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Interactive Tutorials

Optical Sectioning Microscopy

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.

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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.

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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.

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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.

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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.

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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.

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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.

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Confocal versus 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.

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Spinning Disk Fundamentals

Explore how light passes through the pinholes on a spinning disk microscope to produce multiple excitation beams that are swept across the specimen as the disk spins. The Nipkow disk is located in a conjugate image plane and scans with approximately 1000 individual light beams.

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Yokogawa Spinning Disk

The most advanced design in spinning disk instruments was engineered by Yokogawa Electric Corporation of Japan and implemented in a series of increasingly complex disk scanning units. This tutorial examines the operating principles of the Yokogawa scanning units.

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Pinhole Crosstalk in Spinning Disk Microscopy

Axial resolution in spinning disk microscopy is largely defined by the size of the pinhole or slit and the separation distances between these apertures. This tutorial demonstrates how fluorescence removed from the focal plane can generate pinhole crosstalk.

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Microlens Arrays in Spinning Disk Microscopy

The amount of light transmitted through the Nipkow disk in spinning disk microscopy is determined by the diameter of the pinhole or slit and the distance between these apertures. This tutorial explores how the amount of light passed through a disk can be increased by using microlens arrays on the upper disk in a two-disk system.

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Camera Exposure and Disk Speed in Spinning Disk Microscopy

In spinning disk microscopy using a Yokogawa scan head, the camera exposure times are dependent upon the intensity of fluorescence emission gathered by the specimen and vary widely from one sample to another, and the disk rotation speed must be carefully adjusted to match the camera exposure time.

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