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Title: Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest cellSTORM - Cost-effective Super-Resolution on a Cellphone using dSTORM
Authors: Chen, S.-Y.Bestvater, F.Heintzmann, RainerCremer, Christoph
Publishers Version: https://doi.org/10.1364/OE.26.030009
Issue Date: 2018
Published in: Optics Express 26 (2018), Nr. 23
Publisher: Washington D.C. : Optical Society of America
Abstract: Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.
Keywords: single molecule localization microscopy; SMLM; subcellular structures
DDC: 620
License: CC BY 4.0 Unported
Link to License: https://creativecommons.org/licenses/by/4.0/
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