ZEISS Campus

Education in Microscopy and Digital Imaging

Superresolution Microscopy

A wide array of new and exciting methodologies have recently been introduced that are now collectively termed superresolution microscopy and feature both lateral and axial resolution measured in the tens of nanometers and even less. The common thread in all of the these new techniques is that they are able to resolve features beneath the diffraction limit by switching fluorophores on and off sequentially in time so that the signals can be recorded consecutively. Among the methods that improve resolution by PSF modification, the most important techniques are referred to by the acronyms STED (stimulated emission depletion), GSD Ground State Depletion, and SSIM (saturated structured illumination microscopy). Techniques that rely on the detection and precise localization of single molecules include PALM (photoactivated localization microscopy) and STORM (stochastic optical reconstruction microscopy).

Superresolution Microscopy

Introduction

The broad range of superresolution techniques available today are moving optical imaging of biological specimens into the realm traditionally held by electron microscopy. Rapidly evolving techniques are continually altering the perspective of this emerging field with regards to imaging in cell biology.

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Superresolution Microscopy

Photoactivated Localization Microscopy (PALM)

The principle behind photoactivated localization microscopy and related techniques (STORM and FPALM) rests on a combination of imaging single fluorophores (single-molecule imaging) along with the controlled activation and sampling of sparse subsets of these labels in time.

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Superresolution Microscopy

Practical Aspects of PALM Imaging

A number of practical considerations are mission-critical in achieving the best single-molecule localization images using PALM and related methodology. The most important considerations include instrument configuration parameters, choice of fluorescent probes, molecular density, contrast ratio, and specimen preparation.

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Superresolution Microscopy

Superresolution Structured Illumination Microscopy (SR-SIM)

An advanced imaging technique where increased resolution is obtained by discarding traditional Köhler illumination and replacing it with non-uniform excitation light patterns that feature sinusoidal intensity variations in one or more dimensions coupled with powerful image reconstruction techniques.

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