Article(id=1315743837604135038, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315743733992243595, articleNumber=null, orderNo=null, doi=10.3788/gzxb20265501.0129001, pmid=null, cstr=32255.14.gzxb20265501.0129001, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1753113600000, receivedDateStr=2025-07-22, revisedDate=null, revisedDateStr=null, acceptedDate=1758816000000, acceptedDateStr=2025-09-26, onlineDate=1791627376222, onlineDateStr=2026-10-10, pubDate=1769270400000, pubDateStr=2026-01-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1791627376222, onlineIssueDateStr=2026-10-10, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1791627376222, creator=13701087609, updateTime=1791627376222, updator=13701087609, issue=Issue{id=1315743733992243595, tenantId=1146029695717560320, journalId=1315121910733963395, year='2026', volume='55', issue='1', pageStart='1', pageEnd='316', issueExtLink='null', onlineDate='null', pubDate='1769270400000', pubDateStr='2026-01-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1791627351520, creator='13701087609', updateTime=1791627555456, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1315744589420540350, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315743733992243595, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1315744589420540351, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315743733992243595, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=298, endPage=306, ext={EN=ArticleExt(id=1315743838103257216, articleId=1315743837604135038, tenantId=1146029695717560320, journalId=1315121910733963395, language=EN, title=Enhancement of Raman Signals in Scattering Media via Spectrally Resolved Variance Optimization​, columnId=1315743838019371135, journalTitle=Acta Photonica Sinica, columnName=Scattering, runingTitle=null, highlight=null, articleAbstract=

Raman microscopy, a label-free detection technique that identifies targets through the intrinsic vibrational spectra of samples, is highly significant in biomedicine, especially for early disease diagnosis. Its ability to avoid exogenous labels offers distinct advantages when studying biological tissues. However, biological tissues and complex materials have strong optical scattering properties, which disrupt light propagation by randomizing their paths. This severely limits the detection depth of traditional Raman techniques—often restricting imaging to superficial layers, rarely exceeding 100 μm in most biological tissues—and impairs signal contrast, as scattered light mix with target signals, resulting in blurred or noisy outputs. To tackle these critical limitations, this study presents an innovative wavefront shaping method based on spectrally resolved variance optimization.​

The core of this method is establishing a sophisticated wavefront feedback mechanism that combines Raman spectroscopy with speckle variance analysis. The detailed process is as follows: First, the experimental sample is carefully designed to simulate in vivo conditions, consisting of Raman-active particles embedded in a scattering medium. This medium is engineered to replicate the forward scattering characteristics of biological tissues, such as those in epithelial layers or soft tissues, ensuring the results are relevant to real-world applications. The Raman particles, chosen for their biological significance, show a unique and distinct Raman peak at 1 200 cm⁻¹, a wavelength range less prone to autofluorescence interference in biological samples. Using an imaging spectrometer with high spatial and spectral resolution, the system simultaneously captures both the spatial distribution (2D speckle patterns) and spectral signatures of Raman signals. A custom-developed algorithm then filters these data to isolate speckle signals specifically at the 1 200 cm⁻¹ peak. This targeted selection minimizes interference from background noise in non-target wavebands—like autofluorescence from cellular components or stray light—thus extracting speckle contrast parameters suitable for quantitative analysis.​

Next, an iterative optimization algorithm is used to build a closed-loop feedback system, a key innovation of this approach. In each iteration, the Spatial Light Modulator (SLM), a device with high phase modulation precision, dynamically adjusts the wavefront of the input light. This adjustment causes changes in the excited speckles, rearranging their distribution within the scattering medium. At the same time, the system monitors Raman speckle variance in real time, with maximizing variance as the main objective. By continuously refining the phase modulation depth of the SLM—adjusting individual pixels to counteract wavefront distortions from the scattering medium—the input light is gradually guided to focus on a single Raman particle in the medium. The optimization process goes through 3 000 iterations, a number determined by preliminary tests to balance convergence speed and accuracy, ensuring stable and reliable focusing of excitation energy on the target particle. A critical point for this enhancement is the strict linear relationship between the intensity of spontaneous Raman signals and the excitation light intensity: when the focused excitation light reaches maximum intensity, the Raman signal is proportionally amplified to its peak level. Additionally, to address potential focal deformation caused by light absorption by Raman particles—a common issue in high-intensity focusing—a pair of galvanometers rotate the incident light at small angles (within the optical memory effect range). Using the memory effect principle, where speckles translate as a whole without changing their distribution under small-angle rotations, the system obtains clear, undistorted focused spots from the translated speckle patterns.​

Experimental results confirm the effectiveness of this method it achieves a 6-fold enhancement of Raman signals in scattering media, outperforming many existing techniques. The spatial resolution reaches the micrometer scale, allowing precise targeting of individual particles—essential for distinguishing between cellular or subcellular structures in biological samples. Furthermore, the method enables non-invasive focusing on single Raman particles with high specificity: background noise outside the 1200 cm⁻¹ peak is enhanced by only~30%, showing effective suppression of non-target signals. This specificity comes from the spectral filtering step, which limits optimization to the target Raman peak.​

In summary, this method provides a new technical solution for enhancing weak Raman signals in scattering environments, addressing long-standing challenges in deep-tissue imaging. Its ability to support label-free imaging in deep biological tissues, along with high signal enhancement and precise spatial focusing, makes it a valuable tool in biomedicine. Potential applications include early diagnosis of diseases like cancer—where subtle Raman spectral changes precede morphological alterations—and sensitive detection of biomarkers, such as proteins or metabolites, in their native tissue environments. By overcoming limitations in depth and signal strength, this approach advances the practical use of Raman microscopy in clinical and research settings.

, authors=Bingxin TIAN1, Haiqing ZHANG1, Xiangbing ZHU1, Qidi HU1, Gang XIONG1, Bingcai LIU2, authorsList=Bingxin TIAN, Haiqing ZHANG, Xiangbing ZHU, Qidi HU, Gang XIONG, Bingcai LIU, authorCompany=null, correspAuthors=Bingcai LIU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1315743839722258572, articleId=1315743837604135038, tenantId=1146029695717560320, journalId=1315121910733963395, language=CN, title=基于光谱解析方差优化的散射介质内拉曼信号增强技术, columnId=1315743838187143297, journalTitle=光子学报, columnName=散射, runingTitle=null, highlight=null, articleAbstract=

生物组织及复杂材料的强光学散射特性导致光子传播路径随机化,严重制约了传统拉曼技术的探测深度与信号对比度。针对这一问题,提出一种基于光谱解析方差优化的波前整形方法,通过构建拉曼光谱与散斑方差相结合的波前反馈机制,实现散射介质内拉曼目标聚焦和微弱信号增强。实验结果表明:该方法可实现单个拉曼粒子的非侵入性聚焦,且拉曼信号增强6倍。这一成果可用于疾病早期诊断与生物标志物检测,同时为在深层生物组织内实现无标记成像提供技术支撑。

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Laboratoire Kastler Brossel, 2020., articleTitle=Light control in scattering media and computational fluorescence imaging: towards microscopy deep inside biological tissues, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1315743839961333901, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, xref=1, ext=[AuthorCompanyExt(id=1315743839969722510, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, companyId=1315743839961333901, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1School of Information Engineering, Shaanxi A&F Technology University, Yangling 712100, China), AuthorCompanyExt(id=1315743839978111119, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, companyId=1315743839961333901, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1陕西农林职业技术大学 信息工程学院,杨凌 712100)]), AuthorCompany(id=1315743840053608592, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, xref=2, ext=[AuthorCompanyExt(id=1315743840061997201, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, companyId=1315743840053608592, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2School of Opto-electronical Engineering, Xi'an Technological University, Xi'an 710021, China), AuthorCompanyExt(id=1315743840070385810, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, companyId=1315743840053608592, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2西安工业大学 光电工程学院,西安 710021)])], figs=[ArticleFig(id=1315743842473722043, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, language=EN, label=Fig.1, caption=Schematic diagram of wavefront shaping based on Raman spectrally resolved variance optimization, figureFileSmall=OOndnDcoG+OR9v7ExpKG0Q==, figureFileBig=W3P6J3ykI7/JkOu4EH3wWA==, tableContent=null), ArticleFig(id=1315743842553413820, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, language=CN, label=图1, caption=基于拉曼光谱解析方差优化的波前整形原理, figureFileSmall=OOndnDcoG+OR9v7ExpKG0Q==, figureFileBig=W3P6J3ykI7/JkOu4EH3wWA==, tableContent=null), ArticleFig(id=1315743842746351805, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315743837604135038, language=EN, label=Fig.2, caption=Optimization process of a single iteration (the 800th iteration). 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基于光谱解析方差优化的散射介质内拉曼信号增强技术
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田冰心 1 , 张海庆 1 , 朱相兵 1 , 胡启迪 1 , 熊刚 1 , 刘丙才 2
光子学报 | 散射 2026,55(1): 298-306
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光子学报 |散射 2026 , 55 (1) : 298 -306
基于光谱解析方差优化的散射介质内拉曼信号增强技术
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田冰心1 , 张海庆1, 朱相兵1, 胡启迪1, 熊刚1, 刘丙才2
作者信息
  • 1陕西农林职业技术大学 信息工程学院,杨凌 712100
  • 2西安工业大学 光电工程学院,西安 710021
通讯作者:
作者简介:
Enhancement of Raman Signals in Scattering Media via Spectrally Resolved Variance Optimization​
Bingxin TIAN1 , Haiqing ZHANG1, Xiangbing ZHU1, Qidi HU1, Gang XIONG1, Bingcai LIU2
Affiliations
  • 1School of Information Engineering, Shaanxi A&F Technology University, Yangling 712100, China
  • 2School of Opto-electronical Engineering, Xi'an Technological University, Xi'an 710021, China
出版时间: 2026-01-25 doi: 10.3788/gzxb20265501.0129001
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生物组织及复杂材料的强光学散射特性导致光子传播路径随机化,严重制约了传统拉曼技术的探测深度与信号对比度。针对这一问题,提出一种基于光谱解析方差优化的波前整形方法,通过构建拉曼光谱与散斑方差相结合的波前反馈机制,实现散射介质内拉曼目标聚焦和微弱信号增强。实验结果表明:该方法可实现单个拉曼粒子的非侵入性聚焦,且拉曼信号增强6倍。这一成果可用于疾病早期诊断与生物标志物检测,同时为在深层生物组织内实现无标记成像提供技术支撑。

生物成像  /  拉曼信号增强  /  波前整形  /  散射介质  /  拉曼散射

Raman microscopy, a label-free detection technique that identifies targets through the intrinsic vibrational spectra of samples, is highly significant in biomedicine, especially for early disease diagnosis. Its ability to avoid exogenous labels offers distinct advantages when studying biological tissues. However, biological tissues and complex materials have strong optical scattering properties, which disrupt light propagation by randomizing their paths. This severely limits the detection depth of traditional Raman techniques—often restricting imaging to superficial layers, rarely exceeding 100 μm in most biological tissues—and impairs signal contrast, as scattered light mix with target signals, resulting in blurred or noisy outputs. To tackle these critical limitations, this study presents an innovative wavefront shaping method based on spectrally resolved variance optimization.​

The core of this method is establishing a sophisticated wavefront feedback mechanism that combines Raman spectroscopy with speckle variance analysis. The detailed process is as follows: First, the experimental sample is carefully designed to simulate in vivo conditions, consisting of Raman-active particles embedded in a scattering medium. This medium is engineered to replicate the forward scattering characteristics of biological tissues, such as those in epithelial layers or soft tissues, ensuring the results are relevant to real-world applications. The Raman particles, chosen for their biological significance, show a unique and distinct Raman peak at 1 200 cm⁻¹, a wavelength range less prone to autofluorescence interference in biological samples. Using an imaging spectrometer with high spatial and spectral resolution, the system simultaneously captures both the spatial distribution (2D speckle patterns) and spectral signatures of Raman signals. A custom-developed algorithm then filters these data to isolate speckle signals specifically at the 1 200 cm⁻¹ peak. This targeted selection minimizes interference from background noise in non-target wavebands—like autofluorescence from cellular components or stray light—thus extracting speckle contrast parameters suitable for quantitative analysis.​

Next, an iterative optimization algorithm is used to build a closed-loop feedback system, a key innovation of this approach. In each iteration, the Spatial Light Modulator (SLM), a device with high phase modulation precision, dynamically adjusts the wavefront of the input light. This adjustment causes changes in the excited speckles, rearranging their distribution within the scattering medium. At the same time, the system monitors Raman speckle variance in real time, with maximizing variance as the main objective. By continuously refining the phase modulation depth of the SLM—adjusting individual pixels to counteract wavefront distortions from the scattering medium—the input light is gradually guided to focus on a single Raman particle in the medium. The optimization process goes through 3 000 iterations, a number determined by preliminary tests to balance convergence speed and accuracy, ensuring stable and reliable focusing of excitation energy on the target particle. A critical point for this enhancement is the strict linear relationship between the intensity of spontaneous Raman signals and the excitation light intensity: when the focused excitation light reaches maximum intensity, the Raman signal is proportionally amplified to its peak level. Additionally, to address potential focal deformation caused by light absorption by Raman particles—a common issue in high-intensity focusing—a pair of galvanometers rotate the incident light at small angles (within the optical memory effect range). Using the memory effect principle, where speckles translate as a whole without changing their distribution under small-angle rotations, the system obtains clear, undistorted focused spots from the translated speckle patterns.​

Experimental results confirm the effectiveness of this method it achieves a 6-fold enhancement of Raman signals in scattering media, outperforming many existing techniques. The spatial resolution reaches the micrometer scale, allowing precise targeting of individual particles—essential for distinguishing between cellular or subcellular structures in biological samples. Furthermore, the method enables non-invasive focusing on single Raman particles with high specificity: background noise outside the 1200 cm⁻¹ peak is enhanced by only~30%, showing effective suppression of non-target signals. This specificity comes from the spectral filtering step, which limits optimization to the target Raman peak.​

In summary, this method provides a new technical solution for enhancing weak Raman signals in scattering environments, addressing long-standing challenges in deep-tissue imaging. Its ability to support label-free imaging in deep biological tissues, along with high signal enhancement and precise spatial focusing, makes it a valuable tool in biomedicine. Potential applications include early diagnosis of diseases like cancer—where subtle Raman spectral changes precede morphological alterations—and sensitive detection of biomarkers, such as proteins or metabolites, in their native tissue environments. By overcoming limitations in depth and signal strength, this approach advances the practical use of Raman microscopy in clinical and research settings.

Biological imaging  /  Raman signal enhancement  /  Wavefront shaping  /  Scattering media  /  Raman scattering
田冰心, 张海庆, 朱相兵, 胡启迪, 熊刚, 刘丙才. 基于光谱解析方差优化的散射介质内拉曼信号增强技术. 光子学报, 2026 , 55 (1) : 298 -306 . DOI: 10.3788/gzxb20265501.0129001
Bingxin TIAN, Haiqing ZHANG, Xiangbing ZHU, Qidi HU, Gang XIONG, Bingcai LIU. Enhancement of Raman Signals in Scattering Media via Spectrally Resolved Variance Optimization​[J]. Acta Photonica Sinica, 2026 , 55 (1) : 298 -306 . DOI: 10.3788/gzxb20265501.0129001
拉曼显微技术作为一种利用样品固有振动光谱识别目标的无标记探测技术,在生物医学,特别是疾病的早期诊断中发挥着重要的作用[1-3]。然而由于自发拉曼信号十分微弱,强度大约为瑞利散射的千分之一,几乎比荧光强度弱10个数量级,因此对自发拉曼信号的探测十分困难。此外,光在生物组织的传输过程中存在散射、吸收和光学像差等现象,使得传统的拉曼探测技术都受到探测深度和空间分辨率的限制[4-5]。目前一些通过物理手段到达目标平面的拉曼光探测方法在一定程度上增加了探测深度,例如基于光纤的内窥镜技术[6-8]能够借助光纤束的光信号传输与成像功能实现对体内腔道的微创观察,但存在成像分辨率受光纤束像素数量限制的问题。表面增强拉曼散射技术[9-14]可以通过金属基底表面的拉曼光吸收体的局部能量沉积使拉曼信号得到增强,但增强实验可重复性差。最新的研究中,基于线性反馈的波前整形技术通过一个成像光谱仪记录自发拉曼信号,实现了散射介质内拉曼信号的增强[15],但这种方法利用拉曼信号强度作为反馈信号诱导增强,无法对拉曼粒子聚焦,且信号增强效率低(强度仅提高25%)。一种非线性反馈的波前整形技术,即对发射信号的空间散斑分布进行成像,而非仅对积分后的总信号进行成像[16-18],通过优化信号散斑的方差,能够在非稀疏样品中实现线性荧光的非侵入性聚焦,成功规避了上述局限性,但实验仅用于荧光信号。
为了实现对散射介质内拉曼信号的增强,本文提出了一种基于光谱解析方差优化算法的拉曼信号增强技术。这种技术利用成像光谱仪获取拉曼信号的空间-光谱信息,通过光谱解析寻找有效的拉曼散斑,并计算拉曼散斑方差,建立反馈机制,从而实现对散射介质中拉曼目标的聚焦以及拉曼信号的增强。
光谱解析方差优化算法以反馈式波前整形算法为理论基础,通过整合拉曼特征光谱的空间散斑信息与散斑方差优化原理,利用空间光调制器(Spatial Light Modulator, SLM)的相位调制功能,实现对外部输入光场的迭代优化。
光谱解析方差优化算法基于反馈式的波前整形算法,融合拉曼特征光谱的空间散斑信息及散斑方差的优化原理,利用SLM的相位调制功能实现对外部输入光进行迭代优化的方法。
基于拉曼散射的激发特性可知,自发拉曼信号强度与激发光强度呈线性关系。但由于拉曼散射的弱激发特性,拉曼信号本身强度较低;同时,在散射过程中,除目标物产生的拉曼信号外,样本背景环境也会在目标区域产生干扰信号。这些信号的叠加使得经过散射介质的拉曼信号被背景噪声掩盖,难以被有效探测。
为获取可探测的对比度并降低噪声干扰,本方法采用成像光谱仪记录二维空间散斑信息,并将其在光谱维度展开,形成空间-光谱维度的二维图像。通过在该二维图像中选取单一拉曼共振峰,可有效滤除背景噪声,从而获得较高的初始散斑对比度,其原理如图1所示。具体过程如下:激光光源经SLM进行相位调制后,由显微物镜聚焦于散射介质(Scattering Medium, SM)中,以照明其中的拉曼目标物。被激发的拉曼光沿反向传播,经二向色镜(Dichroic Mirror, DM)后被探测器接收,进而在整个波长及空间范围内形成随机散斑。利用散斑的窄带区域,并对该区域进行光谱维度解析,可生成具有二维空间分辨率的图像,其中λ方向代表波长,y方向代表空间位置。此外,DM可反射拉曼光并过滤背景信号。
由图1可知,激光经SLM照射拉曼目标后,会产生全波长范围的相位可控散斑,此时拉曼信号与背景信号的对比度较低,其对比度可表示为Cm=1/Nmedium+Ntarget,其中,Nmedium为背景散粒噪声中的粒子数量,Ntarget为目标粒子数量。为获取可测量的对比度,利用成像光谱仪从散斑数据中选取拉曼谱峰位置处的散斑信息,以产生单一拉曼共振。该共振在特定拉曼位移范围内形成散斑图案,此时散斑对比度得以提升,即Ct=1/Ntarget。根据散射介质的光谱散斑相关带宽特性,当拉曼光谱的去相关带宽大于拉曼光谱跨度时,拉曼散斑具有较高相似性;反之,拉曼散斑差异显著。但无论何种情况,通过从同一目标的多个光谱峰处提取空间散斑,均能实现对比度的增强。
当拉曼目标被激发时,尽管目标源数量N未知,但散斑的对比度C与散斑强度I可通过探测器获取。一方面,对比度C能够反映产生散斑的光源数量信息;另一方面,与激发强度呈线性关系的总拉曼信号强度I可提供激发光强度信息。
由空间标准差σ与对比度C、散斑强度I的关系σ=C×I可知,通过优化空间标准差σ,不仅可实现对激发目标源数量的优化,还能获得最大信号强度。并且,当仅有单个粒子被激发时,对比度C与强度I均会达到最大值,进而使σ达到最大值。反之,若能通过相位调制将σ优化至最大值,则该过程本质上等同于聚焦过程。基于上述原理,本研究建立了基于散斑标准差的反馈机制;又因散斑空间方差为标准差的平方(Var=σ2),为简化计算流程,实验中采用散斑空间方差作为反馈信号。
为进一步明晰SLM的相位优化机制,推导散斑空间方差与激发光强度Iexc的关联。设Ix,y,Φ为所有被激发粒子产生的散斑信号总和,则其表达式为
Ix,y,Φ=∑IexckΦikx,y
式中,IexckΦ=aksinΦ+θ+ck表示在第k个目标位置上的激发散斑强度,Iexc是相位ϕ的正弦函数。ikx,y表示第k个目标发出的散斑空间信息。对于线性激发光,信号强度I与激发光强度 Iexc是正比关系。
散斑方差作为分布在期望值(或均值)周围的值的离散程度的度量,其数学定义为VarIx,y,Φ=E(Ix,y,Φ-E(Ix,y,Φ)2)。实验中,散斑强度Ix,y,Φ通过探测器获取的空间散斑图像获取,其散斑方差表示为
VarIx,y,Φ=Var∑IexckΦikx,y=∑IexckΦ2Varikx,y=∑aksin Φ+θ+ck2Varikx,y=∑ak2sin2 Φ+θ+2akcksinΦ+θ+ck2Varikx,y
对于固定的输入模式和散射介质,式(2)右侧最后一项可以看作一个常数[19],因此将式(2)化简为
VarIx,y,Φ=Asin2Φ+θA+BsinΦ+θB+C
式中,A、θA、B、θB和C都是常数。
由式(3)可见,散斑方差与激发光强度呈二阶非线性关系。这表明,即便目标的响应为线性信号,其散斑方差仍会产生非线性反馈信号,该反馈信号能够对入射光进行优化,从而使其聚焦于单个目标之上。
基于散斑方差的非线性聚焦原理,光谱解析方差优化算法通过获取具有可探测对比度的空间拉曼散斑,计算其空间方差,持续搜索使方差达到最大值时SLM上的最优相位模式,进而优化入射波前相位,实现照明光在单个拉曼目标上的聚焦,其原理如图1所示。本研究中,采用成像光谱仪直接替代相机采集拉曼信号,该成像光谱仪输出经光谱解析的二维拉曼图像,其中横坐标为拉曼位移,纵坐标为空间位置。对空间坐标进行求和处理后可得到拉曼光谱,该光谱虽为多个发光源的叠加谱,但仍能保留微弱的拉曼峰值。
由于拉曼信号强度与激发光强度成正比关系,设Iexcx',y'为目标平面上不同位置的激发光强度,则单个拉曼粒子形成的拉曼散斑强度可表示为激发光强度与散射介质对该拉曼粒子散射响应的乘积,即σλ,NIexcx',y',其中σλ,N为散射介质对不同波长、不同位置拉曼目标的散射响应。最终形成的拉曼散斑是各目标发射拉曼信号的非相干叠加,其强度表达式为
IRamanλ,y=∑Nσλ,NIexcx',y'
式中,λ为拉曼信号的光谱维度,y为探测器的空间位置,N表示拉曼粒子数量,x',y'表示粒子在目标平面的空间位置。
寻找拉曼峰值处的空间散斑作为拉曼目标的有效散斑,设Δλp代表有效散斑的波长范围。在运算过程中,仅对Δλp范围内的散斑计算方差值,以此构建波前整形的反馈机制,实现对激发散斑波前相位的优化。
反馈信号的表达式为
F=Var∑ΔλpIRamanΔλp,y
实际上,每种物质的拉曼光谱均存在其特征拉曼峰值,且峰值数量可为一个或多个。因此,Δλp可由多个离散谱段组成。对于每个谱段,拉曼共振会产生相应的拉曼散斑。基于光谱解析的方差优化算法,通过对选定光谱区域内所有拉曼散斑进行叠加得到空间叠加的散斑分布,再将这一叠加散斑的方差作为反馈信号,用于优化输入波前。由此可见,该反馈信号同样适用于更为复杂的拉曼光谱信号。
利用反馈信号F构建波前整形迭代算法,将SLM按像素组划分为若干网格,在每次迭代过程中,依据Hadamard模式对半数网格进行调制,每个网格的取值定义为1或0。其中,取值为1的像素组需加载相位,相位从0到2π被划分为6个相位阶,并依次加载至SLM上;取值为0的网格作为参考,不进行调制。每加载一次相位,通过光谱仪采集对应的拉曼图像,并计算拉曼散斑的方差。在单次迭代中,方差呈正弦特性变化。找到使方差达到最大值的相位确定为本次迭代的最优相位,将其加载到SLM上并与前一次迭代的相位叠加,形成新的相位掩模。当完成对整个Hadamard基的优化后,算法从第一次迭代开始重新循环,直至方差达到最大值时终止。此时,激发散斑在散射介质中形成焦点,并聚焦于单个拉曼粒子。
基于上述优化算法,实验中在SLM(像素规格为600×800)上加载256个Hadamard基,开展3 000次迭代运算。单次迭代耗时约2 s,其中成像光谱仪的曝光时间为300 ms。
图2显示了第800次迭代的优化过程。根据输入光束的大小与位置,选取SLM上的有效像素(200×200)进行优化。其中,图2(a)和(d)分别为SLM上有效像素在单次迭代中的叠加相位与当前相位。通过相位调制,可获得相应的激发散斑,如图2(c)所示。图2(f)为对应的拉曼散斑轮廓,基于该散斑轮廓计算得到的散斑方差被用作反馈信号。图2(e)为此次迭代过程中不同相位步数下的反馈信号,按照优化原理,在单次迭代中,寻找使反馈信号达到最大值时的相位作为本次迭代的最优相位,并将其加载到SLM上。因此,从图2(e)中可确定最大值对应的相位,将其加载到SLM上作为本次迭代的最优相位。在下一次迭代中重复上述过程,直至优化终止。最终,在整个优化过程中可观察到反馈信号持续增大,如图2(b)所示。
不同迭代次数对相位调制深度存在差异,图3展示了迭代次数分别为60、200和600时相位调制深度曲线变化。由图可知,随着优化过程的推进,当迭代次数达到600时,相位调制曲线呈现出显著的正弦变化趋势,且调制深度也随之增加。同时,从这些曲线可以看出,在初始迭代阶段(#60),二阶非线性占据主导地位;经过多次迭代后(#200和#600),调制曲线逐渐呈现正弦变化特征,这是由于目标信号主要由单个目标发射,这再次验证了第1节所述的方差优化原理。
实验中使用532 nm连续激光作为光源,经过SLM相位调制形成激发散斑,通过一个4f透镜结构成像到一个放大倍数为40×,数值孔径NA为0.8的显微物镜的后孔径上,照射实验样本。实验样本由拉曼活性粒子与散射介质组成,可模拟生物组织的前向散射特性。该拉曼活性粒子选用直径为1 μm的单晶金刚石微颗粒,具有单一、尖锐且无背景干扰的特征拉曼峰(位于1 200 cm-1,半高宽<2 cm-1),无旁瓣干扰,可有效避免多峰叠加对光谱解析的干扰。散射介质是通过蚀刻盖玻片(170 μm)一侧的表面制成,不仅能够满足生物显微镜的短工作距离的要求,且透过该散射介质的散斑能够充分扩散,没有弹道光,同时具有一个小的散斑轮廓,以确保在高NA物镜的情况下仍然有足够的光收集效率。
通过迭代优化算法,每次迭代均会诱导激发散斑发生动态变化,不同的激发散斑照射实验样本形成不同的拉曼散斑信号。经过3 000次迭代后,由观察相机获取激发散斑图像,如图4所示。图4(a)为优化前的激发散斑,样本中的4个拉曼粒子(以白色圆圈标注)隐藏于散射介质内。由图可见,优化前的激发散斑随机照射在拉曼粒子上。当优化结束时,原本随机分布的激发散斑已明显聚焦于单个拉曼粒子,如图4(b)所示。但图4(b)中的焦点不够清晰,这是由于拉曼粒子对光的吸收作用导致焦点发生显著变形。为此,本实验通过一对振镜在光学记忆效应范围内对入射光进行小角度旋转。依据光学记忆效应原理,在该效应范围内旋转入射光时,散斑的分布特性保持不变,仅会发生整体平移。因此,从平移后的散斑中可观察到清晰的焦点,如图4(c)所示。
在实现聚焦的同时,被激发的拉曼光由成像光谱仪接收,其拉曼信号在成像光谱仪中表现为二维拉曼信号图像,如图5所示。图5(a)与图5(b)分别为优化前后的拉曼信号二维图像,其中纵坐标为成像光谱仪的空间位置,横坐标为拉曼位移。
依据光谱解析方差优化算法的原理,选取拉曼光谱峰值处的散斑信号作为运算对象以计算反馈信号,进而优化输入光波前。由图可知,优化后的拉曼信号图像在峰值处的能量显著增强,而背景信号相对减弱。对比优化前后的拉曼光谱,如图5(c),可见优化后的拉曼光谱呈现出明显的谱峰,经计算,拉曼光谱峰值处的信号强度提升约6倍,而背景噪声增强仅约30%,该增强效果达到目前较高水平。结果表明,本算法能够有效筛选优化对象,使运算过程仅针对目标进行,对目标以外的信号无显著优化作用。
实验中,成像光谱仪狭缝位置会影响其探测到的各拉曼粒子散斑强度(因不同位置粒子的散斑存在空间偏移):当粒子排列方向与狭缝方向一致时,光谱仪接收的各粒子散斑强度贡献相同,空间上为强度相当的信号叠加;二者方向偏移时,各粒子散斑强度贡献差异大,空间上为强弱悬殊的信号叠加,会影响波前整形优化效果,因此保持目标粒子空间相对位置与狭缝方向一致,是客观验证算法的关键。同时,光栅色散特性也会影响单个拉曼粒子的信号强度,针对光栅不同色散阶数的研究显示:仅照射单个粒子时,光栅零阶信号的散斑强度大于一阶,但二者散斑对比度无明显差异;照射两个粒子时,一阶信号的散斑强度虽仍较小,却比零阶信号具有更大对比度,而散斑对比度对优化至关重要,且实验样本通常含多个目标,因此实验中采用光栅一阶信号。
由实验结果和分析可知,基于光谱解析的方差优化算法能够通过光学聚焦的方法对生物组织内的微弱信号进行增强。即使在实验过程中受到成像光谱仪的狭缝以及光栅的选择等实验条件的限制,在一定程度上会影响实验结果,但相信经过进一步研究,这种方法能够应用在临床环境中。
从实验结果来看,本研究提出的光谱解析方差优化机制,可有效实现散射介质内拉曼信号的显著增强,为拉曼探测在复杂场景中的应用提供了关键技术支撑,但该机制在迭代优化效率方面仍有提升空间。这主要源于两方面原因:一方面,当前实验采用方形Hadamard模式对SLM像素进行迭代调制,而该模式与物镜后焦面、激光照明区域的圆形光学结构不匹配,易导致模式边界超出照明区域,既产生无效调制,又造成激光能量浪费;另一方面,当前采用的8步相移虽能保障相位精度,却因单次迭代中相位加载和信号采集的操作次数较多,增加了整体耗时。为此,未来可从两方面开展优化研究:一是重构迭代模式,采用圆形Hadamard模式对SLM进行相位调制,该模式能使调制区域精准适配圆形光学结构,在避免能量浪费、提升有效激发能量的同时,确保波前优化的收敛性不受影响;二是简化相位调制步长,考虑用4步相移替代8步相移以缩短单次迭代耗时,但需通过实验重点验证4步相移的调制深度,避免因调制深度不足导致迭代次数反增,最终实现效率与精度的平衡。
本研究提出了一种基于光谱解析方差优化算法的自发拉曼信号增强方法。该方法借助反馈式波前整形技术,首先利用成像光谱仪获取拉曼信号的空间信息与光谱信息,通过选择拉曼光谱峰值处的散斑计算反馈信号,以减少背景信号干扰并获取可测量的散斑对比度;再通过方差优化算法,利用SLM的相位调制功能对外部输入光进行优化,使激发光聚焦于拉曼粒子,由于自发拉曼信号强度与激发光强度呈线性关系,当聚焦后的激发光强度达到最大值时,拉曼信号也随之增强至最大值,从而实现了自发拉曼信号的增强(6倍),为疾病早期诊断与生物标志物检测,以及在深层生物组织内实现无标记成像提供新的路径和技术支撑。

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doi: 10.3788/gzxb20265501.0129001
  • 接收时间:2025-07-22
  • 首发时间:2026-10-10
  • 出版时间:2026-01-25
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  • 收稿日期:2025-07-22
  • 录用日期:2025-09-26
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    1陕西农林职业技术大学 信息工程学院,杨凌 712100
    2西安工业大学 光电工程学院,西安 710021

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