Although most images captured with the microscope are two-dimensional with lateral coordinates in the plane perpendicular to the optical train, the fact remains that most biological materials (including cells, tissues, and whole organisms) are highly three-dimensional. The emergence of laser scanning confocal microscopy and related techniques, including spinning disk, structured illumination, aperture correlation, total internal reflection, multiphoton, deconvolution, and superresolution, has enabled investigators to obtain thin slices from thick specimens by removing the contribution of out-of-focus light in each image plane, rather than actual physical sectioning of the specimen. Referred to as optical sectioning, the resulting image planes provide a high level of contrast and permit three-dimensional reconstructions by computationally combining image data from a collection or stack of images.
Introduction to Aperture Correlation Microscopy - Aperture correlation microscopy combines the light efficiency of structured illumination (SIM) with the acquisition speed of a spinning disk confocal instrument. The technique promises to push SIM into new applications where high temporal resolution is mission-critical.
Optical Sectioning Microscopy - Traditional widefield fluorescence microscopy produces images of thick specimens that often contain a high level of background signal, which dramatically obscures specimen detail and reduces contrast. To obtain crisp and sharp images, optical sections can be generated using either computational (deconvolution) or structured illumination techniques. This interactive tutorial explores the basic concept of optical sectioning using an animated cell model.
Structured Illumination Microscopy: ZEISS ApoTome Basics - Optical sections through thick specimens can be obtained in widefield fluorescence microscopy using structured illumination, as has been implemented in the ApoTome auxiliary device manufactured by ZEISS. This tutorial examines the necessary optical elements to equip a widefield microscope for structured illumination and presents typical image stacks obtained with the ApoTome.
Structured Illumination Microscopy: ZEISS ApoTome Operation - The basic concept behind the ZEISS ApoTome is the use of an evenly spaced grid in the aperture plane to serve as a mask through which the specimen is illuminated. The grid is inserted into the light path of the microscope and uses the epi-illuminator lens system to project a shadow of the grid lines into sharp focus, superimposed on the specimen, in the objective focal plane.
Optical Sectioning with Structured Illumination - Among the numerous advantages of structured illumination microscopy is the ability to produce crisp and distinct optical sections having a thickness that coincides with the objective resolution. This interactive tutorial explores optical sectioning with the ZEISS ApoTome.
VivaTome Basics - In aperture correlation microscopy, the final image is calculated in three steps: image extraction and mirroring followed by registration of both images and, finally, the actual calculation of the optical section itself. In the registration step, distortions as mapped in a previous calibration step are corrected between the two imaging beam paths.
VivaTome Optical Train - In the ZEISS VivaTome, a rotating disk having a defined grating pattern is located in one of the microscope conjugate image planes. Excitation light is directed through this disk, and the transparent regions on the disk are placed very close together so that approximately 50-percent transmission efficiency through the disk is achieved.
Optical Sectioning with Aperture Correlation - Aperture correlation microscopy combines the light efficiency of structured illumination with the acquisition speed of a spinning disk confocal instrument. This interactive tutorial simulates a virtual aperture correlation microscope.
Comparison of Confocal and Widefield Microscopy - Laser scanning confocal microscopy is capable of producing the highest out-of-focus discrimination of all routine optical sectioning techniques. This interactive tutorial explores optical sectioning with confocal microscopy and compares these sections to the results obtained with widefield fluorescence.
Optical Sectioning - The ability to image thin sections without having to mechanically slice a thick specimen is afforded by optical sectioning microscopy. Sections are achieved by eliminating the excitation and detection of fluorescence that originates in regions removed from the focal plane. The reviews listed in this section should be available to students and investigators who have access to subscriptions through their host institutions.
Deconvolution Microscopy - Over the past several decades, deconvolution microscopy has become a mainstream image processing tool for deciphering the substructure of living and fixed specimens in three dimensions. Routinely applied to widefield optical sections, as well as those obtained in confocal and structured illumination, the technique has benefited from the continued development of advanced algorithms and turnkey systems. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of deconvolution.
Laser Scanning Confocal Microscopy - A majority of the literature pertaining to review articles on laser scanning confocal microscopy has been published in textbooks, edited article collections, and symposia, with only an intermittent sprinkling of papers in the scientific journals. The reviews listed in this section should be available to those students and investigators who have access to subscriptions through their host institutions.
Multiphoton Microscopy - The application of nonlinear excitation techniques to the imaging of synthetic fluorophores and fluorescent proteins in biology and medicine has witnessed increasing attention over the past several years, primarily due to the introduction of turnkey pulsed laser systems coupled to advanced instrumentation. The references described in this section contain review articles and original research reports on multiphoton microscopy with emphasis on the theoretical background, microscope configuration, specimen preparation, deep tissue imaging, and numerous applications.
Spinning Disk Confocal Microscopy - Spinning disk confocal microscopy is rapidly emerging as the technique of choice for investigation of dynamics in living cells. Modern commercial instruments and high-performance camera systems are capable of providing high acquisition speeds with acceptable contrast and minimal photobleaching at the low light levels available with this technique. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of spinning disk confocal microscopy.
Structured Illumination for Optical Sectioning - Often referred to as a "poor man's confocal microscope", structured illumination is emerging as a powerful technique for optical sectioning in widefield microscopy at high resolution. Although current implementations are limited in speed and multi-channel acquisition by the requirement of capturing multiple images, new technological innovations are occurring rapidly in this field. The references listed in this section point to original research reports and review articles that should provide the starting point for a thorough understanding of structured illumination.
Aperture Correlation Microscopy - Aperture correlation microscopy is a structured illumination technique that employs a specialized spinning disk having a grid pattern to acquire images in both transmitted and reflected light modes. The resulting images are calculated using a series of algorithms after acquisition. The references listed in this section point to original research reports and review articles on aperture correlation microscopy.
ZEISS ApoTome 3D Image Gallery - The ZEISS ApoTome employs structured illumination to generate optical sections of thick specimens that exhibit dramatically increased sharpness and contrast. The digital images in this gallery are reconstructions of animal and plant tissue optical sections acquired with the ApoTome coupled to a metal halide light source.
ZEISS LSM 700 Image Gallery - The LSM 700 laser scanning confocal microscope is a member of the seventh generation of confocal instruments from Carl Zeiss. The digital images presented in this gallery were obtained using a LSM 700 using a 40x apochromatic objective to examine tissue sections ranging in thickness between 10 and 40 micrometers.