Article(id=1315741649020498440, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315741514865689424, articleNumber=null, orderNo=null, doi=10.3788/gzxb20265503.0311002, pmid=null, cstr=32255.14.gzxb20265503.0311002, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1756137600000, receivedDateStr=2025-08-26, revisedDate=null, revisedDateStr=null, acceptedDate=1766332800000, acceptedDateStr=2025-12-22, onlineDate=1791626854423, onlineDateStr=2026-10-10, pubDate=1774368000000, pubDateStr=2026-03-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1791626854423, onlineIssueDateStr=2026-10-10, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1791626854423, creator=13701087609, updateTime=1791626854423, updator=13701087609, issue=Issue{id=1315741514865689424, tenantId=1146029695717560320, journalId=1315121910733963395, year='2026', volume='55', issue='3', pageStart='1', pageEnd='378', issueExtLink='null', onlineDate='null', pubDate='1774368000000', pubDateStr='2026-03-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1791626822438, creator='13701087609', updateTime=1791626932547, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1315741977111540218, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315741514865689424, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1315741977111540219, tenantId=1146029695717560320, journalId=1315121910733963395, issueId=1315741514865689424, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=288, endPage=297, ext={EN=ArticleExt(id=1315741649511232010, articleId=1315741649020498440, tenantId=1146029695717560320, journalId=1315121910733963395, language=EN, title=Fourier Light Field Microscopic Imaging of the Three-dimensional Structure and Color Changes of Butterfly Scales, columnId=1315741649251185161, journalTitle=Acta Photonica Sinica, columnName=Imaging Systems, runingTitle=null, highlight=null, articleAbstract=

The structure and coloration of butterfly scales play a crucial role in regulating their biological behavior. Accurate three-dimensional imaging of these structures is essential for understanding their functional mechanisms. Traditional imaging techniques usually may suffer from limitations such as sample damage, slow imaging speed, and inaccurate three-dimensional reconstruction. To address these issues, this study employed Fourier Light Field Microscopy (FLFM) to image the three-dimensional structural details of butterfly scales. This imaging method requires no slicing or mounting of samples, thereby preserving their native structural integrity, avoiding damage during imaging, allowing repeated use of the same specimen, and significantly reducing sample consumption. In principle, it can perform the three-dimension imaging in real time by taking the same advantage of single shot three-dimensional imaging and image reconstruction of light field microscopy. Moreover, compared to the conventional light field microscopy, the FLFM can increase the lateral spatial resolution thus the reconstruction quality of the three-dimensional image. We found that FLFM is particularly suitable for delicate biological samples such as butterfly wings and provides reliable technical support for studying their color and structural characteristics.

Here, the representative samples including pigmentary scales, structural color scales, and scale-free areas of different kinds of butterflies were imaged under 10X and 50X objectives with a consistent lighting condition. After introducing the optical system and the imaging mechanism of the FLFM, the three-dimensional reconstruction method based on refocusing and deconvolution algorithms is described. The experimental FLFM setup consisted of a DOIT3DMicro Fourier light-field module mounted on an Olympus IX73 inverted microscope, and the illumination was provided in both transmission and reflection modes depending on the sample type. The refocusing algorithm adopts a shift-and-sum procedure exploiting the conjugate relation between aperture stop and MLA, while the deconvolution algorithm models the system point spread function via wavefront propagation theory for high-resolution reconstruction.

The method is verified by imaging with a resolution target sample and another sample of distributed beads the resolution target image demonstrates the lateral spatial resolution about 8 μm and the beads sample image demonstrate the depth differentiation capability around 60 μm. Finally, the imaging results for the butterfly scales demonstrate that FLFM can clearly reveal morphological characteristics, angle-dependent color variations, and spatial distribution patterns of different scale types. Imaging at 10X magnification verified the feasibility of this technology for visualizing scale structures: the Papilio wing sample shows the relative wide black and white stripes with the boundary somewhat smeared, the Morpho helenor wing sample shows the fine well-organized colorful structures and the color can change from the green blue tone to the blue violet tone, and the Haetera piera wing sample shows shrunk scales and sparsely distributed cilia. Imaging at 50X magnification further revealed significant differences in scale morphology, color formation mechanisms, and optical properties between Catopsilia pomona and Morpho helenor: the color of the Catopsilia pamona wing sample appears mainly due to the pigment structures and does not change when viewing from different angles, while the Morpho helenor wing sample show typical structured color and the hue change apparently by varying the incident light angles or the viewing perspectives.

Therefore, this study demonstrates the advantage and potential wide application prospect of the FLFM in imaging the samples with large depth-of-field. Through careful examination and comparison analysis, we observed the various fine details of the three-dimensional structures of the butterfly wing scales which is very helpful for understanding the different color formation mechanism of the scales. We expect that the FLFM not only can find important applications for studying the structured color of the butterfly wings, but also can provide the experiment support for the further exploration and investigation of biomimetic materials.

, authors=Liang LIANG, Er OUYANG, Zeyu TAN, Hui FANG, authorsList=Liang LIANG, Er OUYANG, Zeyu TAN, Hui FANG, authorCompany=null, correspAuthors=Hui FANG, 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=1315741653357408792, articleId=1315741649020498440, tenantId=1146029695717560320, journalId=1315121910733963395, language=CN, title=蝴蝶鳞片三维结构及其颜色变化的傅里叶光场显微成像研究, columnId=1315741649624478219, journalTitle=光子学报, columnName=成像系统, runingTitle=null, highlight=null, articleAbstract=

现有的蝴蝶显微成像方法对样品具有一定的破坏性且成像慢,傅里叶光场显微具有实时大景深成像及多角度可视化的特点,可以用来研究蝴蝶鳞片的三维结构。采用傅里叶光场显微镜,在10×和50×物镜下对不同蝴蝶样品进行了成像对比,展示了蝴蝶色素色鳞片和结构色鳞片的三维结构差异,以及其颜色在不同角度下变化的不同特征。该研究展现了傅里叶光场显微技术在蝴蝶鳞片研究方面的独特应用前景。

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梁俍,

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Imaging parameters of the FLFM system at 10X and 50X magnification at 525 nm

, figureFileSmall=null, figureFileBig=null, tableContent=
Microscope magnificationNumber of element images NResolution ρ/µmDepth of field DOF/µmField of view FOV/µm
10×58.69278.81 562.5
50×31.81412.6312.5
), ArticleFig(id=1315741659137159752, tenantId=1146029695717560320, journalId=1315121910733963395, articleId=1315741649020498440, language=CN, label=表1, caption=

525 nm波长下FLFM系统在10X及50X的放大倍率下的各成像参数

, figureFileSmall=null, figureFileBig=null, tableContent=
Microscope magnificationNumber of element images NResolution ρ/µmDepth of field DOF/µmField of view FOV/µm
10×58.69278.81 562.5
50×31.81412.6312.5
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蝴蝶鳞片三维结构及其颜色变化的傅里叶光场显微成像研究
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梁俍 , 欧阳尔 , 谭泽宇 , 方晖
光子学报 | 成像系统 2026,55(3): 288-297
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光子学报 |成像系统 2026 , 55 (3) : 288 -297
蝴蝶鳞片三维结构及其颜色变化的傅里叶光场显微成像研究
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梁俍 , 欧阳尔, 谭泽宇, 方晖
作者信息
  • 1深圳大学 微纳光电子学研究院 纳米光子学研究中心,深圳 518060
通讯作者:
作者简介:
Fourier Light Field Microscopic Imaging of the Three-dimensional Structure and Color Changes of Butterfly Scales
Liang LIANG , Er OUYANG, Zeyu TAN, Hui FANG
Affiliations
  • 1Nanophotonics Research Center, Institute of Microscale Optoelectronics, Shenzhen University, Shenzhen 518060, China
出版时间: 2026-03-25 doi: 10.3788/gzxb20265503.0311002
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现有的蝴蝶显微成像方法对样品具有一定的破坏性且成像慢,傅里叶光场显微具有实时大景深成像及多角度可视化的特点,可以用来研究蝴蝶鳞片的三维结构。采用傅里叶光场显微镜,在10×和50×物镜下对不同蝴蝶样品进行了成像对比,展示了蝴蝶色素色鳞片和结构色鳞片的三维结构差异,以及其颜色在不同角度下变化的不同特征。该研究展现了傅里叶光场显微技术在蝴蝶鳞片研究方面的独特应用前景。

傅里叶光场显微  /  蝴蝶鳞片  /  三维结构  /  色素色  /  结构色

The structure and coloration of butterfly scales play a crucial role in regulating their biological behavior. Accurate three-dimensional imaging of these structures is essential for understanding their functional mechanisms. Traditional imaging techniques usually may suffer from limitations such as sample damage, slow imaging speed, and inaccurate three-dimensional reconstruction. To address these issues, this study employed Fourier Light Field Microscopy (FLFM) to image the three-dimensional structural details of butterfly scales. This imaging method requires no slicing or mounting of samples, thereby preserving their native structural integrity, avoiding damage during imaging, allowing repeated use of the same specimen, and significantly reducing sample consumption. In principle, it can perform the three-dimension imaging in real time by taking the same advantage of single shot three-dimensional imaging and image reconstruction of light field microscopy. Moreover, compared to the conventional light field microscopy, the FLFM can increase the lateral spatial resolution thus the reconstruction quality of the three-dimensional image. We found that FLFM is particularly suitable for delicate biological samples such as butterfly wings and provides reliable technical support for studying their color and structural characteristics.

Here, the representative samples including pigmentary scales, structural color scales, and scale-free areas of different kinds of butterflies were imaged under 10X and 50X objectives with a consistent lighting condition. After introducing the optical system and the imaging mechanism of the FLFM, the three-dimensional reconstruction method based on refocusing and deconvolution algorithms is described. The experimental FLFM setup consisted of a DOIT3DMicro Fourier light-field module mounted on an Olympus IX73 inverted microscope, and the illumination was provided in both transmission and reflection modes depending on the sample type. The refocusing algorithm adopts a shift-and-sum procedure exploiting the conjugate relation between aperture stop and MLA, while the deconvolution algorithm models the system point spread function via wavefront propagation theory for high-resolution reconstruction.

The method is verified by imaging with a resolution target sample and another sample of distributed beads the resolution target image demonstrates the lateral spatial resolution about 8 μm and the beads sample image demonstrate the depth differentiation capability around 60 μm. Finally, the imaging results for the butterfly scales demonstrate that FLFM can clearly reveal morphological characteristics, angle-dependent color variations, and spatial distribution patterns of different scale types. Imaging at 10X magnification verified the feasibility of this technology for visualizing scale structures: the Papilio wing sample shows the relative wide black and white stripes with the boundary somewhat smeared, the Morpho helenor wing sample shows the fine well-organized colorful structures and the color can change from the green blue tone to the blue violet tone, and the Haetera piera wing sample shows shrunk scales and sparsely distributed cilia. Imaging at 50X magnification further revealed significant differences in scale morphology, color formation mechanisms, and optical properties between Catopsilia pomona and Morpho helenor: the color of the Catopsilia pamona wing sample appears mainly due to the pigment structures and does not change when viewing from different angles, while the Morpho helenor wing sample show typical structured color and the hue change apparently by varying the incident light angles or the viewing perspectives.

Therefore, this study demonstrates the advantage and potential wide application prospect of the FLFM in imaging the samples with large depth-of-field. Through careful examination and comparison analysis, we observed the various fine details of the three-dimensional structures of the butterfly wing scales which is very helpful for understanding the different color formation mechanism of the scales. We expect that the FLFM not only can find important applications for studying the structured color of the butterfly wings, but also can provide the experiment support for the further exploration and investigation of biomimetic materials.

Fourier light field microscopy  /  Butterfly scales  /  Three-dimensional structure  /  Pigmented color  /  Structural color
梁俍, 欧阳尔, 谭泽宇, 方晖. 蝴蝶鳞片三维结构及其颜色变化的傅里叶光场显微成像研究. 光子学报, 2026 , 55 (3) : 288 -297 . DOI: 10.3788/gzxb20265503.0311002
Liang LIANG, Er OUYANG, Zeyu TAN, Hui FANG. Fourier Light Field Microscopic Imaging of the Three-dimensional Structure and Color Changes of Butterfly Scales[J]. Acta Photonica Sinica, 2026 , 55 (3) : 288 -297 . DOI: 10.3788/gzxb20265503.0311002
蝴蝶翅微结构因其重要的自然功能作用,一直以来都受到研究者的广泛关注[1, 2]。蝴蝶翅的鳞片状微结构形成了五彩斑斓的图案,在蝴蝶的求偶、警示和躲避害虫、栖息地适应等方面起着至关重要的作用,还对体温和飞行功能调节以及水分调节方面产生影响[3]。不同的蝴蝶演化出了独特的鳞片微结构,以适应其特定的生存需求,有些蝴蝶翅鳞片的显示色可随观察角度而变化,这种光学特性与鳞片上形成的光子晶体微结构有关[4-5]。深入研究这些鳞片微结构在仿生学研究方面有着重要价值,其独特的光学特性启发了多种新型材料和器件的开发,例如抗反射涂层材料、彩色显示器、彩色传感器等[6-8]。
目前,为研究这些鳞片微结构而获取高分辨率精细图像所采用的主要成像技术包括扫描电子显微镜(Scanning Electron Microscope, SEM)和共聚焦显微镜(Confocal Laser Scanning Microscope, CLSM)[9-10]。但这些技术也存在一定的不足,如SEM需要在真空环境下进行且需要进行样品金属涂覆和冷冻干燥处理,这可能会对原本较为脆弱的蝴蝶翅膀造成不同程度的破坏[11];而CLSM在成像深度方面有一定的限制,对深层结构的成像能力相对较弱,而且成像速度较慢[12]。
近年来,光场显微镜(Light Field Microscopy, LFM)为生物样品高分辨率三维成像提供了一种独特的新选择,LFM主要是通过微透镜阵列(Microlens Array, MLA)周期性排列实现对光场的调制,同时捕获从样品中射出的光线的空间和角度信息,将物体的三维信息编码进了二维图像中,从获取的具有时空和角度信息的元素图像中使用算法解码重建出标本的3D信息。LFM能够在一次拍摄中捕获光场的深度和角度信息,因此适用于实时观察3D快速动态样品[13-14]。目前,LFM方法在生物样品中主要应用于两个方向,一是亚细胞结构的可视化,如对细胞中过氧化氢酶体与线粒体作用的跟踪[15-16],对免疫细胞的动态成像及对癌细胞反应的监测等[17];二是对活体动物的动态图像捕捉,如对秀丽隐杆线虫的高速筛选[18-19],对斑马鱼大脑神经元活动的捕捉[20-22]及对小鼠血流成像等。而傅里叶光场显微镜(Fourier Light Field Microscopy, FLFM)是在传统LFM之上发展演变而来[23-26]:在传统LFM中MLA是放置在显微物镜的成像面上,而在FLFM中则是把微透镜阵列放置到了显微物镜的傅里叶平面上,这种改进明显地提高了成像的分辨率[27]。FLFM保留了LFM的主要优点,即只需通过一次电荷耦合元件(Charge-Coupled Device, CCD)图像拍摄,就可以由特定的重建算法来重建大景深样品的三维结构图像,这极大地缩短了成像时间,减小了对样品的光损害,容易实现对样品的实时动态三维观测[28]。传统的光场技术的主要问题是横向分辨率和角度分辨率之间的权衡,角度信息的获得是以失去横向分辨率为代价的,而横向分辨率是显微镜的一个基本参数。而FLFM则通过提供了横向偏移不变性,避免了重建分辨率的不均匀性,提高了横向分辨率,将并三维物品的角度实时可视化[29-32]。
FLFM近年来发展迅速,已成为较为成熟的技术。2017年,CONG Lin等采用两组微透镜阵列来扩展传统FLFM的景深[20]。2019年,GUO Changliang等提出一种精确的波动光学模型,实现了更好的重建效果[33]。2021年,HUA Xuanwen等提出了一种混合点扩散函数(Point Spread Function, PSF)来提高反卷积算法的性能[16]。2023年,HUANG Haixin等在傅里叶光场成像系统中引入超分辨光学涨落成像技术,进一步提高了重建的三维空间分辨率[34]。本研究中,采用西班牙瓦伦西亚大学3D成像和显示实验室的光场显微成像模块,相较于其他FLFM系统,该模块提供了一种更便利且快速的成像方法。只需要在传统的相机端口插入附件,即可进行调试和拍摄,且其重建算法快速高效,方便对蝴蝶鳞片进行实时成像[29]。
为解决传统成像技术存在对样品的破坏性、成像速度慢、无法准确还原蝴蝶翅的三维结构等缺点,本研究将FLFM应用到对蝴蝶翅鳞片结构的成像中,以达到在样品无须染色或其他制备过程的自然状态下,获取由多观察角度和多成像深度综合信息所构成的三维成像,相对于一般需要通过纵向扫描获取三维图像的光学成像技术而言成像时间更短。利用FLFM进行成像的一个重要优势在于,无须对蝴蝶样品进行切割或装载于载玻片,能够在自然状态下观察样品的三维结构,从而避免对鳞片微结构的破坏。此外,样品可以多次重复利用,减少实验中的样品消耗。这种非侵入性的成像方式特别适合蝴蝶翅膀这种结构复杂的脆弱生物样品,能够为其精细的色彩和结构研究提供更可靠的技术支持。
本文对柑橘凤蝶、海伦闪蝶和黄晶眼蝶三种样本分别在10×和50×的放大倍率下进行了FLFM成像,实验中在相同光照条件下同时获取了多个观测角度的成像,以此着重分析了色素色鳞片与结构色鳞片的差异。这些成像分析结果证明了将FLFM应用于蝴蝶翅鳞片结构研究的可行性,展现了其在相关研究方向进行进一步推广应用的潜力。
本研究所使用的FLFM系统由傅里叶光场成像模块(DOIT3DMicro,Doitplenoptic Inc, Digital Optical Imaging Technologies S.L,SPANISH)装载在奥林巴斯IX73倒置显微镜上构成,该实验系统成像光路示意如图1。其中IX73显微镜的主要由显微物镜(10×/0.25或50×/0.7)、孔径光阑(Aperture Stop, AS)、筒镜L1(焦距为180 mm)共同组成。图1中蓝色方框中的部分是傅里叶光场成像模块的光路图,其中包括视场光阑、傅里叶透镜L2、MLA、CMOS成像传感器。与传统LFM不同,FLFM不再将MLA直接放置在显微镜成像面上,而是通过4f中继系统(L1和L2)将显微物镜的孔径光阑与MLA共轭,将MLA放置在了显微物镜的傅里叶平面上[13]。视场光阑的作用是避免视图之间的交叠,使得MLA后焦面上的CMOS相机可以采集到不同角度的正交视图[14]。
为确保每个视图在传感器上不发生交叠,场阑∅FS的大小必须与MLA的间距相匹配,而视场也与其间距相关
∅FS=f2fMLA
FOV=1MTp
式中,f2是傅里叶透镜L2的焦距,fMLA则是微透镜的焦距,p是微透镜的间距,FLFM系统的总放大率MT由
MT=MOBfMLAf2
给出,其中MOB是物镜的有效放大率。而FLFM系统中孔径光阑的大小可以由物镜的给出的参数求出,即
∅AS=2NAfOB
式中,fOB是显微物镜的焦距,NA是物镜的数值孔径,由生产商标定。孔径光阑的大小也决定了合适的元素图像(Elemental Images, EIs)的数量N。
N∙p=f2f1 
式中,f1是筒镜透镜L1的焦距。在FLFM中,物镜的有效数值孔径NAef与微透镜数量n有关。
NAef=NAn
捕获的视图分辨率为
ρEI=maxλ2NAef+δ MT,2δ MT
式中,λ是从三维物体上入射的光的波长,δ是传感器中的像素大小。景深的理论值为
DOF=λNAef2+δ MTNAef
根据以上公式,FLFM系统在10×及50×的放大倍率下的各成像参数如表1所示(根据FLFM系统设计波长525 nm计算所得)。
实验采取了透射式和反射式照明两种方式,其中透射式照明选择了汞灯(U-LH1000L-3,OLYMPUS),反射式照明则采用了长波通二向色镜滤光片立方体(DMLP425R,thorlabs)控制通过光波段为440~700 nm。
本研究主要采用了两种重建算法:一是DOIT模块所配备软件的重聚焦算法[35],二是在oLaf[36]的功能基础上加了彩色图像处理功能的去卷积算法,重建算法如图2所示。DOIT重新聚焦算法也称为偏移和求和(Shift and Sum,S&S)算法,是EIs像素的移位和求和的结果。图2(a)中,通过AS和MLA之间的共轭关系,还原了在AS处的投影MLA。图中不同颜色表示光束穿过不同微透镜的路径,每个微透镜不同角度穿过不重叠区域的光。图2(b)展示了重聚焦的过程,当所有EI无相对移位(ns=0)叠加时,生成参考面(Reference Object Plane,ROP)的强度分布。对于目标深度位置zR,需将各EI移位ns个像素后叠加,其中深度和位移量满足线性关系
zR=nsδropσp
式中,δrop=δ/MT为传感器像素在ROP的等效尺寸(δ为物理像素尺寸,MT为系统横向放大率)。σp=p/N为MLA在传感器面的投影间距(p为MLA物理间距,N为单张EI的像素边长)。通过AS-MLA共轭关系,将原始光场图像分解为N×N个EI,每个EI对应不同视角的光线信息。根据图2(a)中绿色框中的光路图可知,EI可以通过相对移动叠加裁切以重聚焦各深度的图像。在ROP上的重建图像是FLFM捕获EI的直接叠加而得,而在标准面前后的重建图则是通过EI图位移对应的像素数ns后叠加获得,对于目标深度zR计算位移量ns=(zR∙σp)/δrop,将各个EI位移像素叠加后最终生成重聚焦图像。
oLaf去卷积算法通过系统PSF建模实现高分辨率重建。其核心流程和公式如图2(c)所示,首先基于波前传播模型计算PSF矩阵:根据Rayleigh-Sommerfeld理论[37]计算点光源在物镜前焦面的波前分布,经Debye标量积分得到物镜后焦面波前[38],精确描述AS与MLA间的波传播;入射MLA的波前为AS孔径的放大版,经MLA调制后再次通过Rayleigh-Sommerfeld衍射传播至传感器平面[39],最终生成PSF矩阵。样品Iobject经过FLFM成像系统的PSF卷积后形成观测图像IFLFM,即IFLFM=Iobject*PSF,其中*代表卷积运算。算法通过IFLFM与PSF矩阵逆向求解,重建真实三维结构。针对蝴蝶鳞片的彩色特性,算法对RGB三通道独立进行反卷积后融合,最终重建出高精度彩色三维结构。
为了检验FLFM系统的成像性能,采用透射光照明的方式,在10×物镜下用DOIT重聚焦算法重建对美空1951分辨率板进行了成像,以检测其分辨率极限。此外,对160 µm红黑双层小球进行了成像和重建,以确保其景深和重建能力适用于蝴蝶样品的复杂结构,这些测试结果如图3所示。
通过重聚焦算法得到分辨率板的FLFM焦平面的重建图后,对分辨率板第五组的进行光强对比分析。如图3(c)所示,在10%对比度的条件下能够识别出第5~6组,其线宽为8.77 µm,与理想分辨率8.69 µm接近。与理想分辨率值有差距的一个重要原因是使用了长波通玻片,实际上入射波长比设计波长偏大,因此导致了分辨率有所降低。蝴蝶鳞片的尺度在几十到上百微米范围内,该FLFM系统所达到的分辨率可以对用来其进行成像研究。如图3(d)所示,对深度为160 µm红黑双层小球的重建是在273.6 µm的体积深度上进行的。通过重建的图像可以看到FLFM系统在其景深范围的良好重聚焦效果,在-205.2 µm到-136.8 µm的深度范围内能清晰分辨红色小球,而在0~68.4 µm的深度范围内更能清晰分辨出黑色小球。综上所述,这两个实验评估了FLFM在研究厚样品三维信息的可行性,表明其适用于如蝴蝶等复杂生物样品的成像研究。
蝴蝶翅的颜色是由其鳞片的特性决定的,包括鳞片的大小、形状、排列方式和反射光线的方式,这些因素共同影响了翅膀的色彩。根据鳞片与光相互作用的方式,可以把蝴蝶鳞片产生的颜色分成两大类:色素色和结构色。首先,色素色的显现取决于光的波长和色素的成分,是由鳞片中的色素吸收特定波长和反射特定波长的光线而产生的。由于色素的氧化和还原反应可能会改变其性质,因此可能导致色素色的变化、褪色或消失。结构色的显现则取决于鳞片表面的微观结构,主要是由于鳞片表面及内部对光线的折射、干涉和散射作用,而使得翅面在不同观察角度下呈现出多样的颜色。对比色素色来说,结构颜色不受化学作用的影响,因此具有极高的稳定性。
在本研究中,根据蝴蝶的独特的微结构特征,选择了三种不同类型的样品进行分析:
1)色素色鳞片样品:柑橘凤蝶和杏菲粉蝶,柑橘凤蝶黑白条纹源于鳞片中的黑色素,而杏菲粉蝶的黄色则是由于类胡萝卜色素造成的。
2)结构色鳞片样品:海伦闪蝶,这种样品通过鳞片微结构分别形成了明亮的蓝色调,适合研究结构色的特性。
3)无鳞片蝴蝶样品:黄晶眼蝶,其鳞片因先天性萎缩而显露出翅膜,翅膀呈现出略带色素沉着的半透明特征,并在某些区域展现出薄膜干涉现象。
对这三种样品的研究能够揭示蝴蝶鳞片在颜色和外观方面影响的三种主要机制:色素色、结构色以及鳞片缺失对颜色呈现的影响。这些蝴蝶种类在色彩、翅纹理及表面微结构上存在显著差异,因此非常适合作为分析对象,用于探讨FLFM的成像能力。
首先使用10X显微物镜对分别代表色素鳞片、结构鳞片和无鳞片的柑橘凤蝶、海伦闪蝶和黄晶眼蝶进行了成像,实验结果如图4所示。本实验采用DOIT重聚焦算法进行重建,其重建的体积深度为136.8 µm,步长为34.2 µm,重建了5个深度的图像。通过FLFM系统获取4-5-4的EI图、对焦平面的重建结果和重建深度图,进一步对这三种蝴蝶翅的结构特征进行详细的比较和分析。
柑橘凤蝶的翅面呈现出明显的黑白相间条纹,而根据图4(a)对柑橘凤蝶的反射光照明FLFM成像结果可知,其条纹界限不是那么清晰,在交接处黑白鳞片相互参杂。这些鳞片大致呈现出中间较宽两边收窄的椭圆形,整体的高低起伏较小。在焦平面上的重建图像中,能够清晰地观察到大部分鳞片的表面细节。为了进一步观察鳞片的高度差,采用深度热力图方法可视化了重建结果的深度图。对比三种蝴蝶样品的深度图可以看出,柑橘凤蝶翅膀的深度在这三种蝴蝶中最大,顶端的白色鳞片与底部的黑色鳞片之间的深度差约为110 µm,黑色鳞片层比白色鳞片层之间距离约为25 µm。白色鳞片之间的起伏较大,约为50 µm,而黑色鳞片的起伏则相对平缓,约为20 µm。
图4(b)展现了海伦闪蝶的反射光照明FLFM成像结果。在FLFM系统捕获的图像中,海伦闪蝶的鳞片呈现出绚丽的金属色彩,色调从蓝绿色渐变至蓝紫色。从整体看,结构鳞片之间的高度差相对较小,鳞片端部呈现出略带圆弧的方形,由于鳞片之间存在部分交叠,整体结构呈现出高度错落约60 µm的梳状排列。这种排列导致鳞片之间的倾斜角度和光线入射角度各不相同,从而产生了颜色的微妙变化。根据图4(b)中右上角位置可以观察到,在一些蓝绿色的鳞片中,夹杂着蓝紫色鳞片,增添了视觉的层次感。在焦平面重建图像中,可以看到稀疏的半透明表层鳞片,透过这些鳞片可以隐约观察到基层鳞片的轮廓。
图4(c)展现的是对黄晶眼蝶的透射光照明FLFM成像结果。从图中可以观察到,黄晶眼蝶的鳞片完全萎缩,略带色素的透明翅膜得以完全显现,因此采用了透射式照明方式来获取其FLFM图像。黄晶眼蝶的翅面上生长着许多的长短不一的纤毛,这些纤毛的分布及其抬起的角度与其他蝴蝶的鳞片的导管相似,但也存在许多杂乱的小纤毛。通过FLFM采集的多个角度图像,能够重建纤毛结构之间的遮挡关系,从而更深入地理解其翅膀的结构特征。从深度图可以看出,蝴蝶翅膀结构深度较前两者更小,纤毛的根部位于翅膜中间(黄色圆点部分),而翅膜之间高低起伏变化不大,约为20 µm。
为探究不同角度下蝴蝶色素鳞片和结构鳞片的变化,通过反射光照明,在50×物镜下的FLFM系统对杏菲粉蝶的色素鳞片和海伦闪蝶的结构鳞片进行了观察和比较,并采用oLaf去卷积算法进行重建以获得更高的分辨率,分析了它们在大小、形状、排列方式以及光学特性等方面的差异,如图5所示。
通过在17 µm的深度重建杏菲粉蝶的FLFM图像(如图5(a)),可以清晰观察到其鳞片结构呈现为两层,表层鳞片与基层鳞片在形状上十分相似,都以方形片状交错规则排列。其鳞片端部具有3个齿裂,而基层鳞片颜色则相对略深。杏菲粉蝶的鳞片相对较小,表面平坦,几乎呈现扁平片状。部分表层鳞片出现萎缩和脱落现象,脱落的鳞片颜色较其他在蝶翅上的鳞片颜色更浅,这与其色素的不稳定分解有关。如图5(c)所示,海伦闪蝶同样具有两层鳞片,并在49.3 µm的深度重建了海伦闪蝶(图5(d))。对比可知,海伦闪蝶表层鳞片与基层鳞片之间的距离更大,约为30 µm。海伦闪蝶的鳞片相对更大且端部的弯曲更为明显,尽管其鳞片的齿裂数量更多,但整体边缘则显得更加平滑。在图5(d)的32.3 µm深度重建图中,可以观察到表层鳞片呈半透明状态,几乎不含色素,而且大部分已萎缩,覆盖率在80%到50%之间。而在表层鳞片覆盖的区域,亮度更高,颜色呈现显著的蓝色。
通过对比图5(a)和(c)的FLFM捕获的2-3-2EI图发现,由于杏菲粉蝶颜色是由色素造成,因此相对单一,几乎看不到除黄色之外的其他颜色。在不同的EI中,同一个鳞片的颜色几乎不发生任何改变。而海伦闪蝶的结构色由干涉形成,对角度比较具有依赖性,在其同一个鳞片中可同时看到蓝绿色、紫色和黑。并且在切换不同EI视角时,可以观察到一个区域在不同角度下颜色会发生变化。从左下到右上,基层鳞片颜色又黄绿向蓝绿再到黑色渐变。而表层鳞片由于没有黑色素,从左下到右上呈现黄绿到蓝绿色,再到深蓝的变化,主要是对基层鳞片产生颜色进行了映射加强。
本文利用傅里叶光场显微镜在10×和50×物镜下,展示了色素鳞片、结构鳞片和无鳞片蝴蝶样品成像结果。对比了蝴蝶色素鳞片和结构鳞片的三维结构特点,以及结构色在不同角度下颜色的变化的性质。通过10×物镜下的FLFM系统,对色素鳞片、结构鳞片和无鳞片的翅膀结构进行了形状特征、颜色变化及深度关系的对比分析,初步验证了FLFM对蝴蝶鳞片结构成像的可行性。通过50×物镜下的FLFM系统,观察到杏菲粉蝶和海伦闪蝶在鳞片的构造、颜色形成机制和光学特性上存在显著差异。海伦闪蝶的结构色既可以由光线入射角的变化而变化,也可以由观察者的视角的变化而变化。而FLFM通过EI实时观察三维物体的各个角度的优势展现了其在研究角度依赖性结构色蝴蝶样品的应用前景。目前本文仅对静态的样品标本进行了研究,如果能将该技术应用于活体成像,将更能体现出FLFM快速动态成像的优势。
研究结果显示,傅里叶光场显微系统具有低光损伤、实时性的优势,而角度可视化的特性与蝴蝶结构色鳞片的特性相匹配,能够清晰展示结构鳞片的微观细节和色彩特性,使得FLFM为深入研究蝴蝶鳞片结构提供了一种高效的3D显微成像方法。此外,本研究展现了傅里叶光场显微技术在角度依赖性的大景深样品成像方面的优势和广阔的应用前景。通过系统的样品比较分析,深入探讨了结构鳞片的独特之处,为理解蝴蝶色彩形成的生物学机制提供了重要的视角。综上所述,本研究不仅展示了傅里叶光场显微镜在蝴蝶结构色研究中的重要应用,还为相关领域的进一步探索和仿生材料设计提供了理论基础和实验支持。

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doi: 10.3788/gzxb20265503.0311002
  • 接收时间:2025-08-26
  • 首发时间:2026-10-10
  • 出版时间:2026-03-25
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  • 收稿日期:2025-08-26
  • 录用日期:2025-12-22
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    1深圳大学 微纳光电子学研究院 纳米光子学研究中心,深圳 518060

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2种不同金属材料的力学参数
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species
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total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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