Pub Date : 1997-09-01DOI: 10.1002/1361-6374(199709)5:3<153::AID-BIO9>3.3.CO;2-3
X. Gan, C. Sheppard, M. Gu
yOptoelectronic Imaging Group, Department of Applied Physics, Victoria Universityof Technology, PO Box 14428, MCMC, VIC 8001, AustraliazDepartment of Physical Optics, University of Sydney, NSW 2006, AustraliaSubmitted 9 January 1997, accepted 26 June 1997Abstract. The effect of the Fresnel diffraction on the imaging properties of confocalmicroscopy with one circular aperture and one annular aperture is investigated. Theresults show that the Fresnel diffraction patterns alter the axial imaging properties moreseriously than the transverse imaging properties. The axial response of such a confocalmicroscope is sharpened, but severely distorted.Keywords: Fresnel diffraction, annular pupil, confocal microscopy1. IntroductionAnnular aperture is a widely used spatial filter in confocalmicroscopy. It gives some improvement in transverseresolution, but a degradation in axial resolution appears ina confocal system with an annular structure [1,2]. Recentresearch suggests that the use of annular apertures as spatialfilters can suppress the scattered photons [3]. For example,a reflection-mode confocal system with an annular aperturein the illumination path is reported to show a significantenhancement in signal-to-noise ratio for imaging through ahighly scattering medium [4].In practice, an annular aperture cannot be placed exactlyat the back focal plane of an objective. In this case,Fresnel diffraction by the annular aperture exists. Fresneldiffraction of a finite-sized aperture can produce bright anddark fringes because of the interference of the waveletscontributed from points over the aperture [5,6]. Theintensity modulation caused by the interference fringes canalter the imaging properties of confocal microscopy, sincethe effective pupil function of the objective illuminatedby the Fresnel patterns changes accordingly, resulting inthe alteration of the intensity in the focal region [7–10].Therefore, it is important to investigate the effect of Fresneldiffraction on the imaging performance of a confocalsystem with an annular aperture.This paper is organized as follows. Section 2 presentsthe Fresnel diffraction of an annular aperture. The effectof Fresnel diffraction patterns of an annular aperture on theimaging properties of confocal microscopy is discussed insection 3.2. Fresnel diffraction of an annular apertureIf an aperture is placed in the back focal plane of anobjective then focusing is described by Debye theory. TheFresnel number is infinity in this situation. However, ifthe aperture is placed away from the back focal plane by adistance
1 .维多利亚科技大学应用物理系光电成像组，澳大利亚，MCMC, VIC 8001, PO Box 14428;悉尼大学物理光学系，新南威尔士州，2006，澳大利亚，1997年1月9日提交，1997年6月26日接受。研究了菲涅耳衍射对单圆孔和单环孔共聚焦显微镜成像特性的影响。结果表明，菲涅耳衍射模式对轴向成像特性的影响比对横向成像特性的影响更大。这种共聚焦显微镜的轴向响应是尖锐的，但严重扭曲。关键词:菲涅耳衍射，环形瞳孔，共聚焦显微镜环形孔径是共聚焦显微镜中广泛使用的空间滤光片。它在横向分辨率上有所提高，但在具有环形结构的共焦系统中出现轴向分辨率的退化[1,2]。最近的研究表明，使用环形孔径作为空间滤波器可以抑制散射光子[3]。例如，据报道，在照明路径上带有环形孔径的反射模式共聚焦系统在通过高散射介质成像时，信噪比显著提高[4]。在实际应用中，环形光圈不能精确地放置在物镜的后焦平面上。在这种情况下，存在环形孔径的菲涅耳衍射。有限孔径的菲涅耳衍射可以产生明亮和黑暗的条纹，因为来自孔径上的点的小波的干涉[5,6]。干涉条纹引起的强度调制影响了共聚焦显微镜的成像特性，因为被菲涅耳图案照射的物镜的有效瞳孔功能会发生相应的变化，从而导致焦点区域的强度发生变化[7-10]。因此，研究菲涅耳衍射对环形孔径共焦系统成像性能的影响具有重要意义。本文组织如下。第2节给出了环形孔径的菲涅耳衍射。讨论了环形孔径菲涅耳衍射图对共聚焦显微镜成像特性的影响。环形光阑的菲涅耳衍射如果在物镜的后焦平面上放置一个光阑，那么用德拜理论来描述聚焦。在这种情况下，菲涅耳数是无穷大。但是，如果光圈离后焦平面有一段距离
{"title":"Effects of Fresnel diffraction on confocal imaging with an annular lens","authors":"X. Gan, C. Sheppard, M. Gu","doi":"10.1002/1361-6374(199709)5:3<153::AID-BIO9>3.3.CO;2-3","DOIUrl":"https://doi.org/10.1002/1361-6374(199709)5:3<153::AID-BIO9>3.3.CO;2-3","url":null,"abstract":"yOptoelectronic Imaging Group, Department of Applied Physics, Victoria Universityof Technology, PO Box 14428, MCMC, VIC 8001, AustraliazDepartment of Physical Optics, University of Sydney, NSW 2006, AustraliaSubmitted 9 January 1997, accepted 26 June 1997Abstract. The effect of the Fresnel diffraction on the imaging properties of confocalmicroscopy with one circular aperture and one annular aperture is investigated. Theresults show that the Fresnel diffraction patterns alter the axial imaging properties moreseriously than the transverse imaging properties. The axial response of such a confocalmicroscope is sharpened, but severely distorted.Keywords: Fresnel diffraction, annular pupil, confocal microscopy1. IntroductionAnnular aperture is a widely used spatial filter in confocalmicroscopy. It gives some improvement in transverseresolution, but a degradation in axial resolution appears ina confocal system with an annular structure [1,2]. Recentresearch suggests that the use of annular apertures as spatialfilters can suppress the scattered photons [3]. For example,a reflection-mode confocal system with an annular aperturein the illumination path is reported to show a significantenhancement in signal-to-noise ratio for imaging through ahighly scattering medium [4].In practice, an annular aperture cannot be placed exactlyat the back focal plane of an objective. In this case,Fresnel diffraction by the annular aperture exists. Fresneldiffraction of a finite-sized aperture can produce bright anddark fringes because of the interference of the waveletscontributed from points over the aperture [5,6]. Theintensity modulation caused by the interference fringes canalter the imaging properties of confocal microscopy, sincethe effective pupil function of the objective illuminatedby the Fresnel patterns changes accordingly, resulting inthe alteration of the intensity in the focal region [7–10].Therefore, it is important to investigate the effect of Fresneldiffraction on the imaging performance of a confocalsystem with an annular aperture.This paper is organized as follows. Section 2 presentsthe Fresnel diffraction of an annular aperture. The effectof Fresnel diffraction patterns of an annular aperture on theimaging properties of confocal microscopy is discussed insection 3.2. Fresnel diffraction of an annular apertureIf an aperture is placed in the back focal plane of anobjective then focusing is described by Debye theory. TheFresnel number is infinity in this situation. However, ifthe aperture is placed away from the back focal plane by adistance","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"48 1","pages":"153-158"},"PeriodicalIF":0.0,"publicationDate":"1997-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90263248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-09-01DOI: 10.1002/1361-6374(199709)5:3<134::AID-BIO7>3.3.CO;2-8
M. Kinjo, G. Nishimura
{"title":"Fluorescence correlation spectroscopy as a detection tool of point mutation in genes","authors":"M. Kinjo, G. Nishimura","doi":"10.1002/1361-6374(199709)5:3<134::AID-BIO7>3.3.CO;2-8","DOIUrl":"https://doi.org/10.1002/1361-6374(199709)5:3<134::AID-BIO7>3.3.CO;2-8","url":null,"abstract":"","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"11 1","pages":"134-138"},"PeriodicalIF":0.0,"publicationDate":"1997-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72646904","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-09-01DOI: 10.1002/1361-6374(199709)5:3<105::AID-BIO4>3.3.CO;2-H
C. Eggeling, L. Brand, C. Seidel
The efficiency of detecting a single fluorescent coumarin dye molecule in aqueous solution by one-photon excitation (OPE) at 350 nm as well as by coherent two-photon excitation (TPE) at 700 nm is studied. The photostability, which is crucial for single molecule detection (SMD), is determined at a low irradiance for various coumarin derivatives using a ‘cell-bleaching’ method. The yields of photobleaching for these coumarins in aqueous solution are in the order of 10−3 to 10−4. Thus, most of the dyes are sufficiently stable to allow SMD. However, for SMD in a fluorescence microscope a high quasi-CW irradiance (at least 104 W cm−2) is necessary for efficient OPE by a pulsed, frequency doubled titanium:sapphire laser. Detailed investigations on the dye Coumarin-120 using fluorescence correlation spectroscopy (FCS), different repetition rates of the laser and transient absorption spectroscopy (TRABS) gave clear evidence that OPE at a high irradiance results in two-step photolysis via the first electronic excited singlet and triplet state, S1 and T1, producing dye radical ions and solvated electrons. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Using coherent TPE for single molecule detection, saturation of the fluorescence was observed for a high quasi-CW irradiance (108 W cm−2), which may also be caused by photobleaching. Furthermore, TPE is deteriorated by other competing nonlinear processes (e.g. continuum generation in the solvent), which only occur above a threshold irradiance (7 × 107 W cm−2). Nevertheless, TPE allows an efficient detection of single Coumarin-120 molecules in water. Using a maximum likelihood estimator, we are also able to identify single dye molecules via their characteristic fluorescence lifetime of 4.8 ± 1.2 ns.
研究了350 nm单光子激发(OPE)和700 nm相干双光子激发(TPE)在水溶液中检测单个荧光香豆素染料分子的效率。光稳定性对单分子检测(SMD)至关重要，它是使用“细胞漂白”方法在低辐照度下测定各种香豆素衍生物的。这些香豆素在水溶液中光漂白的产率为10−3 ~ 10−4。因此，大多数染料都足够稳定，可以进行SMD。然而，对于荧光显微镜下的SMD，需要高准连续波辐照度(至少104 W cm−2)才能通过脉冲倍频钛:蓝宝石激光器实现有效的OPE。利用荧光相关光谱(FCS)、不同激光重复率和瞬态吸收光谱(TRABS)对染料香豆素-120进行了详细的研究，清楚地表明，在高辐照度下，OPE通过第一电子激发单重态和三重态S1和T1进行了两步光解，产生染料自由基离子和溶剂化电子。因此，这种额外的光漂白途径限制了OPE的适用辐照度。使用相干TPE进行单分子检测，在高准连续波辐照度(108 W cm−2)下观察到荧光饱和，这也可能是由光漂白引起的。此外，TPE还会受到其他非线性过程的影响(例如溶剂中连续统的产生)，这些过程仅发生在阈值辐照度(7 × 107 W cm−2)以上。然而，TPE可以有效地检测水中单个香豆素-120分子。使用最大似然估计器，我们还能够通过单个染料分子的特征荧光寿命(4.8±1.2 ns)来识别它们。
{"title":"Laser-induced fluorescence of coumarin derivatives in aqueous solution: Photochemical aspects for single molecule detection","authors":"C. Eggeling, L. Brand, C. Seidel","doi":"10.1002/1361-6374(199709)5:3<105::AID-BIO4>3.3.CO;2-H","DOIUrl":"https://doi.org/10.1002/1361-6374(199709)5:3<105::AID-BIO4>3.3.CO;2-H","url":null,"abstract":"The efficiency of detecting a single fluorescent coumarin dye molecule in aqueous solution by one-photon excitation (OPE) at 350 nm as well as by coherent two-photon excitation (TPE) at 700 nm is studied. The photostability, which is crucial for single molecule detection (SMD), is determined at a low irradiance for various coumarin derivatives using a ‘cell-bleaching’ method. The yields of photobleaching for these coumarins in aqueous solution are in the order of 10−3 to 10−4. Thus, most of the dyes are sufficiently stable to allow SMD. However, for SMD in a fluorescence microscope a high quasi-CW irradiance (at least 104 W cm−2) is necessary for efficient OPE by a pulsed, frequency doubled titanium:sapphire laser. Detailed investigations on the dye Coumarin-120 using fluorescence correlation spectroscopy (FCS), different repetition rates of the laser and transient absorption spectroscopy (TRABS) gave clear evidence that OPE at a high irradiance results in two-step photolysis via the first electronic excited singlet and triplet state, S1 and T1, producing dye radical ions and solvated electrons. Hence, this additional photobleaching pathway limits the applicable irradiance for OPE. Using coherent TPE for single molecule detection, saturation of the fluorescence was observed for a high quasi-CW irradiance (108 W cm−2), which may also be caused by photobleaching. Furthermore, TPE is deteriorated by other competing nonlinear processes (e.g. continuum generation in the solvent), which only occur above a threshold irradiance (7 × 107 W cm−2). Nevertheless, TPE allows an efficient detection of single Coumarin-120 molecules in water. Using a maximum likelihood estimator, we are also able to identify single dye molecules via their characteristic fluorescence lifetime of 4.8 ± 1.2 ns.","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"136 1","pages":"105-115"},"PeriodicalIF":0.0,"publicationDate":"1997-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87770679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-06-01DOI: 10.1002/1361-6374(199706)5:2<82::AID-BIO5>3.3.CO;2-6
J. Barron, A. Liptay
A method is presented for measuring 3-D plant growth using the optical flow computed on an image sequence of a growing corn seedling. Each image in the sequence consists of two views of the same seedling; one view of the corn seedling is front-on while the second view is a orthogonal view (at 90°) of the seedling made by projecting the plant's orthogonal image onto a mirror oriented at 45° with respect to the camera. We compute 3-D velocity (motion) of the corn seedling's tip by using a simple extension of the 2-D motion constraint equation used in optical flow analysis. This method is an extension of the work presented by Barron and Liptay where optical flow was used to measure the 2-D growth (in the vertical plane) of a corn seedling.
{"title":"Measuring 3‐D plant growth using optical flow","authors":"J. Barron, A. Liptay","doi":"10.1002/1361-6374(199706)5:2<82::AID-BIO5>3.3.CO;2-6","DOIUrl":"https://doi.org/10.1002/1361-6374(199706)5:2<82::AID-BIO5>3.3.CO;2-6","url":null,"abstract":"A method is presented for measuring 3-D plant growth using the optical flow computed on an image sequence of a growing corn seedling. Each image in the sequence consists of two views of the same seedling; one view of the corn seedling is front-on while the second view is a orthogonal view (at 90°) of the seedling made by projecting the plant's orthogonal image onto a mirror oriented at 45° with respect to the camera. We compute 3-D velocity (motion) of the corn seedling's tip by using a simple extension of the 2-D motion constraint equation used in optical flow analysis. This method is an extension of the work presented by Barron and Liptay where optical flow was used to measure the 2-D growth (in the vertical plane) of a corn seedling.","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"98 1","pages":"82-86"},"PeriodicalIF":0.0,"publicationDate":"1997-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88124097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-06-01DOI: 10.1002/1361-6374(199706)5:2<65::AID-BIO3>3.3.CO;2-5
G. Landini, J. Rigaut
{"title":"A method for estimating the dimension of asymptotic fractal sets","authors":"G. Landini, J. Rigaut","doi":"10.1002/1361-6374(199706)5:2<65::AID-BIO3>3.3.CO;2-5","DOIUrl":"https://doi.org/10.1002/1361-6374(199706)5:2<65::AID-BIO3>3.3.CO;2-5","url":null,"abstract":"","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"80 1","pages":"65-70"},"PeriodicalIF":0.0,"publicationDate":"1997-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76975500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-06-01DOI: 10.1002/1361-6374(199706)5:2<71::AID-BIO4>3.3.CO;2-D
M. Pitter, J. G. Walker
{"title":"The effect of spatial filtering on the performance of time‐of‐flight imaging systems","authors":"M. Pitter, J. G. Walker","doi":"10.1002/1361-6374(199706)5:2<71::AID-BIO4>3.3.CO;2-D","DOIUrl":"https://doi.org/10.1002/1361-6374(199706)5:2<71::AID-BIO4>3.3.CO;2-D","url":null,"abstract":"","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"211 1","pages":"71-81"},"PeriodicalIF":0.0,"publicationDate":"1997-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82828709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-03-01DOI: 10.1002/1361-6374(199703)5:1<9::AID-BIO2>3.3.CO;2-1
A. D. Scully, R. Ostler, D. Phillips, P. O'Neill, K. Townsend, A. Parker, A. MacRobert
The potential for the application of fluorescence lifetime imaging (FLIM) microscopy to studies of photosensitization mechanisms in photodynamic therapy (PDT) has been investigated. The fluorescence microscope incorporates a standard inverted optical microscope, a picosecond pulsed dye-laser excitation source, and an intensified CCD camera detector capable of being gated on a sub-nanosecond timescale. Fluorescence lifetime images resulting from multi-component analysis of sub-nanosecond gated fluorescence images of monolayer V79-4 Chinese hamster lung fibroblasts stained with disulphonated aluminium phthalocyanine (AlPcS2), a photosensitizer used in PDT, are presented. The results of these measurements are discussed in terms of the intracellular localization of the sensitizer. Preliminary results from multi-component FLIM of V79-4 cells multiply stained with AlPcS2 and a potential intracellular pH lifetime probe, 5(+6)-carboxynaphthofluorescein, are also presented.
{"title":"Application of fluorescence lifetime imaging microscopy to the investigation of intracellular PDT mechanisms","authors":"A. D. Scully, R. Ostler, D. Phillips, P. O'Neill, K. Townsend, A. Parker, A. MacRobert","doi":"10.1002/1361-6374(199703)5:1<9::AID-BIO2>3.3.CO;2-1","DOIUrl":"https://doi.org/10.1002/1361-6374(199703)5:1<9::AID-BIO2>3.3.CO;2-1","url":null,"abstract":"The potential for the application of fluorescence lifetime imaging (FLIM) microscopy to studies of photosensitization mechanisms in photodynamic therapy (PDT) has been investigated. The fluorescence microscope incorporates a standard inverted optical microscope, a picosecond pulsed dye-laser excitation source, and an intensified CCD camera detector capable of being gated on a sub-nanosecond timescale. Fluorescence lifetime images resulting from multi-component analysis of sub-nanosecond gated fluorescence images of monolayer V79-4 Chinese hamster lung fibroblasts stained with disulphonated aluminium phthalocyanine (AlPcS2), a photosensitizer used in PDT, are presented. The results of these measurements are discussed in terms of the intracellular localization of the sensitizer. Preliminary results from multi-component FLIM of V79-4 cells multiply stained with AlPcS2 and a potential intracellular pH lifetime probe, 5(+6)-carboxynaphthofluorescein, are also presented.","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"147 1","pages":"9-18"},"PeriodicalIF":0.0,"publicationDate":"1997-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85620062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-03-01DOI: 10.1002/1361-6374(199703)5:1<1::AID-BIO1>3.3.CO;2-S
P. C. Ke, X. Gan, J. Szajman, S. Schilders, M. Gu
{"title":"Optimizing the strength of an evanescent wave generated from a prism coated with a double‐layer thin‐film stack","authors":"P. C. Ke, X. Gan, J. Szajman, S. Schilders, M. Gu","doi":"10.1002/1361-6374(199703)5:1<1::AID-BIO1>3.3.CO;2-S","DOIUrl":"https://doi.org/10.1002/1361-6374(199703)5:1<1::AID-BIO1>3.3.CO;2-S","url":null,"abstract":"","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"1 1","pages":"1-8"},"PeriodicalIF":0.0,"publicationDate":"1997-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89963707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1997-03-01DOI: 10.1002/1361-6374(199703)5:1<40::AID-BIO4>3.3.CO;2-N
Z. Kam, D. Agard, J. Sedat
A modified optical system for the light microscope has been devised in order to remotely shift the focal plane and to manipulate the point spread function for any given objective lens. An adjustable telescope system is inserted into the microscope tube so as to move the intermediate image position, thus achieving two goals of fundamental importance for the three-dimensional imaging of biological samples. First, it allows the focus to be rapidly varied without actually moving the objective lens. This permits high throughput three-dimensional microscopy of living specimens. Secondly, it makes possible the compensation of objective lens spherical aberration. This distortion is especially significant when high numerical aperture objectives are utilized to image deep into thick specimens.
{"title":"Three‐dimensional microscopy in thick biological samples: A fresh approach for adjusting focus and correcting spherical aberration","authors":"Z. Kam, D. Agard, J. Sedat","doi":"10.1002/1361-6374(199703)5:1<40::AID-BIO4>3.3.CO;2-N","DOIUrl":"https://doi.org/10.1002/1361-6374(199703)5:1<40::AID-BIO4>3.3.CO;2-N","url":null,"abstract":"A modified optical system for the light microscope has been devised in order to remotely shift the focal plane and to manipulate the point spread function for any given objective lens. An adjustable telescope system is inserted into the microscope tube so as to move the intermediate image position, thus achieving two goals of fundamental importance for the three-dimensional imaging of biological samples. First, it allows the focus to be rapidly varied without actually moving the objective lens. This permits high throughput three-dimensional microscopy of living specimens. Secondly, it makes possible the compensation of objective lens spherical aberration. This distortion is especially significant when high numerical aperture objectives are utilized to image deep into thick specimens.","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"36 1","pages":"40-49"},"PeriodicalIF":0.0,"publicationDate":"1997-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86785647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bio-speckle flowmetry for measuring the retinal blood flow velocity is described. The measuring principle is briefly discussed in comparison with the laser Doppler method. The basic properties of the photon correlation measurements including reproducibility were experimentally investigated with a rotating ground glass disk and for the normal human retina. The error was estimated to be less than 20% for in vivo measurements. By using a glass capillary model, the reciprocal of correlation time was calibrated to the mean flow velocity with a consideration of the effects of the vessel diameter and the background reflectance. The blood flow volume rate in the human retina was estimated by using the calibrated velocity and the vessel diameter. The results compared well with those reported in the literature, and show the usefulness of this flowmetry for clinical diagnostic purposes.
{"title":"Bio-speckle flowmetry for retinal blood flow diagnostics","authors":"Yoshihisa Aizu, Toshimitsu Asakura, Kouji Ogino, Toshiaki Sugita, Yasuyuki Suzuki, Kanjiro Masuda","doi":"10.1002/1361-6374(199612)4:4<254::AID-BIO4>3.0.CO;2-7","DOIUrl":"https://doi.org/10.1002/1361-6374(199612)4:4<254::AID-BIO4>3.0.CO;2-7","url":null,"abstract":"<p>Bio-speckle flowmetry for measuring the retinal blood flow velocity is described. The measuring principle is briefly discussed in comparison with the laser Doppler method. The basic properties of the photon correlation measurements including reproducibility were experimentally investigated with a rotating ground glass disk and for the normal human retina. The error was estimated to be less than 20% for <i>in vivo</i> measurements. By using a glass capillary model, the reciprocal of correlation time was calibrated to the mean flow velocity with a consideration of the effects of the vessel diameter and the background reflectance. The blood flow volume rate in the human retina was estimated by using the calibrated velocity and the vessel diameter. The results compared well with those reported in the literature, and show the usefulness of this flowmetry for clinical diagnostic purposes.</p>","PeriodicalId":100176,"journal":{"name":"Bioimaging","volume":"4 4","pages":"254-267"},"PeriodicalIF":0.0,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/1361-6374(199612)4:4<254::AID-BIO4>3.0.CO;2-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72338567","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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