Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy

Kavya, M and Regmi, Raju and Mondal, Partha P (2013) Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy. In: REVIEW OF SCIENTIFIC INSTRUMENTS, 84 (9).

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Abstract

Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4 pi geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as compared to 690 nm of the state-of-art confocal system. Reconstruction of experimentally obtained 2PE - 4pi data of green fluorescent protein (GFP)-tagged mitocondrial network shows near elimination of artifacts arising out of side-lobes. Proposed technique may find interesting application in fluorescence microscopy, nano-lithography, and cell biology. (C) 2013 AIP Publishing LLC.

Item Type:	Journal Article
Publication:	REVIEW OF SCIENTIFIC INSTRUMENTS
Publisher:	AMER INST PHYSICS
Additional Information:	copyright for this article belongs to AIP
Department/Centre:	Division of Physical & Mathematical Sciences > Instrumentation Appiled Physics
Date Deposited:	14 Nov 2013 10:46
Last Modified:	14 Nov 2013 10:46
URI:	http://eprints.iisc.ac.in/id/eprint/47756

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