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High resolution imaging in three dimension is important for biological research. The RESOLFT (Reversible Saturable Optical Fluorescence Transitions) fluorescent microscopy is one technique which can achieve lateral super-resolution imaging. Two-photon microscopy naturally generate high resolution in the longitudinal direction with less background compared to single photon excitation. We combine these two methods to realize three-dimensional high-resolution imaging. This super-resolution method also is limited in imaging speed. We use a spatial light modulator (SLM) as a flexible phase mask of the microscopy. It is used to compensate the system aberration, as well as increasing the imaging speed. The parallel scanning generates multiple super-resolution focuses as an array or in arbitrary positions by phase retrieval calculation. This microscopy combined with SLM control could applied to high throughput 3D imaging or multiple spots tracking in high-resolution.
Super Resolution Optical Imaging and Microscopy Extremely comprehensive resource containing cutting-edge and practical knowledge of super-resolution optical imaging This book covers both the basic principles and specific technical details of super-resolution microscopy techniques. It covers the criteria to choose different fluorophores for various SRM methods and critically assesses the nitty-gritty of associated problems that are often encountered in practical applications. A progressive guide to designing the next generation of advanced fluorophores to meet the goal of advanced SR imaging studies is also put forward. Written by two well-qualified authors, the book contains exclusive content to enhance readers’ understanding on innovation of newer SRM technologies. Sample topics covered in the book include: Optical techniques, fluorescent probe design, and algorithm development Recent highlight and breakthroughs in biology using SRM methods The overall success of SRM in biological inventions The future direction and scope of the field This book is an invaluable resource for chemists and researchers/scientists involved in designing newer fluorescent materials for SRM studies. It can also assist biologists engaged in advanced biological studies using SRM by guiding them through sample preparation, image processing, and precautions to be taken in practical imaging studies.
Fluorescence Microscopy: Super-Resolution and other Novel Techniques delivers a comprehensive review of current advances in fluorescence microscopy methods as applied to biological and biomedical science. With contributions selected for clarity, utility, and reproducibility, the work provides practical tools for investigating these ground-breaking developments. Emphasizing super-resolution techniques, light sheet microscopy, sample preparation, new labels, and analysis techniques, this work keeps pace with the innovative technical advances that are increasingly vital to biological and biomedical researchers. With its extensive graphics, inter-method comparisons, and tricks and approaches not revealed in primary publications, Fluorescence Microscopy encourages readers to both understand these methods, and to adapt them to other systems. It also offers instruction on the best visualization to derive quantitative information about cell biological structure and function, delivering crucial guidance on best practices in related laboratory research. Presents a timely and comprehensive review of novel techniques in fluorescence imaging as applied to biological and biomedical research Offers insight into common challenges in implementing techniques, as well as effective solutions
This monograph focuses on modern femtosecond laser microscopes for two photon imaging and nanoprocessing, on laser tweezers for cell micromanipulation as well as on fluorescence lifetime imaging (FLIM) in Life Sciences. The book starts with an introduction by Dr. Wolfgang Kaiser, pioneer of nonlinear optics and ends with the chapter on clinical multiphoton tomography, the novel high resolution imaging technique. It includes a foreword by the nonlinear microscopy expert Dr. Colin Sheppard. Contents Part I: Basics Brief history of fluorescence lifetime imaging The long journey to the laser and its use for nonlinear optics Advanced TCSPC-FLIM techniques Ultrafast lasers in biophotonics Part II: Modern nonlinear microscopy of live cells STED microscopy: exploring fluorescence lifetime gradients for super-resolution at reduced illumination intensities Principles and applications of temporal-focusing wide-field two-photon microscopy FLIM-FRET microscopy TCSPC FLIM and PLIM for metabolic imaging and oxygen sensing Laser tweezers are sources of two-photon effects Metabolic shifts in cell proliferation and differentiation Femtosecond laser nanoprocessing Cryomultiphoton imaging Part III: Nonlinear tissue imaging Multiphoton Tomography (MPT) Clinical multimodal CARS imaging In vivo multiphoton microscopy of human skin Two-photon microscopy and fluorescence lifetime imaging of the cornea Multiscale correlative imaging of the brain Revealing interaction of dyes and nanomaterials by multiphoton imaging Multiphoton FLIM in cosmetic clinical research Multiphoton microscopy and fluorescence lifetime imaging for resection guidance in malignant glioma surgery Non-invasive single-photon and multi-photon imaging of stem cells and cancer cells in mouse models Bedside assessment of multiphoton tomography
Zu dem Thema gibt es viele Publikationen, die von Experten für Experten geschrieben wurden. Dieses Buch wendet sich insbesondere an Studenten höherer Semester und Forscher, denen das Hintergrundwissen der Physik fehlt, um neuartige Verfahren der Fluoreszenzmikroskopie zu verstehen. Die zweite Auflage wartet mit neuen Kapiteln und einer erweiterten Einführung auf. Der Schwerpunkt liegt auf der hochauflösenden und Einzelmolekül-Mikroskopie. Jedes Kapitel wurde von einem anerkannten Experten des Fachgebiets geschrieben und sorgfältig überarbeitet, um so die Entwicklungen der letzten Jahre wiederzugeben.
Image-based single cell analysis is essential to study gene expression levels and subcellular functions with preserving the native spatial locations of biomolecules. However, its low throughput has prevented its wide use to fundamental biology and biomedical applications which require large cellular populations in a rapid and efficient fashion. Here, we report a 2.5D microcopy (2.5DM) that significantly improves the image acquisition rate while maintaining high-resolution and single molecule sensitivity. Unlike serial z-scanning in conventional approaches, volumetric information is simultaneously projected onto a 2D image plane in a single shot by engineering the fluorescence light using a novel phase pattern. The imaging depth can be flexibly adjusted and multiple fluorescent markers can be readily visualized. We further enhance the transmission efficiency of 2.5DM by ~2-fold via configuring the spatial light modulator used for the phase modulation in a polarization-insensitive manner. Our approach provides a uniform focal response within a specific imaging depth, allowing to perform quantitative high-throughput single-molecule RNA measurements for mammalian cells over a 2x2 mm2 region within an imaging depth of ~5 [micrometer] in less than 10 min and immunofluorescence imaging at a volumetric imaging rate of >30 Hz with significantly reduced light exposure. With implementation of an adaptive element, our microscope provides an extra degree of freedom in correcting aberrations induced by specimens and optical components, showing its capability of imaging thick specimens with high-fidelity of preserving volumetric information with fast imaging speed. We also demonstrate multimodal imaging that can be switched from 2.5DM to a 3D single-molecule localization imaging platform by encoding the depth information of each emitter into the shape of point spread function, which enables us to obtain a resolution of
Second-harmonic generation (SHG) microscopy has shown great promise for imaging live cells and tissues, with applications in basic science, medical research, and tissue engineering. Second Harmonic Generation Imaging offers a complete guide to this optical modality, from basic principles, instrumentation, methods, and image analysis to biomedical a