Super-resolution microscopy

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3D-SIM-1 NPC Confocal vs 3D-SIM detail

3D-SIM-1 NPC Confocal vs 3D-SIM

3D-SIM-2 Nucleus prophase 3d rotated

3D-SIM-3 Prophase 3 color

3D-SIM-4 Anaphase 3 color

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Opthalmology AMD Super Resolution Cremer

Super-resolution microscopy refers to a form of light microscopy that allows for imaging at a resolution higher than the diffraction limit of light. The diffraction limit, approximately half the wavelength of light, restricts the resolution of conventional fluorescence microscopy to about 200 nanometers. Super-resolution techniques break this barrier, enabling the observation of structures and dynamics at the nanoscale.

History

The concept of super-resolution microscopy was developed to overcome the limitations imposed by the diffraction limit, first described by Ernst Abbe in 1873. Significant advancements were made in the late 20th and early 21st centuries, with techniques such as Stimulated Emission Depletion (STED) microscopy, Photoactivated Localization Microscopy (PALM), and Stochastic Optical Reconstruction Microscopy (STORM) leading the way. These methods have been recognized with numerous awards, including the 2014 Nobel Prize in Chemistry, which was awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for the development of super-resolved fluorescence microscopy.

Principles and Methods

Super-resolution microscopy encompasses several techniques, each based on unique principles to bypass the diffraction limit.

STED Microscopy

STED microscopy uses a de-excitation laser beam to selectively inhibit fluorescence in specific regions of the sample, effectively narrowing the point of emission and increasing resolution beyond the diffraction limit.

PALM and STORM

Both PALM and STORM are based on the precise localization of individual fluorescent molecules. These techniques rely on the stochastic activation and imaging of single molecules, allowing for their positions to be determined with high accuracy. The images are then reconstructed from the accumulated positions of many molecules.

Structured Illumination Microscopy (SIM)

SIM enhances resolution by illuminating the sample with a series of high-frequency patterns. The resulting moiré patterns are used to reconstruct an image with resolution beyond the conventional limits.

Applications

Super-resolution microscopy has revolutionized biological and medical research by providing unprecedented insights into cellular structures, protein interactions, and dynamic processes at the molecular level. It is widely used in neuroscience, cell biology, and virology, among other fields.

Challenges and Future Directions

Despite its advantages, super-resolution microscopy faces challenges such as high light doses that can damage biological samples, complex sample preparation, and extensive computational requirements for image reconstruction. Ongoing research aims to address these issues, improve accessibility, and expand the capabilities of super-resolution techniques.

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