雙光子顯微鏡在小動(dòng)物活體光學(xué)成像中的研究進(jìn)展①

張文豪，李建軍，楊德剛，楊明亮，杜良杰，高峰，劉長彬，李大鵬，胡安明，蔡暢

雙光子顯微鏡在小動(dòng)物活體光學(xué)成像中的研究進(jìn)展①

張文豪1,2a,3,4，李建軍1,2a,3,4，楊德剛1,2a,3,4，楊明亮1,2a,3,4，杜良杰1,2a,3,4，高峰1,2a,3,4，劉長彬1,2a,3,4，李大鵬1,2a,3,4，胡安明1,2b，蔡暢1,2a,3,4

雙光子顯微鏡結(jié)合了激光掃描共聚焦顯微鏡和雙光子激發(fā)技術(shù)。雙光子熒光顯微鏡具有光損傷小、漂白區(qū)域小、穿透能力強(qiáng)、高分辨率、熒光收集率高、圖像對(duì)比度高、可實(shí)現(xiàn)暗場(chǎng)成像、適合多標(biāo)記復(fù)合測(cè)量等多種特點(diǎn)，可應(yīng)用于小動(dòng)物活體光學(xué)成像，在腫瘤免疫治療、基因治療、干細(xì)胞研究、藥物篩選與評(píng)價(jià)、活體脊髓損傷成像等領(lǐng)域研究中有廣泛應(yīng)用。

雙光子顯微鏡；活體光學(xué)成像；小動(dòng)物；綜述

[本文著錄格式] 張文豪，李建軍，楊德剛，等.雙光子顯微鏡在小動(dòng)物活體光學(xué)成像中的研究進(jìn)展[J].中國康復(fù)理論與實(shí)踐, 2017,23(1):37-41.

CITED AS:Zhang WH,Li JJ,Yang DG,et al.Research progress of two-photon microscopy in small animals in vivo imaging(review)[J].Zhongguo Kangfu Lilun Yu Shijian,2017,23(1):37-41.

1931年，Maria G?ppert-Mayer在博士論文中第一次提出原子或分子雙光子激發(fā)的理論假設(shè)。20世紀(jì)60年代初，Kaiser、Garret以及Abella等利用當(dāng)時(shí)剛發(fā)明的激光技術(shù)，在晶體中觀察到雙光子吸收現(xiàn)象。1976年，Berns第一次報(bào)道活細(xì)胞中的雙光子效應(yīng)。1990年Denk等將雙光子激發(fā)現(xiàn)象應(yīng)用到激光共聚焦掃描顯微鏡中，制造出雙光子激光共聚焦顯微鏡[1]。相對(duì)于其他成像技術(shù)，如超聲(ultrasound)、計(jì)算機(jī)斷層(computed tomography,CT)、磁共振成像(magnetic resonance imaging,MRI)、正電子衍射斷層(positron-emission tomography, PET)、單光子衍射計(jì)算機(jī)斷層(single-photon-emission computed tomography,SPECT)等技術(shù)，活體光學(xué)成像具有許多獨(dú)特的優(yōu)點(diǎn)：操作簡便、結(jié)果直觀、測(cè)量快速、靈敏度高以及費(fèi)用低廉等[2]，尤其是可在活體內(nèi)實(shí)現(xiàn)對(duì)分子事件的動(dòng)態(tài)、實(shí)時(shí)、連續(xù)監(jiān)測(cè)，并且能夠揭示生物分子相互作用過程的時(shí)間、空間關(guān)系[3-11]。

活體動(dòng)物光學(xué)成像(in vivo optical imaging)主要采用生物發(fā)光(bioluminescence)與熒光(fluorescence)兩種技術(shù)[12-15]。生物發(fā)光主要用熒光素酶(luciferase)基因標(biāo)記細(xì)胞或DNA，而熒光技術(shù)則采用熒光報(bào)告基團(tuán)(GFP、RFP、Cyt及dyes等)進(jìn)行標(biāo)記[16-18]。相對(duì)于傳統(tǒng)的單光子成像，雙光子成像在生物厚組織，如活體腦組織成像中具有較高的空間分辨率、信噪比以及較低的組織損傷性等優(yōu)勢(shì)。近年來，雙光子熒光顯微鏡(two-photon fluorescence microscopy,TPM)在醫(yī)學(xué)領(lǐng)域得到廣泛應(yīng)用[19-21]。TPM使用紅外波段的超快激光作為光源，利用光學(xué)非線性效應(yīng)實(shí)現(xiàn)對(duì)樣品的三維、四維甚至實(shí)時(shí)監(jiān)測(cè)[22-23]。由于紅外光對(duì)生物組織的殺傷作用相對(duì)較小，因此可利用此技術(shù)對(duì)生物樣品進(jìn)行活體動(dòng)態(tài)觀察；同時(shí)由于長波長激光在組織中具有很高的穿透深度，雙光子熒光成像具有成像深度大的特點(diǎn)[24]。TPM對(duì)生物樣品成像具備光漂白性小、光毒性小、穿透性強(qiáng)等優(yōu)點(diǎn)，一經(jīng)問世即很快應(yīng)用于活體動(dòng)物醫(yī)學(xué)領(lǐng)域的研究中，尤其適用于對(duì)小動(dòng)物模型(如斑馬魚、果蠅、小鼠、大鼠)進(jìn)行長時(shí)間內(nèi)反復(fù)多次動(dòng)態(tài)活體成像[25-30]。TPM已成為現(xiàn)代生命科學(xué)研究的重要工具，并帶來革命性的變化。本文重點(diǎn)綜述TPM在小動(dòng)物活體光學(xué)成像中的研究應(yīng)用。

1 成像原理

傳統(tǒng)激光共聚焦顯微鏡有兩大局限。①光漂白現(xiàn)象：因?yàn)楣簿劢沟尼樋妆仨氉銐蛐?，以獲得高分辨率的圖像，而孔徑小又會(huì)擋掉很大部分從樣品發(fā)出的熒光，包括從焦平面發(fā)出的熒光，相應(yīng)的，激發(fā)光必須足夠強(qiáng)，以獲得足夠的信噪比；而高強(qiáng)度的激光會(huì)使熒光染料在連續(xù)掃描過程中迅速褪色，熒光信號(hào)會(huì)隨著掃描進(jìn)程變得越來越弱。②光毒性作用：在激光照射下，許多熒光染料分子會(huì)產(chǎn)生諸如單態(tài)氧或自由基等細(xì)胞毒素，所以實(shí)驗(yàn)中要限制掃描時(shí)間和激發(fā)光的光功率密度，以保持樣品活性。光漂白和光毒現(xiàn)象很大程度限制了活性樣品的研究，尤其是動(dòng)態(tài)觀察活性樣品生長、發(fā)育過程的各個(gè)階段[31-32]。

熒光顯微技術(shù)的發(fā)展經(jīng)歷了從定性到定量、從二維到三維成像的過程，即從傳統(tǒng)熒光顯微鏡(fluorescence microscope, FM)到以單光子激光共焦掃描顯微鏡(single-photon laser con-focal scanning microscope,SPLCSM)為代表的空間分辨熒光顯微技術(shù)的發(fā)展。FM收集的是樣品的整體熒光，無法獲得準(zhǔn)確的定位和定量信息。該技術(shù)采用場(chǎng)光源，難以區(qū)分樣品內(nèi)不同部位的熒光信號(hào)，也難以再現(xiàn)樣品內(nèi)熒光物質(zhì)的原有存在狀態(tài)。由于FM是二維成像，較厚樣品切片后無法觀察活體樣品內(nèi)熒光物質(zhì)的動(dòng)態(tài)變化過程。

雙光子激發(fā)的基本原理是[33-34]，在高光子密度下，熒光分子可以同時(shí)吸收2個(gè)長波長的光子，在經(jīng)過很短時(shí)間，即所謂激發(fā)態(tài)壽命后，發(fā)射出一個(gè)波長較短的光子。其效果和使用一個(gè)波長為長波長一半的光子去激發(fā)熒光分子相同。由于雙光子激發(fā)需要很高的光子密度，為了不損傷細(xì)胞，TPM使用高能量鎖模脈沖激光器。這種激光器發(fā)出的激光具有很高的峰值能量和很低的平均能量，其脈沖寬度只有100 fs，而頻率可達(dá)80～100 MHz。

TPM結(jié)合了SPLCSM和雙光子激發(fā)技術(shù)。在使用高數(shù)值孔徑的物鏡將脈沖激光的光子聚焦時(shí)，物鏡的焦點(diǎn)處光子密度最高，雙光子激發(fā)只發(fā)生在物鏡焦點(diǎn)上；觀察標(biāo)本時(shí)，只在焦平面上才有光漂白和光毒性。雙光子的吸收現(xiàn)象也是非線性效應(yīng)，激發(fā)只發(fā)生在物鏡焦點(diǎn)上，所以TPM不需要共聚焦針孔，提高了成像亮度和信噪比[35]。

2 成像特點(diǎn)

雙光子吸收率依賴于兩個(gè)入射光子在空間和時(shí)間上的重合程度。雙光子吸收截面很小，只有在具有很大光子流量的區(qū)域，熒光團(tuán)才會(huì)被激發(fā)。因此所用激光器多為鈦激光器，可以達(dá)到ps或fs級(jí)的掃描速度，且具有非常高的峰值功率和較低的平均功率，從而可以減小或消除光漂白和光毒作用帶來的不利影響。而在一個(gè)很小的范圍提供高密度光子，保證了雙光子的同時(shí)激發(fā)。

雙光子激發(fā)過程如下。在激光照射下，基態(tài)熒光原子或分子同時(shí)吸收兩個(gè)光子而成激發(fā)態(tài)。如還原型煙酰胺腺嘌嶺二核苷酸(nicotinamide adenine dinucleotide H,NADH)酶，在單光子激發(fā)時(shí)，在350 nm光的激發(fā)下產(chǎn)生450 nm熒光；而在雙光子激發(fā)時(shí)，可采用溫和的紅外或近紅外光，如750 nm激光下得到450 nm熒光。這既避免了紫外光對(duì)樣品的傷害和使用紫外光學(xué)元件的許多限制，又可延長對(duì)活體生物樣品的觀察時(shí)間，為研究氨基酸、蛋白質(zhì)和神經(jīng)遞質(zhì)等提供了獨(dú)特而重要的方法[33-34]。

SPLCSM在FM技術(shù)基礎(chǔ)上加裝激光掃描，可在連續(xù)、固定范圍內(nèi)進(jìn)行小功率掃描，記錄動(dòng)態(tài)變化，共軛聚焦裝置可獲得清晰圖像[36]。光路中兩個(gè)共焦小孔的設(shè)置消除了焦平面以外雜散光的干擾，提高了分辨率和成像質(zhì)量。該技術(shù)實(shí)現(xiàn)了熒光物質(zhì)在亞細(xì)胞水平的定位，可無損傷地對(duì)較厚樣品逐層連續(xù)掃描，并重構(gòu)其組織或細(xì)胞的三維立體結(jié)構(gòu)。雖然SPLCSM克服了FM技術(shù)的部分不足，但仍有一定的不足：①激光能光漂白熒光物質(zhì)，共焦小孔在阻擋焦平面外雜散光的同時(shí)亦阻擋了部分本應(yīng)該接收到的來自焦平面的光；為了獲得足夠的信噪比，需要很強(qiáng)的激發(fā)光，熒光信號(hào)也會(huì)隨著掃描進(jìn)程而變得越來越弱；②許多熒光染料分子在激光照射下會(huì)產(chǎn)生細(xì)胞毒素，限制了掃描時(shí)間和激光光源的強(qiáng)度；③由于細(xì)胞成分散射，短波長的激發(fā)光不易穿透標(biāo)本。

將雙光子技術(shù)與各種顯微鏡技術(shù)相結(jié)合，在生物醫(yī)學(xué)領(lǐng)域的應(yīng)用中更能發(fā)揮其潛力。與SPLCSM相比，TPM具有以下優(yōu)點(diǎn)：①具有高空間局域性和高分辨率；②雙光子熒光遠(yuǎn)離激發(fā)波長，避免了激發(fā)光對(duì)熒光探測(cè)的影響，能實(shí)現(xiàn)暗場(chǎng)成像，散射產(chǎn)生的背景噪聲小，圖像對(duì)比度高；③焦點(diǎn)以外不發(fā)生漂白現(xiàn)象；④可用紅外或近紅外激光作為光源，紅外光在生物組織中穿透力強(qiáng)，能對(duì)生物組織的深層成像觀察。

3 主要應(yīng)用領(lǐng)域

近年來，隨著活體光學(xué)成像設(shè)備的進(jìn)展以及轉(zhuǎn)基因動(dòng)物研究的興起，國內(nèi)外科研機(jī)構(gòu)已經(jīng)將小動(dòng)物活體光學(xué)成像技術(shù)廣泛應(yīng)用于腫瘤免疫治療、基因治療、干細(xì)胞研究、藥物篩選與評(píng)價(jià)、活體脊髓損傷成像等領(lǐng)域，并取得許多成果[37-39]。

3.1 活體監(jiān)測(cè)腫瘤的生長與轉(zhuǎn)移

傳統(tǒng)腫瘤研究方法主要局限于肉眼觀察、處死動(dòng)物后進(jìn)行大體解剖或組織學(xué)觀察等，無法動(dòng)態(tài)觀察整個(gè)腫瘤事件。利用TPM研究腫瘤細(xì)胞凋亡具有獨(dú)特優(yōu)點(diǎn)，可避免由于處死動(dòng)物而造成的組間差異，節(jié)省動(dòng)物成本，并能動(dòng)態(tài)監(jiān)測(cè)腫瘤在體內(nèi)的生長和轉(zhuǎn)移，使分子水平的研究能在更接近活體的環(huán)境中進(jìn)行，可以作為細(xì)胞分析的完整應(yīng)用工具[40-41]。

實(shí)時(shí)進(jìn)行高清晰度熒光成像，TPM低光漂白和光毒性掃描和檢測(cè)技術(shù)最大限度地減少光對(duì)活細(xì)胞的傷害；能直接快速地測(cè)量各種癌癥模型中腫瘤的生長和轉(zhuǎn)移，并可對(duì)癌癥治療中癌細(xì)胞的變化進(jìn)行實(shí)時(shí)觀測(cè)和評(píng)估；活體生物發(fā)光成像能夠無創(chuàng)傷地定量檢測(cè)小鼠整體原位瘤、轉(zhuǎn)移瘤及自發(fā)瘤。

3.2 監(jiān)測(cè)基因治療中基因的表達(dá)

基因治療包括在體內(nèi)將一個(gè)或多個(gè)感興趣基因及其產(chǎn)物安全而有效的傳遞到靶細(xì)胞。可應(yīng)用熒光素酶基因作為報(bào)告基因用于載體的構(gòu)建，觀察目的基因是否能夠在試驗(yàn)動(dòng)物體內(nèi)持續(xù)高效和組織特異性表達(dá)[42]。這種非侵入方式具有容易準(zhǔn)備、低毒性及輕微免疫反應(yīng)的優(yōu)點(diǎn)。熒光素酶基因也可以插入脂質(zhì)體包裹的DNA分子中，用來觀察以脂質(zhì)體為載體的DNA運(yùn)輸和基因治療情況。

基因表達(dá)研究目的基因何時(shí)、何種刺激下表達(dá)。將熒光素酶基因插入目的基因啟動(dòng)子下游，并穩(wěn)定整合于實(shí)驗(yàn)動(dòng)物染色體中，形成轉(zhuǎn)基因動(dòng)物模型[43]。利用其表達(dá)的熒光素酶與底物作用，產(chǎn)生生物發(fā)光，反應(yīng)目的基因的表達(dá)情況，從而實(shí)現(xiàn)對(duì)目的基因的研究。

3.3 干細(xì)胞應(yīng)用研究

干細(xì)胞光學(xué)標(biāo)記的常用方法有：①利用螢火蟲熒光素酶(Firefly Luciferase)作為報(bào)告基因，通過轉(zhuǎn)基因技術(shù)體外轉(zhuǎn)染干細(xì)胞；②通過親脂性熒光染料直接標(biāo)記干細(xì)胞；③從已構(gòu)建好的生物發(fā)光轉(zhuǎn)基因動(dòng)物中提取干細(xì)胞，所提取干細(xì)胞即具備生物發(fā)光特性。

總體來說，應(yīng)用TPM成像技術(shù)進(jìn)行干細(xì)胞研究主要集中于以下幾個(gè)方面：①監(jiān)測(cè)干細(xì)胞的移植、存活和增殖；②示蹤干細(xì)胞在體內(nèi)的分布和遷移；③多能誘導(dǎo)干細(xì)胞、腫瘤干細(xì)胞等新興研究[44-50]。

3.4 藥物的篩選與評(píng)價(jià)

藥物先導(dǎo)化合物的篩選與評(píng)價(jià)通常處于藥物發(fā)現(xiàn)和開發(fā)的瓶頸位置，基于現(xiàn)代分子生物學(xué)、細(xì)胞生物學(xué)技術(shù)以及現(xiàn)代儀器自動(dòng)化技術(shù)產(chǎn)生的高通量篩選(high throughput screening, HTS)的快速發(fā)展，代表了現(xiàn)代發(fā)現(xiàn)藥物先導(dǎo)化合物的主要趨勢(shì)。HTS是指運(yùn)用自動(dòng)化篩選系統(tǒng)，在短時(shí)間內(nèi)、在特定的篩選模型上，完成數(shù)以千計(jì)甚至萬計(jì)的樣品活性測(cè)試。TPM可提供靶基因在體內(nèi)的實(shí)時(shí)表達(dá)和對(duì)候選藥物的準(zhǔn)確反應(yīng)，還可以用來評(píng)估候選藥物和其他化合物的毒性，為藥物在疾病中的作用機(jī)制及效用提供研究方法[50-51]。

3.5 小動(dòng)物活體脊髓成像

TPM能夠?qū)D(zhuǎn)基因熒光小鼠脊髓軸突、小膠質(zhì)細(xì)胞、鈣離子活性進(jìn)行活體成像；能在長時(shí)間內(nèi)觀察脊髓損傷后軸突退變與再生、小膠質(zhì)細(xì)胞聚集、鈣離子的動(dòng)態(tài)變化等脊髓損傷后細(xì)胞水平的活體成像，為理解和追蹤脊髓損傷的病理生理過程奠定重要的理論基礎(chǔ)[22,27,52-55]。Zhang等[56-57]應(yīng)用TPM進(jìn)行甲波尼龍對(duì)小鼠脊髓損傷效應(yīng)的活體成像研究，并且進(jìn)行了甲波尼龍治療脊髓損傷時(shí)間窗的探討性研究。

4 總結(jié)與展望

在過去二十多年里，TPM成像技術(shù)的應(yīng)用范圍迅速增加，使我們對(duì)活細(xì)胞生理、病理和藥理領(lǐng)域的認(rèn)識(shí)得到前所未有的進(jìn)步[58]。

盡管TPM的應(yīng)用越來越廣泛，但仍有一些問題需要解決：①只能對(duì)熒光成像；②由于使用紅外和近紅外光源，樣品可能會(huì)受到熱損傷；③受昂貴的超快激光器限制，TPM的價(jià)格和維護(hù)成本還比較高。

該技術(shù)下一步發(fā)展可能涉及紅外染料，因?yàn)槠浼ぐl(fā)光波長更長，并能減少組織對(duì)光的吸收和折射，從而實(shí)現(xiàn)更深層組織的成像。隨著激光成像技術(shù)、計(jì)算機(jī)成像技術(shù)、分子探針、熒光標(biāo)記技術(shù)、高靈敏度探測(cè)技術(shù)、計(jì)算機(jī)圖像處理等技術(shù)系統(tǒng)裝備，以及醫(yī)學(xué)應(yīng)用等方面的飛速發(fā)展，TPM技術(shù)會(huì)得到更大提升和更廣泛的應(yīng)用。TPM將不僅用于基礎(chǔ)研究和藥物研發(fā)，也將可能會(huì)作為新技術(shù)擴(kuò)展到臨床檢驗(yàn)及藥物治療領(lǐng)域。

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Research Progress of Two-photon Microscopy in SmallAnimals in Vivo Imaging(review)

ZHANG Wen-hao1,2a,3,4,LI Jian-jun1,2a,3,4,YANG De-gang1,2a,3,4,YANG Ming-liang1,2a,3,4,DU Liang-jie1,2a,3,4,GAO Feng1,2a,3,4,LIU Chang-bin1,2a,3,4,LI Da-peng1,2a,3,4,HU An-ming1,2b,CAI Chang1,2a,3,4
1.Capital Medical University School of Rehabilitation Medicine,Beijing 100068,China;2.a.Department of Spinal and Neural Function Reconstruction;b.Department of Neurosurgery,China Rehabilitation Research Center,Beijing Bo'ai Hospital,Beijing 100068,China;3.Center of Neural Injury and Repair,Beijing Institute for Brain Disorders,Beijing 100068,China;4.Beijing Key Laboratory of Neural Injury and Rehabilitation,Beijing 100068,China

LI Jian-jun.E-mail:crrc100@163.com

Two-photon microscopy is a new technique which combines laser scanning con-focal microscopy and two-photon excitation technique.Two-photon fluorescence microscopy has the advantages of little light damage,small bleaching area,strong penetrability,high resolution,high fluorescence collection efficiency,and high image contrast.It is suitable for dark field imaging and multi-labeled compound measurement,and has been widely used in small animals in vivo optical imaging,such as research for tumour,gene therapy,stem cells,drug development,spinal cord injury,etc.

two-photon microscopy;in vivo optical imaging;small animals;review

10.3969/j.issn.1006-9771.2017.01.09

R446.8

A

1006-9771(2017)01-0037-05

2016-10-19

2016-10-26)

1.國家自然科學(xué)基金項(xiàng)目(No.81272164)；2.中央級(jí)公益性科研院所基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(No.2015CZ-6)；3.中國康復(fù)研究中心課題(No.2012-1;No.2013-7)。

1.首都醫(yī)科大學(xué)康復(fù)醫(yī)學(xué)院，北京市100068；2.中國康復(fù)研究中心北京博愛醫(yī)院，a.脊柱脊髓神經(jīng)功能重建科；b.神經(jīng)外科，北京市100068；3.北京腦重大疾病研究院神經(jīng)損傷與修復(fù)研究所，北京市100068；4.北京市神經(jīng)損傷與康復(fù)重點(diǎn)實(shí)驗(yàn)室，北京市100068。作者簡介：張文豪(1991-)，男，漢族，河南柘城縣人，碩士研究生，主要研究方向：脊柱脊髓損傷的康復(fù)與治療。通訊作者：李建軍(1962-)，男，漢族，教授，主任醫(yī)師，博士研究生導(dǎo)師，主要研究方向：骨科及脊柱脊髓損傷的康復(fù)與治療。E-mail:crrc100@163.com。