Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest
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...
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| Main Authors: | , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
[12 Nov 2018]
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| In: |
Optics express
Year: 2018, Volume: 26, Issue: 23, Pages: 30009-30020 |
| ISSN: | 1094-4087 |
| DOI: | 10.1364/OE.26.030009 |
| Online Access: | Verlag, Volltext: https://doi.org/10.1364/OE.26.030009 Verlag, Volltext: https://www.osapublishing.org/oe/abstract.cfm?uri=oe-26-23-30009 
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| Author Notes: | Shih-Ya Chen, Felix Bestvater, Wladimir Schaufler, Rainer Heintzmann, and Christoph Cremer |
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| 520 | |a 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. | ||
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