Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage

Venkataraman Krishnaswami; Giulia M. R. De Luca; Ronald M. P. Breedijk; Cornelis J. F. Van Noorden; Erik M. M. Manders; Ron A. Hoebe

doi:10.1117/12.2251329

17 February 2017 Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage

Venkataraman Krishnaswami, Giulia M. R. De Luca, Ronald M. P. Breedijk, Cornelis J. F. Van Noorden, Erik M. M. Manders, Ron A. Hoebe

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Proceedings Volume 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV; 100700G (2017) https://doi.org/10.1117/12.2251329
Event: SPIE BiOS, 2017, San Francisco, California, United States

Abstract

Fluorescence microscopy is an important tool in biomedical imaging. An inherent trade-off lies between image quality and photodamage. Recently, we have introduced rescan confocal microscopy (RCM) that improves the lateral resolution of a confocal microscope down to 170 nm. Previously, we have demonstrated that with controlled-light exposure microscopy, spatial control of illumination reduces photodamage without compromising image quality. Here, we show that the combination of these two techniques leads to high resolution imaging with reduced photodamage without compromising image quality. Implementation of spatially-controlled illumination was carried out in RCM using a line scanning-based approach. Illumination is spatially-controlled for every line during imaging with the help of a prediction algorithm that estimates the spatial profile of the fluorescent specimen. The estimation is based on the information available from previously acquired line images. As a proof-of-principle, we show images of N1E-115 neuroblastoma cells, obtained by this new setup with reduced illumination dose, improved resolution and without compromising image quality.

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Venkataraman Krishnaswami, Giulia M. R. De Luca, Ronald M. P. Breedijk, Cornelis J. F. Van Noorden, Erik M. M. Manders, and Ron A. Hoebe "Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage", Proc. SPIE 10070, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXIV, 100700G (17 February 2017); https://doi.org/10.1117/12.2251329

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KEYWORDS

Image quality

Signal detection

Confocal microscopy

Microscopy

Image resolution

Microscopes

Cameras

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