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

• Published in: Review of scientific instruments 2013-09, Vol.84 (9), p.093704-093704
• Main Authors: M, Kavya, Regmi, Raju, Mondal, Partha P
• Format: Article
• Language: English
• Subjects: Microscopy, Fluorescence, Multiphoton - instrumentation; Microscopy, Fluorescence, Multiphoton - methods; Models, Theoretical
• ISSN: 1089-7623
• DOI: 10.1063/1.4820922

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 4π 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.