Adaptable single molecule localization microscopy (aSMLM) for superresolution optical fluorescence imaging

Authors:

Joshi, P., & Mondal, P. P.

Keywords:

MINFLUX

Abstract:

Single-molecule imaging over a large area is beneficial for understanding interlinked intracellular biophysical processes and cell–cell interaction. To study, the interrogation system requires real-time adaptability of the field-of-view (FOV). We developed a widefield non-scanning system (aSMLM) that consists of an autotunable illumination system. The 4f-autotunable optical sub-system (combination of auto-tunable lens and objective lens) is introduced in the illumination path to enable change of focus at the specimen plane (located at the working distance of the objective lens). The combined incident parallel beam (of wavelengths, 405 and 561 nm) is converged/diverged by the 4f sub-system, resulting in a change of focal spot at the working distance. The spot is essentially a defocussed field with an order increase in FOV (14.79 to 316.31 ?m2) and demonstrates better field homogeneity. However, the most important point is the tunability of the FOV in this range. A dedicated control unit is engaged to facilitate a rapid shift of focus (at a rate of 200 Hz), resulting in desirable spot-size (FOV). However, the detection subsystem is a 4f-system that collects light emerging from the specimen plane (located at the working distance of objective lens) and produces an image at the focus of tube-lens. The technique is further explored to study single-molecule (Dendra2-HA) clusters in transfected NIH3T3 cells that demonstrate its efficiency over a variable FOV. A near-uniform illumination of desired FOV is achieved along with a threefold increase in the number of detected single molecules. We anticipate that the proposed aSMLM technique may find immediate application in the emerging field of single-molecule biophysics and fluorescence microscopy.

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