, the mean< n > and the variancevar(n) are equal). Under this hypothesis, thevariancevar(S) is a linear function of the mean< S> (with slopeg = ∂var(S)/∂< S>).

As shown in Fig. 2(a),var(S) is indeed a linear function of< S>, in agreement with aPoisson photoelectron statistics. The mean number of photoelectrons< n > per pixel was de-duced from the relationship< n >≈< S>2/Var(S), which histogram is presented in Fig. 2(b).It demonstrates that an average of about 1 photoelectron per10µspixel dwell time is detected at415nm for an average laser excitation intensity< I >∼ 300mW/µm2 at 830nm(< I >=< P>/Swith S= πw2

xy). Despite such a very small signal, we will show that the optical nonlinear

Fig. 2. (color online) : (a) Linear relationship between SHGsignal mean and variance ;(b) Histogram of the mean number of photoelectrons< n > per pixel. (c) Axoneme pho-todamage induced by repetitive laser scanning (23.5×23.5 µm2). The number representsthe image scan number. (d) Axoneme mean SHG intensity as a function of scan number.The continuous line represent the best fit with a logistic function.



properties of axonemes can be addressed. During scanning, the SHG intensity delivered bythe axonemes was found to decrease (Fig. 2(c)), as a result ofphotodamage. The evolutionof the mean SHG intensity as a function of the number of scanst is presented in Fig. 2(d).The decrease is strongly nonlinear, and the data are well fitted by the logistic function [17]f (n,k, t1/2) = 1/[1+exp(k(t− t1/2))], wherek ≈ 0.65 determines the curvature of the curveandt1/2 ≈ 8 is the scan number at which the intensity is halved.

This photodamage process limits the number of images that can be acquired, thus the numberof polarizations that can be addressed. Then, if a decrease of ∼10-20% of the intensity is ac-cepted, a maximum of 6 polarizations (6 scans) can be used to keep a detection level around onephotoelectron per image pixel. This justifies the use of OF-SHM that requires only 4 images toreconstruct orientation fields and estimateρ whenξ = 1, or 6 images to further estimateξ .

A curved axoneme was selected to illustrate the effectiveness of OF-SHM using 4 polariza-tionsnπ/4. The 4 images of Figs. 3(a1)-(a4) show the strong sensitivity of the SHG contrastto laser polarization. As expected, the isotropic image of Fig. 3(b) show that the SHG inten-sity is roughly independent of the local orientation of the axonemes, with higher intensitiesat axoneme crossings. The orientation field of Fig. 3(c), where each small bar represents theorientation of theχ (2) symmetry axis, is clearly tangent to the axoneme direction.The correla-tion between the axoneme direction andχ (2) symmetry axis is presented in Figs. 3(d),(e). Thedata are well aligned on the bissectrix, with correlation coefficientsR= 0.998 andR= 0.999respectively for 4 or 6 polarizations. Again, this shows that OF-SHM gives the orientation withhigh reliability, despite the very low SHG signal level.

Fig. 3. (color online) : OF-SHM studies of axonemes (512x512images, zoom 10X, fullscale 23.5µm). (a1-a4) A set of 4 SHG polarization images indicated by the double whitearrows; (b) isotropic image U; (c) orientation field represented by bars directed along thesymmetry axis ofχ(2). For clarity, only a few bars are represented; (d)-(e) Correlationbetween the orientationω of the axonemes, andφ of the principal axis ofχ(2) for 4 (d) or6 (e) polarizations. Lines represent the bissectrices.

We finally address the problem of the estimation ofξ andρ . To this end, up to 119 individualnon-overlapping linear axonemes of different orientations were selected, with lengths of typi-cally 4±1µm. The mean intensity of each axoneme for each polarizationnπ/6 was calculated.To improve the reliability of the nonlinear least-square fitting procedure with Eq. 1, we usedour finding that the orientation of an axoneme is parallel to its χ (2) symmetry axis. Angleφ was



thus imposed in Eq. 1, lowering the number of fitting parameters to 3 (∼ χxzx, ∼ χzzz,∼ χzxx)for 6 data points. Thus, the system is overdetermined.

Two methods were used. In the first one, the mean intensity of each axoneme was fittedwith Eq. 1 as a function of laser polarization. Typical fits are presented in Fig. 4(a), and thehistograms of the nonlinear coefficients appear in Fig. 4(b). The histograms are well fittedwith gaussians, and we obtainedξ = 1.2±0.1, ρ = 3.9±0.4. The second method makes theassumption that the 119 selected axonemes have equivalent nonlinear optical properties, thusproviding an almost homogeneous distribution of orientations. We then constructed a mastercurve of the intensity of each axoneme normalized to its isotropic valueU as a function ofθ .To avoid bias, the orientation angles were selected to obtain a uniform distribution when thebin width is 5◦. The resulting curve is presented in Fig. 4(c). The intensity is clearly maximumwhen the laser polarization is aligned along the axoneme. The best fit with Eq. 1 leads toξ = 1.1± 0.2 andρ = 3.9± 0.5 (R = 0.997). Another estimation with the same set of datawas obtained by averaging this master curve over intensities binned every 5◦ (Fig. 4(d)), givesξ = 1.2±0.2, ρ = 3.7±0.4 (R= 0.93). All these methods give consistent results. To the bestof our knowledge, this is the first time these quantities havebeen experimentally determinedfor axonemes.

Fig. 4. Determination ofξ and ρ by OF-SHM with 6 polarizationnπ/6. (a) Examplesof fits of the SHG polarization data derived from 3 axonemes ofdifferent orientations. (b)Histograms of the ratiosχαβγ/X, whereX = χzxx+χxzx+χzzz. Continuous lines representsthe best fit with gaussians. (c) Master curve obtained from the intensities as a function ofθfor all the axonemes. (d) Mean intensity curve obtained by binning the intensity over binsof 5◦ width. Continuous lines are best fits with Eq. 1.

Interestingly, the ratioξ = 1.1± 0.2 is close to unity, indicating that Kleinman symmetryis, at least, approximately verified, like for collagen and myosin. In a model of axisymmetricsupramolecules built from uniaxial harmonophores with only one nonzero molecular hyper-polarizability componentβzzz, ξ = 1. Moreover,ρ = 2/ tan2 ϕ , whereϕ is the polar angle ofthe harmonophores.ϕ was found consistent with the helix pitch angle in collagen or myosin[18]. Here, we obtainϕ = 35±2◦. Although some proteins, like nexin, form helical structuresin axonemes [15], further ultrastructural work will be needed to assign this angle to a givenstructural feature of axonemes.

In conclusion, we have characterized, for the first time to our best knowledge, the nonlinearoptical properties of axonemes. This opens the possibilityof SHM characterization of suchsupramolecular structuresin vivo.

Acknowledgments

This work was supported by Region Bretagne and Rennes Metropole, and CNRS project ”Interface Physique-Chimie-Biologie : soutien a la prise de risque ” for CO and YLG. The workwas supported by Agence Nationale de la Recherche (ANR) for CH and DC.



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Second harmonic microscopy of axonemes