Introduction

Time- and space-resolved molecular information on organs, tissues and cells is important in advancing our understanding of life. Broadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy has enabled fast, quantitative and multi-vibrational mode imaging of tissues and living cells1. Although Raman spectrum reflects not only molecular composition but also molecular structures, this strong feature is still underutilized for CARS microspectrscopy. The aim of the present study is to extend CARS microspectroscopy to investigate not only biological molecular compositions but also molecular structures. One approach is development of an analytical method of CARS spectra to extract protein secondary structures. The Another approach is an extension of CARS microspectroscopy with adding a new optical process, coherent Stokes Raman scattering (CSRS).

1. Protein Secondary Structure Imaging with CARS microspcetroscopy

CARS microspectroscopy provides the third-order nonlinear susceptibility χ(3) spectra at high speed. The imaginary part of χ(3) spectra corresponds to spontaneous Raman spectra. It is well known that a spontaneous Raman spectrum of a protein in the fingerprint region (intensity, peak position and bandwidth of characteristic bands) sharply reflects its protein secondary structures. Hence, protein secondary structures are visualized through analyzing the retrieved Im [χ(3)] spectra. In this study, a human hair sample as one of the models consisting mainly of proteins is investigated with CARS microspectroscopy.

Experimental

CARS spectra were obtained from hair samples with and without treatments by chemical reduction and mechanical extension by broadband multiplex CARS microspectrometer1.

Results and Discussion

In order to visualize the spatial distributions of protein secondary structures of hairs, two-dimensional intersection images were constructed (see Fig. 1) of the peak amplitude and the peak position of the amide I band observed in Im[χ(3)] spectra. The peak amplitudes did not change between the untreated and treated hairs. On the other hand, the peak positions were dramatically changed between hairs by the phase transition of secondary structures due to the treatments. It clearly visualizes the treatments induced changes in protein secondary structures and their spatial distributions.

2. Development of Broadband Multiplex CARS/CSRS Microspectroscopic System

Figure 2 shows the energy diagram of nondegenerate CARS and CSRS with three different laser fields, ω1, ω2 and ω3. Ordinary (degenerate) CARS and CSRS scheme is obtained for ω1 = ω3. If a broadband laser source is used as ω2, a broadband Raman coherence is generated. With the third field ω3, broadband CARS and CSRS spectra are produced simultaneously at ωCARS = ω3 + ω1 - ω2 and ωCSRS = ω3 - ω1 + ω2. Because the CARS and CSRS spectra do not overlap with each other, they can be detected simultaneously by a multichannel spectrometer.

Experimental

The constructed apparatus is schematically shown in Fig. 3. The laser source is a sub-ns microchip Nd:YAG laser, which is also used as a seed laser to generate supercontinuum (SC). The fundamental output (1064 nm), the SC (1.1 ~ 1.7 μm), and the second harmonic (532 nm) of 1064 nm are used for ω1, ω2, and ω3, respectively. The coherent Raman signal generated from the sample is spectrally filtered with a dual (532/1064 nm) notch filter. The signal beam is guided into a polychromator, and detected by a CCD camera. The measured CARS/CSRS spectra are intensity-corrected by a non-resonant background from an underneath cover glass measured under the same experimental condition.

Results and Discussion

Figure 4 (a) shows a broadband nondegenerate CARS/CSRS spectrum of toluene. The intensity-corrected CARS spectrum very much resembles the CSRS spectrum, showing that there is no significant electronic resonance effect with ω3 at 532 nm. On the other hand, the CARS/CSRS spectrum of 10 mM β-carotene in toluene shows CARS bands much stronger than the corresponding CSRS bands. This asymmetry is due to the electronic resonance of β-carotene, which has a strong electronic absorption at 490 nm. The developed non-degenerate coherent Raman microspcetroscopic system enables us to obtain simultaneously the broadband CARS and CSRS spectra with quantitative information on the electronic resonance effect.

3. A New Phase Retrieval Method with CARS/CSRS Simultaneous Analysis

The MEM has been recognized as an effective method to retrieve phase information from coherent Raman spectra to obtain Im[X(3)] spectra that correspond to ordinary Raman spectra2. In MEM, an autocorrelation function obtained from a measured coherent Raman spectrum is extrapolated to maximize the information entropy with a fixed maximum lag, the order of pole, as a parameter. The order parameter can be freely selected. Generally, the higher the order is, the better the expected result is, if the measured spectrum contains no noise. Coherent Raman spectra, however, have inevitably non-negligible noise. On the other hand, the lower the order is, the broader the spectral resolution is. Determination of the optimum order of pole without any assumption is desirable in order to extract precise molecular information from coherent Raman spectra in the fingerprint region (intensity, peak position and bandwidth). In this study, I demonstrate a new phase retrieval method through the simultaneous analysis of CARS/CSRS spectra.

Experimental

The broadband CARS/CSRS spectrum of a 50 wt% ethanol aqueous solution was measured with the newly developed system. The intensity-corrected spectrum (data points: N = 1024) was analyzed with the MEM with an order (m < 1024). The Im[X(3)] spectra obtained from CARS and CSRS are theoretically identical with each other under an electronic non-resonant condition. Therefore, it is expected that the optimum order of pole can be determined through the evaluation of the difference between the retrieved Im[X(3)] spectra.

Results and Discussion

Figure 5 (a) shows the observed CARS/CSRS spectrum of the 50 wt% ethanol aqueous solution. Since ethanol has no absorption in the visible wavelength region, the CARS and CSRS spectra must be identical with each other. Figure 5 (b) shows the order of pole dependence of the difference between the MEM retrieved Im[X(3)] spectra obtained from CARS and CSRS. The dependence shows a minimum at m = 688 (see Fig. 5 (b)). In principle, a larger order of pole gives a higher spectral resolution. However, it tends to make the MEM more sensitive to higher frequency noise. Figure 5 (b) therefore shows that 688 is the most appropriate order of pole to make the two Im[X(3)] spectra from CARS and CSRS consistent with each other, while keeping the highest spectral resolution. The retrieved Im[X(3)] spectra for m = 688 and their difference are shown in Figs. 5 (c) and (d), respectively. The same data for m = 800 are shown in Figs. 5 (e) and (f). It is obvious that high artificial sharp peaks are generated for m = 800 (Fig. 5 (f)). Thus, the most appropriate order for the MEM is determined to be 688 by using experimental data only. The simultaneous observation of CARS and CSRS spectra with the constructed system provides us with a new reliable phase retreive method in cohrent Raman spectroscopy.

Conculusion

In this study, the techniques for investigation of bio-molecular structures were developed. They applied to the sudty of proteins in human hairs, the demonstration of the electronic resonance effect of carotenoids under a microspcope and the reliable phase retrieval of CARS spectra of model samples. They will be applied to biological systems for furthur investigation on roles of bio-molecule structures.

Fig. 1. The amide I mode peak amplitude and position images of hair samples; (a) the untreated and (b) the treated hair peak amplitude; (c) the untreated and (d) the treated hair peak position. The scale bar corresponds to 10 μm.

Fig. 2. Energy diagram of nondegenerate CARS and CSRS. ωCARS = ω3 + ω1 - ω2 and ωCSRS = ω3 - ω1 + ω2. ω1 - ω2 = Ω for a vibrational Raman resonance. v denotes the vibrational quantum number.

Fig. 3. Schematic diagram of the setup of broadband multiplex CARS/CSRS microspectroscopy.

Fig. 4. Broadband multiplex CARS/CSRS spectrum of (a) toluene and (b) 10 mM β-carotene in toluene.

Fig. 5. (a) Intensity-corrected CARS/CSRS spectrum of EtOH aq.; (b) Order of pole dependence of the difference between the Im[X(3)] spectra obtained from CARS and CSRS; (c) The retrieved Im[X(3)] spectra for m = 688; (d) The difference Im[X(3)] spectrum for m = 688; (e) Im[X(3)] spectra for m = 800; (f) Difference Im[X(3)] spectrum for m = 800.

本論文は、コヒーレントラマン顕微分光法によって生体試料中の分子の組成に加え、状態の情報を可視化するための技術開発と、その応用について記述されており、全5章から構成される。

第1章では導入として、生体内の分子の情報(組成および状態)を時間、空間分解能を有して観察することが生体組織の理解ために必要であり、そのための強力な手段の1つであるコヒーレントアンチストークスラマン散乱(CARS)顕微分光法を2つのアプローチで拡張し、従来得られている分子組成情報に加え、状態の情報を引き出す生体分子イメージング技術を開発することが本研究の目的として述べられている。

第2章では、CARS顕微分光法によるヒト毛髪内のタンパク質二次構造イメージング法について示されている。CARS顕微分光法で得られるスペクトル中のラマンバンドのピーク位置、幅をマッピングすることで、毛髪内のタンパク質の二次構造の分布を高速かつ高い空間分解能で可視化できることが示されている。未処理の毛髪および化学還元および延伸処理を施した毛髪中の二次構造の変化およびその分布から、毛髪中の繊維からなるコルテックス部に3層からなる多層構造が存在することが初めて見出されている。

第3章では、開発されたCARSとコヒーレントストークスラマン散乱(CSRS)を同時に観察可能なCARS/CSRS顕微分光装置について述べられている。通常、CARSとCSRSスペクトルは同時に取得することは困難であるが、従来のCARS顕微分光装置に異なる波長の光を加えることで発生させられる非縮退CARS/CSRS過程を利用することで、同時取得を可能にしている。CARSとCSRSは、電子共鳴効果がない条件では、同一のスペクトルとなるが、電子共鳴効果がある場合は、同一のスペクトルを与えないことが、開発された装置による標準試料の測定から確認されている。この現象を利用し、開発された装置で同時に得られるCARSとCSRSのスペクトルのラマンバンドの強度比を得ることで、顕微鏡下で取得困難である分子の吸収スペクトルに対応する指標が得られる可能性が示されている。

第4章では、最大エントロピー法(MEM)をCARSとCSRSの2つスペクトルに同時に適用するコヒーレントラマンスペクトルの新しい位相回復法について述べられている。従来利用されてきたMEMによる位相回復法には、任意に選ぶことのできるパラメーター、次数があり、次数により位相回復して得られる自発ラマンスペクトルに対応するスペクトルの形状が変化するという問題があることが指摘されている。電子共鳴効果がない条件ではCARSとCSRSが一致するという性質を利用し、MEMをCARSとCSRSの2つのスペクトルに同時に適用し、位相情報が回復された両者のスペクトルの差の極小値を与える次数を最適な次数と決定できる信頼性の高い位相回復法が提案されている。

第5章は以上の研究成果のまとめである。

本研究において、CARS顕微分光法の拡張により、生体分子の組成に加え、状態をイメージングするための新たな手法が開発された。タンパク質二次構造イメージング法はヒト毛髪に適用され、毛髪内の新たな構造を見出し、本手法が生体機能を理解する上で有用であることが明確に示されている。新たに構築されたCARS/CSRS顕微分光装置を用いて開発された手法は、生体分子の状態を可視化する上で重要な分子の吸収スペクトルに対応する指標、信頼性の高い位相回復法に基づくスペクトル形状を提供し、生体機能の解明に有用な新しいアプローチとなることが期待される。このような新規の観測手法の開発とその有用性を提示した本論文の内容は高く評価できる。

本論文第2 章の主要部分は、The Journal of Physical Chemistry B 誌に受理済み、公表予定(奥野将成、加納英明、徳原志穂美、内藤智、増川克典、Philippe Leproux、Vincent Couderc、〓口宏夫との共著)であるが、論文提出者が主体となって実験および解析を行なっており、その寄与が十分であるので、学位論文の一部とすることに何ら問題はないと判断する。

以上の理由から、論文提出者尾藤宏達に博士(理学)の学位を授与することが適当であると認める。