McGregor et al 2015 Methods.pdf (3.58 MB)
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Post-processing strategies in Image Scanning Microscopy

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journal contribution
posted on 11.05.2015, 12:07 by Juliette E. McGregor, Claire A. Mitchell, Nicholas A. Hartell
Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of 1.41 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and by pinholing around the detected maxima, recover the signal across the whole field of view.

Funding

BBSRC BB/L00691X/1; BB/L024284/1; BB/M012034/1

History

Citation

Methods (2015)

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Biological Sciences/Department of Cell Physiology and Pharmacology

Version

AM (Accepted Manuscript)

Published in

Methods (2015)

Publisher

Elsevier for Academic Press

issn

1046-2023

eissn

1095-9130

Copyright date

2015

Available date

08/05/2016

Publisher version

http://www.sciencedirect.com/science/article/pii/S1046202315001930

Language

en