1,721,087 research outputs found
Time multiplexing and parallelization in multifocal multiphoton microscopy
No full textWe investigate the imaging properties of high-aperture multifocal multiphoton microscopy on the basis of diffraction theory. Particular emphasis is placed on the relationship between the sectioning property and the distance between individual foci. Our results establish a relationship between the degree of parallelization and the axial resolution for both two- and three-photon excitation. In addition, we show quantitatively that if a matrix of temporal delays is inserted between the individual foci, it is, for the first time to our knowledge, possible to solve the classical conflict between the light budget and the sectioning property in three-dimensional microscopy and to provide a virtually unlimited density of foci at best axial resolution
Time-multiplexed multifocal multiphoton microscope
No full textWe resolve the classical conflict between parallelization and axial resolution in three-dimensional fluorescence microscopy through time-multiplexed multifocal multiphoton excitation. A rotating array of microlenses on a disk splits ultrafast laser pulses in such a way that an array of high-aperture foci are created in the sample. Two rigidly mounted corotating glass disks with suitable arrays of holes ensure that adjacent foci illuminate the sample at different lime points. Recordings of biological specimens demonstrate elimination of out-of-focus haze for densely packed foci and concomitant substantial improvement of contrast and resolution
ISM-Assisted Tomographic STED Microscopy Allows for Sample-Gentle Superresolution Imaging
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Modern Statistical Challenges in High-Resolution Fluorescence Microscopy
No full textConventional light microscopes have been used for centuries for the study of small length scales down to approximately 250 nm. Images from such a microscope are typically blurred and noisy, and the measurement error in such images can often be well approximated by Gaussian or Poisson noise. In the past, this approximation has been the focus of a multitude of deconvolution techniques in imaging. However, conventional microscopes have an intrinsic physical limit of resolution. Although this limit remained unchallenged for a century, it was broken for the first time in the 1990s with the advent of modern superresolution fluorescence microscopy techniques. Since then, superresolution fluorescence microscopy has become an indispensable tool for studying the structure and dynamics of living organisms. Current experimental advances go to the physical limits of imaging, where discrete quantum effects are predominant. Consequently, this technique is inherently of a non-Gaussian statistical nature, and we argue that recent technological progress also challenges the long-standing Poisson assumption. Thus, analysis and exploitation of the discrete physical mechanisms of fluorescent molecules and light, as well as their distributions in time and space, have become necessary to achieve the highest resolution possible. This article presents an overview of some physical principles underlying modern fluorescence microscopy techniques from a statistical modeling and analysis perspective. To this end, we develop a prototypical model for fluorophore dynamics and use it to discuss statistical methods for image deconvolution and more complicated image reconstruction and enhancement techniques. Several examples are discussed in more detail, including variational multiscale methods for confocal and stimulated emission depletion (STED) microscopy, drift correction for single marker switching (SMS) microscopy, and sparse estimation and background removal for superresolution by polarization angle demodulation (SPoD). We illustrate that such methods benefit from advances in large-scale computing, for example, from recent tools from convex optimization. We argue that in the future, even higher resolutions will require more detailed models that delve into sub-Poissonian statistics
Fluorescence microscopy with super-resolved optical sections
No full textThe fluorescence microscope, especially its confocal variant, has become a standard tool in cell biology research for delivering 3D-images of intact cells. However, the resolution of any standard optical microscope is at least 3 times poorer along the axis of the lens that in its focal plane. Here, we review principles and applications of an emerging family of fluorescence microscopes, such as Vi microscopes,which improve axial resolution by a factor of seven by employing two opposing lenses. Noninvasive axial sections of 80-160 nm thickness deliver more faithful 3D-images of subcellular features, providing a new opportunity to significantly enhance our understanding of cellular structure and function
Nanoscale photonic imaging
Full text linkThis open access book, edited and authored by a team of world-leading researchers, provides a broad overview of advanced photonic methods for nanoscale visualization, as well as describing a range of fascinating in-depth studies. Introductory chapters cover the most relevant physics and basic methods that young researchers need to master in order to work effectively in the field of nanoscale photonic imaging, from physical first principles, to instrumentation, to mathematical foundations of imaging and data analysis. Subsequent chapters demonstrate how these cutting edge methods are applied to a variety of systems, including complex fluids and biomolecular systems, for visualizing their structure and dynamics, in space and on timescales extending over many orders of magnitude down to the femtosecond range. Progress in nanoscale photonic imaging in Göttingen has been the sum total of more than a decade of work by a wide range of scientists and mathematicians across disciplines, working together in a vibrant collaboration of a kind rarely matched. This volume presents the highlights of their research achievements and serves as a record of the unique and remarkable constellation of contributors, as well as looking ahead at the future prospects in this field. It will serve not only as a useful reference for experienced researchers but also as a valuable point of entry for newcomers
Equivalence of the Huygens-Fresnel and Debye approach for the calculation of high aperture point-spread-functions in the presence of refractive index mismatch.
No full textAs discussed in recent work (Sheppard, C. J. R. & Torok, P., I. Microsc.. 185, 366-384; Torok et al., J. Microsc., 188, 158-172), two approaches have been used extensively for vectorial computations of high aperture confocal point-spread functions when focusing through a dielectric interface. Whereas the equation by Hell, Reiner, Cremer & Stelzer (I. Microsc., 169, 391-405) is based on the Huygens-Fresnel principle, the more recent approach by Torok. Varga Br Booker (J. Opt. Sec. Aln. A, 12, 325-332; J. Opt. SOC. Am. A, 12, 2136-2144) is based on the Debye approximation. While the earlier theory considers a large but finite focal length the second theory is derived for an infinitely high Fresnel number, In a high aperture microscope, a high Fresnel number is equivalent to assuming that the focal length be infinitely large with respect to the wavelength. So far, the two theories are regarded as different, with the one by Torok et al, being rigorous, In this paper, we demonstrate that, if the same conditions are applied, the equation by Torok et ttl, can be analytically derived from that by Hell et nl. Producing the same results, the benefit brought about by the equation by Torok el nl. is improved flexibility and computational speed for cases with azimuthal symmetry
Automatic deconvolution in 4Pi-microscopy with variable phase
No full text4Pi-microscopy doubles the aperture of the imaging system by coherent addition of the wavefronts for illumination and/or detection through opposing objective lenses. This improves the axial resolution 3-7 fold, but the raw data usually features ghost images which have to be removed by image reconstruction. This straightforward procedure is sometimes precluded by imperfect alignment of the instrument or a specimen with strong variations of its refractive index, because the image formation process now depends on the space-variant phase difference between the counter-propagating wavefronts. Here we present a computationally fast method of parametric blind deconvolution that allows for automatic and robust simultaneous estimation of both the object and the phase function in such cases. We verify the performance of our approach on both synthetic and real data. Because the method does not require a-priori knowledge of the phase function it is a major step towards reliable 4Pi-imaging and automatic image restoration by non-expert users
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