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"The objective of this thesis is to present the fabrication of a multiphoton microscope and the underlying theory responsible for its proper functioning. A basic introduction to nonlinear optics will give the necessary knowledge to the reader to understand the optical effects involved. Femtosecond laser pulses will be presented and characterized. Each part of the microscope, their integration and the design of the microscope will be discussed. The basic concepts of laser scanning microscopy are also required to explain the design of the scanning optics. Fast scanning problems and their solutions are also briefly viewed. As a working proof, the first images taken with the microscope will be presented. Fluorescent beads, rat tail tendon, gold nanoparticles and pollen grain images using various nonlinear effects will be shown and discussed." --
This book covers important aspects of modern optical microscopy and image restoration technologies. Instead of pure optical treatment, the book is delivered with the consideration of the scientists who utilize optical microscopy in their daily research. However, enough details are provided in basic imaging principles, optics and instrumentation in microscopy, spherical aberrations, deconvolution and image restoration. A number of microscopic technologies such as polarization, confocal and multi-photon microscopy are highlighted with their applications in biological and materials sciences/engineering.
"The design and development of a nonlinear two-photon, 3-D microscope imaging system, software for manipulation of 2-D and 3-D images, and the operation of a novel 2-D imaging system used for monitoring cultured live cell activity for many hours is reported. Included in the first portion of the thesis is a description of the final assembly of a mode-locked Ti:Sapphire laser producing 10-100 femtosecond pulses at high repetition rate. Also described is the construction of a laser microscope with digitally controlled XY mirror laser beam scanning with linear motor stage Z-stepping. This system was later used to image GFP tagged cultured live cancer cells."--Abstract.
Multimodal non-linear imaging techniques provide non-invasive and potentially in vivo means to investigate tissue with cellular resolution. A particularly promising approach that has garnered attention as of late is the combination of coherent antiStokes Raman scattering (CARS), second harmonic generation (SHG) and two photon excited autofluorescence (TPEF) microscopy. In the first section of this thesis, the diagnostic potential of multimodal non-linear imaging has been demonstrated in the case of head and neck squamous cell carcinoma. The second part of this thesis investigates the feasibility of CARS microscopy for imaging intense bands in the finger-print region (800-1800 cm-1) wherein the presence of multiple overlapping peaks and interference with non-resonant background present challenges. Specifically, the emphasis is on imaging the prominent peaks arising from conjugated C=C double bonds in retinol, tretinoin, [beta]-carotene, and various microalgal pigments. The first CARS fingerprint imaging application in the thesis is concerned with the vitamin A content of liver tissue. Analogously, in a uni-cellular application, CARS has been employed to image carotenoids in the diatoms D. brightwellii and S. turris. As part of the effort in transferring multimodal microscopic technologies to the enduser, the third part of the thesis examines two beam excitation and demultiplexed detection as a means of doubling the speed of laser scanning microscopes based on compact fiber laser sources. Another area of improvement explored is the resolution of the CARS microscopic setup wherein, based on results from numerical studies, a Bessel like beam was employed as one of the excitation arms in the setup to enhance lateral resolution.
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
This book covers various aspects of modern microscopy, with emphasis on multidimensional (three-dimensional and higher) and multimodality microscopy. The topics discussed include multiphoton fluorescent microscopy, confocal microscopy, x-ray microscopy and microtomography, electron microscopy, probe microscopy and multidimensional image processing for microscopy. In addition, there are chapters demonstrating typical microscopical applications, both biological and material.
Confocal microscopes have become valuable tools in many fields of study. This work is intended to serve as a primer for basic confocal microscopy theory as well as a description of the design and construction of a homebuilt system in the Elson Lab at the Washington University School of Medicine. A description of the different designs of confocal microscopes is provided, and the capabilities, benefits, and detriments of each is described. The thought process behind each design decision is explained leading to the final system design. A more in depth description of the final design is provided in Chapters Two and Three, but, in short, the final design is a non-descanned, laser scanning, two-photon confocal microscope. The system is designed to integrate as seamlessly as possible with the existing system and to allow for all existing functionality to remain. When completed, the system will be capable of one- and two-photon fluorescence correlation ii spectroscopy (FCS); low-speed, one-photon confocal imaging using a piezo-stage; and high-speed, two-photon confocal imaging using the laser scanning system. Finally, future directions as well as the limitations of the system are also described. All resources necessary to continue work on the system as well as those necessary to use it are provided. This includes: System diagrams, Optical layout plots, Component data sheets, LabVIEW program. These resources are intended to make using, modifying, and improving the system much simpler.