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Programmable diffractive optics for laser scanning confocal microscopy

Programmable diffractive optics for laser scanning confocal ...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Boruah, B. R. Neil, M. A. A. Imperial Coll London Dept Phys Blackett Lab Photon Grp Prince Consort Rd London SW7 2BW England

ISBN: (纸本)9780819465566

In this work, we describe the design and implementation of a laser scanning confocal microscope with active beam forming optics. We demonstrate dynamic control over intensity and polarization properties of the beam using the technique of programmable diffractive optics. This technique is used facilitate active aberration correction in the beam and to generate radially polarized pupil function in order to get an on-axis axially polarized point spread function. We also develop the high numerical aperture theory to calculate the focal point spread function for a radially polarized pupil function. We describe the design and implementation of a simple vector beam formation unit consisting of a polarizing beam splitter and two right angled prisms in conjunction with a ferro-electric spatial light modulator. We also describe the design and implementation of a beam scanning system comprising of a novel off-axis scanner mirror that maintains registration of the conjugate pupil planes in the system.

关键词： diffractive optics spatial light modulator laser scanning confocal microscope radially polarized beam

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Large-area sub-pixel resolution imaging using a spatial light modulator

Large-area sub-pixel resolution imaging using a spatial ligh...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Ballestad, Anders McFadden, Brian Lane, Pierre McKinnon, Nick MacAulay, Calum Constantinou, Paul Wilson, Brian Yeow, John British Columbia Canc Res Ctr Dept Canc Imaging Vancouver BC V5Z 1L3 Canada Ontario Canc Ctr Toronto ON Canada Univ Waterloo Waterloo ON Canada

ISBN: (纸本)9780819465566

We report results from a proof-of-principle study investigating a technique for high-resolution imaging of large fields of view (FOV). This is achieved through structured illumination of the sample from a laterally replicated spatial light modulator (SLM). By incorporating the SLM into the illumination path of an otherwise conventional microscopy imaging system, we can perform the sampling by using our illumination source instead of our areal detector (camera). The increased resolution is achieved through anti-binning or splitting of the charge-coupled device (CCD) pixels, and the extended FOV is obtained by a lateral replication technique applied to the whole illumination field. With anti-binning, we effectively exceed the sampling resolution limit set by the Nyquist theorem. Also, our lateral replication technique enables us to maintain the same FOV for the increased resolution without the need for adaptive optics or highly corrected lenses far from the optical axis. The two techniques of resolution enhancement and lateral replication of the illumination field could be employed independently, hence offering increased versatility and adaptability for specialized imaging applications. Different imaging modes can be accessed digitally, without the need to change objectives, stitch together individual frames, or move the sample. The resulting imaging modality of this system is quasi-confocal.

关键词： Charge-coupled devices Spatial light modulators image resolution Imaging systems Mirrors Adaptive optics Digital micromirror devices Relays Resolution enhancement technologies microscopy

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Soft computing approach to confocal and two-photon excitation microscopy

Soft computing approach to confocal and two-photon excitatio...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Vicidomini, Giuseppe Mondal, Partha P. Diaspro, Alberto Univ Genoa Dept Phys LAMBS MicroSCoBiO I-16146 Genoa Italy Univ Genoa Dept Comp Sci I-16146 Genoa Italy Abdus Salaam Int Ctr Theoret Phys I-34014 Trieste Italy FIRC Inst Mol Oncol Fdn IFOM I-20139 Milan Italy CNRS Inst Biophys I-16149 Genoa Italy

ISBN: (纸本)9780819465566

Confocal laser scanning (CLS) and two-photon excitation (TPE) microscopy are powerful techniques for 3D imaging of biological samples. Although CLS and TPE microscopy images are better than standard epifluorescence images, they still undergo degradation due to blurring and random noise because of the inherent nature of the physical phenomenon (diffraction and photon counting noise) involved. The aim is to obtain the real object from the degraded noisy image. This problem belongs to inverse problems and is found to be very notorious in nature. Several algorithms such as maximum likelihood (ML) based algorithm, have been proposed to reduce these artifacts. Unfortunately, ML based algorithm tends to generate noise artifacts, so regularization constraints based on some prior knowledge have to be integrated to stabilize the solution. This is termed as maximum a-posteriori (MAP) technique. We propose a MAP approach in which the image field is suitably modeled as Markov random field (MRF), forcing the image distribution to be Gibbs distribution. The prior knowledge is incorporated through the potential function in the Gibbs distribution. We proposed potential functions based on white-noise prior, smoothest prior and fuzzy logic. MAP approach has the advantage of include the available prior knowledge in the restoration procedure. In other words, inclusion of prior knowledge makes the notorious inverse problem well-posed. Various evaluations such as visual inspection and Csiszar I-divergence are performed on the CLS microscopy restored images to study the characteristics of the proposed approach (in both simulated and real data). It is observed that the noise artifacts are considerably reduced and the desired images characteristics (edges and minute features as islets) are retained in the restored images. The algorithm is extended in the third dimension for 3D-image restoration application. The proposed algorithm is found to perform better than existing image restoration alg

关键词： confocal microscopy two-photon excitation microscopy maximum a-posteriori approach FuzzyLogic image-restoration edge-preservation

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Mie scattering model for dual-axes confocal architecture

Mie scattering model for dual-axes confocal architecture

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Wong, Larry K. Mandella, Michael J. Holcomb, Paul Kino, Gordon S. Wang, Thomas D. Stanford Univ Sch Med Div Gastroenterol Stanford CA 94305 USA Stanford Univ Ginzton Lab Stanford CA 94305 USA Breault Res Org Inx Tucson AZ 85715 USA

ISBN: (纸本)9780819465566

Tissue scattering has a significant effect on the image resolution and light collection efficiency in confocal microscopy. The dual-axes (DA) confocal architecture has many advantages including high axial resolution with low numerical aperture lenses and long working distance for use in vivo as a microendoscope. In addition, less scattered light along the illumination path may be collected and introduced as noise. In this paper, we use Monte Carlo tissue scattering simulations to compare the dual-axes and conventional single-axis (SA) configurations. Simulation results show that the axial response for the dual axes configuration varies with pinhole size and optical thickness of scattering media in a way that differs from the single axis architecture. The DA configuration is able to filter out efficiently multiply-scattered photons and out-of-focus light, thus allowing imaging with greater tissue penetration depths to provide vertical cross-sectional images, which has significant implications for in vivo imaging.

关键词： scattering Mie theory confocal microscopy single-axis dual-axes Monte Carlo

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Multi-wavelength Digital Holographic microscopy for sub-micron topography of reflecting specimens

Multi-wavelength Digital Holographic Microscopy for sub-micr...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Montfort, Frederic Charriere, Florian Kuehn, Jonas Colomb, Tristan Cuche, Etienne Emery, Yves Marquet, Pierre Depeursinge, Christian Ecole Polytech Fed Lausanne Imaging & Appl Opt Inst CH-1015 Lausanne Switzerland Lyncee Tec SA PSE A CH-1015 Lausanne Switzerland CHUV Ctr Neurosci Psychiat DP CH-1008 Prilly Switzerland

ISBN: (纸本)9780819465566

The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording at different wavelengths equally separated in the k-domain, of the interference between an off-axis reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefronts. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and a destructive one in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.

关键词： digital holography topography 3D imaging

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Signal post processing in frequency domain OCT and OCM using a filter bank approach

Signal post processing in frequency domain OCT and OCM using...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Hofer, Bernd Povazay, Boris Hermann, Boris Unterhuber, Angelika Matz, Gerald Hlawatsch, Franz Drexler, Wolfgang Cardiff Univ Dept Optometry & Vis Sci Biomed Imaging Grp Cardiff Wales Vienna Univ Technol Inst Commun & Radio Frequency Engn Vienna Austria

ISBN: (纸本)9780819465566

Current signal post processing in spectrally encoded frequency domain (FD) optical coherence microscopy (OCM) and optical coherence tomography (OCT) uses Fourier transforms in combination with non-uniform resampling strategies to map the k-space data acquired by the spectrometer to spatial domain signals which are necessary for tomogram generation. We propose to use a filter bank (FB) framework for the remapping process. With our new approach, the spectrometer is modeled as a critically sampled analysis FB, whose outputs are quantized subband signals that constitute the k-space spectroscopic data. The optimal procedure to map this data to the spatial domain is via a suitably designed synthesis FB which has low complexity. FB theory additionally states that 1) it is possible to find a synthesis FB such that the overall system has the perfect reconstruction (PR) property;2) any processing on critically sampled subband signals (as done in current schemes) results in aliasing artifacts. These perspectives are evaluated both theoretically and experimentally. We determine the analysis FB corresponding to our FD-OCM system by using a tunable laser and show that for our grating-based spectrometer - employing a CCD-line camera - the non-uniform resampling together with FFT indeed causes aliasing terms and depth dependent signal attenuation. Furthermore, we compute a finite impulse response based synthesis FB and assess the desired PR property by means of layered samples. The resulting images exhibit higher resolution and improved SNR compared to the common FFT-based approach. The potential of the proposed FB approach opens a new perspective also for other spectroscopic applications.

关键词： filter bank spectrometer spectroscopy optical coherence microscopy optical coherence tomography frequency domain tunable lasers fourier transform aliasing FFT

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Full-pupil versus divided-pupil confocal line-scanners for reflectance imaging of human skin in vivo

Full-pupil versus divided-pupil confocal line-scanners for r...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XIV

作者： Gareau, Dan Abeytunge, Sanjeewa Rajadhyaksha, Milind Mem Sloan Kettering Canc Ctr 1275 York Ave New York NY 10021 USA

ISBN: (纸本)9780819465566

A full-pupil confocal line-scanning microscope is under development for imaging human skin in vivo in reflectance. The new design potentially offers an alternative to current point- and line-scanners that may simplify the optics, electronics and mechanics, and lead to simpler and smaller confocal microscopes. With a combination of a cylindrical lens and an objective lens, the line-scanner creates a focused line of laser light in the object plane within tissue. An oscillating galvanometric mirror scans the focused line transverse to its axis. The backscattered light from the tissue is de-scanned and focused onto a linear CMOS detector array. Preliminary measurements of the axial line-spread function, with a 30x, 0.9-NA water immersion objective lens and illumination wavelength of 633 mn, determined the optical sectioning to be 10 pm. The new design is simple, requiring only eight optical components. However, the disadvantage is non-confocality in one dimension that results in 20% weaker sectioning than with a pointscanner, and reduced contrast in scattering tissue. The images of standard reflective targets such as a mirror and grating as well as dermis-like scattering target such as paper offer a preliminary glimpse into the performance of the line-scanner. A similar alternative design is the divided-pupil (theta) line-scanner, which provides 50% weaker sectioning than with a point scanner, but better contrast and less speckle due to the theta configuration. Such line scanners may prove useful for routine imaging of humans in clinical settings.

关键词： Confocal microscopy Skin Objectives Reflectivity In vivo imaging Mirrors Tissue optics Scanners Beam splitters Chromatic aberrations

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acquisition of three-dimensional coordinates of objects from axi-vision image data and application to stereoscopic display using integral photography system

Acquisition of three-dimensional coordinates of objects from...

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9th Videometrics conference

作者： Aida, Tahito Yoneda, Akira Uragaki, Takeshi Kawakita, Masahiro Osaka City Univ Grad Sch Engn Sumiyoshi Ku 3-3-138 Sugimoto Osaka 5588585 Japan Japan Broadcasting Corp NHK Sci & Tech Res Labs Setagaya Ku Tokyo 1578510 Japan

ISBN: (纸本)9780819466044

Versatile three-dimensional (3D) image data as in the form of I(X, Y, Z) defined in the camera coordinate system are acquired from sets of color image and depth image, which are simultaneously captured at a video frame rate by a 3D camera named Axi-Vision camera. The X and Y coordinates are calculated from the image coordinates (x, y) on the image plane of CCD based on the perspective projection theory. The Z coordinate can be obtained for each pixel of the color image directly from the depth image without any complex processing. Thus, the 3D image data of objects, I(X, Y, Z), is obtained from the relation between the camera coordinate and the image coordinate systems. A stereoscopic video image display is demonstrated using the 3D image data using an integral photography (IP) system, which combines a 4-inch VGA liquid crystal color display panel and a pinhole array. Aliasing, an issue from mismatch of image qualities between the 3D image data and the IP system, is effectively suppressed by pre-processing the color Axi-Vision image data using a two-dimensional lowpass filter, which is designed based on analyses of the maximum spatial frequency of the 3D image transformed appropriate for the LCD size and the Nyquist spatial frequency of the IP system.

关键词： axi-vision camera three-dimensional image data integral photography stereoscopic display

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GPU-based visualization techniques for 3D microscopic imaging data

GPU-based visualization techniques for 3D microscopic imagin...

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conference on Computational Imaging V

作者： Wang, Qiqi Sun, Yinlong Robinson, J. Paul Purdue Univ Dept Comp Sci W Lafayette IN 47907 USA Purdue Univ Dept Biomed Engn W Lafayette IN 47907 USA

ISBN: (纸本)9780819466112

Three-dimensional (3D) microscopic imaging techniques such as confocal microscopy have become a common tool in measuring cellular structures. While computer volume visualization has advanced into a sophisticated level in medical applications, much fewer studies have been made on data acquired by the 3D microscopic imaging techniques. To optimize the visualization of such data, it is important to consider the data characteristics such as thin data volume. It is also interesting to apply the new GPU (graphics processing unit) technology to interactive volume rendering of the data. In this paper, we discuss several texture-based techniques to visualize confocal microscopy data by considering the data characteristics and with support of GPU. One simple technique generates one set of 2D textures along the axial direction of image acquisition. An improved technique uses three sets of 2D textures in the three principal directions, and creates the rendered image via a weighted sum of the images generated by blending the individual texture sets. In addition, we propose a new approach based on stencil such that textures are blended based on a stencil control. Given the viewing condition, a texel needs to be drawn only when its corresponding projection on the image plane is inside a stencil area. Finally, we have explored the use of multiple-channel datasets for flexible classification of objects. These studies are useful to optimize the visualization of 3D microscopic imaging data.

关键词： volume visualization interactive rendering GPU-based techniques texture blending stencil techniques 3D microscopic imaging confocal microscopy

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Evaluation of a new method for the determination of experimental PSF of a wide-field microscope using white-light and a linear sensor

Evaluation of a new method for the determination of experime...

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conference on Confocal, Multiphoton and Nonlinear Microscopic Imaging III

作者： Macedo, Milton P. Barata, Antonio J. Fernandes, Ana G. Correia, Carlos M. ISEC P-3030199 Coimbra Portugal Univ Coimbra CEI P-3004516 Coimbra Portugal

ISBN: (纸本)9780819467744

In the biology field there is a high demand for three-dimensional (3D) microscopy techniques owing to the need for obtaining depth information. Confocal microscopy is the reference method but it has the drawback of slow image acquisition time. Different configurations were developed to obviate this such as spinning-disk that basically consists in a set of parallel confocal microscopes and line scanning that uses line illumination so the scanning in the fast axis is not required. Amongst other 3D techniques there is the digital microscopy that normally uses a wide-field microscope to acquire 2D images (optical sections) of the specimen containing substantial contributions from out-of-focus portions. To remove this, a computational method is used that is based on the definition of a model for the process of image formation and recording. A compromise should be taken between a more accurate model with best results or a simplified one with a lower computational processing time. This work is based on a laboratory prototype of a wide-field microscope with white-light illumination using a CMOS linear image sensor. It aims at the application of one simplified model that takes advantage of a new method for the determination of experimental point spread function (PSF) that uses a specimen consisting of a set of lines in two-perpendicular directions due to the anisotropy that results from detector geometry. The 1D and 2D PSF were built. A simulation of the application of the model was developed using Matlab ((Mathworks (c)) using different simulated specimens. Its preliminary results will be presented.

关键词： PSF image formation linear sensor confocal microscopy convolution deconvolution

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