An analysis is presented of the image formation in widefield fluorescence microscopy using standard light. The region of support of the resulting optical transfer function is discussed.
ISBN:
(纸本)0819427004
An analysis is presented of the image formation in widefield fluorescence microscopy using standard light. The region of support of the resulting optical transfer function is discussed.
This paper describes a method for the improvement of biological images acquired using a Transmission Electronic Microscope (TEM). Several techniques are presented that deal with noise reduction, artifact removal and n...
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ISBN:
(纸本)0819427004
This paper describes a method for the improvement of biological images acquired using a Transmission Electronic Microscope (TEM). Several techniques are presented that deal with noise reduction, artifact removal and non-uniform illumination correction. Experimental results are shown.
We introduce an optical means of reconstructing the topology of rough objects observed under the microscope or at a relatively high magnification. First, 2D images were obtained under the conventional optical microsco...
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ISBN:
(纸本)0819427004
We introduce an optical means of reconstructing the topology of rough objects observed under the microscope or at a relatively high magnification. First, 2D images were obtained under the conventional optical microscope or under the macroscopic imaging system, focused on differing height of the sample. The depth information was extracted by sensing the focus of each pixel of the conventional image slice. Each sample height associated with the best "focus figure" for each pixel was marked and were later utilized to generate the threedimensional coordinate map.
We describe a method of obtaining optically sectioned fluorescence images in a widefield conventional microscope by interfering two beams on an object so as to illuminate it with a single spatial frequency fringe patt...
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ISBN:
(纸本)0819427004
We describe a method of obtaining optically sectioned fluorescence images in a widefield conventional microscope by interfering two beams on an object so as to illuminate it with a single spatial frequency fringe pattern. images taken at three spatial positions of the fringe pattern are processed in real time to produce optically sectioned images which are substantially similar to those obtained with confocal microscopes.
In this work we discuss the role of polarisation in confocal microscopy. The effect of the optical system on light polarisation is analysed in the absence of a specimen. We also present numerical results on confocal m...
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ISBN:
(纸本)0819427004
In this work we discuss the role of polarisation in confocal microscopy. The effect of the optical system on light polarisation is analysed in the absence of a specimen. We also present numerical results on confocal microscopes imaging small dielectric scatterers and suggest an optimum pinhole size for crossed linear polars. Finally, imaging of large spherical scatterers and contrast optimisation of the optical system is discussed.
In previous studies, we examined three-dimensionalimages of a latex bead containing a surface layer of fluorescence whose thickness was determined by physical sectioning. Confocal images of the bead from six differen...
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ISBN:
(纸本)0819427004
In previous studies, we examined three-dimensionalimages of a latex bead containing a surface layer of fluorescence whose thickness was determined by physical sectioning. Confocal images of the bead from six different microscopes all exhibited a significantly thicker fluorescent shell than actually present. In contrast, deconvolved wide-field images of the bead produced an image with the correct shell thickness. We have now repeated some of these studies using a new latex bead containing a much thinner layer of surface fluorescence. In contrast to earlier studies, confocal images of this bead appear to show an accurate thickness for the fluorescent shell. These particular confocal microscopes (a Biorad 1024 and a Zeiss 510) also yielded essentially aberration free images, which was not the case for some of the earlier microscopes tested. In these recent studies however, deconvolution of the bead appeared less robust than earlier. Of three independent wide-held images of the bead, only one yielded a substantially artifact-free image upon deconvolution.
Imaging thick specimens in 3D transmission confocal modes presents two key problems. The first problem is variable aberrations introduced by changes in refractive index. The second problem is revealed when visualising...
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ISBN:
(纸本)0819427004
Imaging thick specimens in 3D transmission confocal modes presents two key problems. The first problem is variable aberrations introduced by changes in refractive index. The second problem is revealed when visualising acquired data, where thick 3D datasets are difficult to interpret. In this paper we present our emerging solutions to these problems. Aberrations can be classified as simple tip-tilt deflection of the beam, or more complicated higher order aberrations. We discuss our results which demonstrate successful on-the-fly detection and correction for tip-tilt For detecting higher order aberrations, we have chosen to investigate the wavefront curvature sensing technique. The second problem of rendering thick 3D datasets can be solved by extracting features of interest from the background. Simple intensity threshholding is not sufficient for complex biological specimens, and imageprocessing in only two dimensions neglects any threedimensional structure. Use of Kohonen's self-organising map neural network in 3D results in clear segmentation of features for sample chromosome specimens.
Tomographic measurements of the 3D refractive index spatial distribution within optically transparent phase samples with computerized interferometric microscopes are proposed. Phase shifting interferometric microtomog...
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ISBN:
(纸本)0819427004
Tomographic measurements of the 3D refractive index spatial distribution within optically transparent phase samples with computerized interferometric microscopes are proposed. Phase shifting interferometric microtomography applications for the 3D image reconstruction of the blood cells (lymphocytes) are represented. The immersion 100x, N.A.=1.25 objective was used to increase the spatial resolution and observation angle range to 90 degree. ART, combined ART and iterative Gerchberg-Papoulis 3D algorithm were used for the tomogram reconstruction. To determine the accuracy and spatial resolution of the blood cells image reconstruction by means of the interferometric microtomographic method the numerical simulations were implemented.
In our lab have been developed an atomic force microscope (AFIM) for biological and technical applications with improved optical method for measuring of cantilever deflection. We use conventional technique of measurin...
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ISBN:
(纸本)0819427004
In our lab have been developed an atomic force microscope (AFIM) for biological and technical applications with improved optical method for measuring of cantilever deflection. We use conventional technique of measuring the change in angle of light reflected off the cantilever with an additional lens placed between cantilever and four-segment photodiode, The lens forms an image of the cantilever in the photodiode plane. Small sizes of the cantilever image improve the resolution of AFM and reduce the requirements to the optical scheme of the microscope. The lens non-linear transmission of the cantilever deflection is compensated by means of computer program. With the A FM were imaged the DNA of plague microbes (Y. pestis EV) and phages of plague and V. Cholera.
imageacquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in c...
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ISBN:
(纸本)0819427004
imageacquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in confocal laser scanning microscopy (CLSM). The system consists of a 4 channel Leica CLSM equipped with a high resolution z-scanning stage (Leica Lasertechnik) mounted on a xy-motorized stage (Marzhauser). The 3D-MISA software is implemented into the microscope scanning software (Scanware, Leica Lasertechnik) and uses the microscope settings (magnification, zoom) for the movements of the xy-stage. It allows storage and recall of 70 xyz-positions and the automatic 3D-scanning of image arrays between selected xyz-coordinates. The number of images within one array is limited only by the amount of disk space or memory available. Although for most applications the accuracy of the xy-scanning stage is sufficient for a precise alignment of tiled views, the software provides the possibility of an adjustable overlap between two image stacks by shifting the moving steps of the xy-scanning stage. After scanning a tiled image gallery of the extended focus-images of each channel will be displayed on a graphic monitor. In addition, a tiled image gallery of individual focal planes can be created. In summary, the 3D-MISA allows 3D-imageacquisition of coherent regions in combination with high resolution of single images.
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