Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

Martens, Koen J.A.; Jabermoradi, Abbas; Yang, Suyeon; Hohlbein, Johannes

doi:10.1016/j.ymeth.2020.07.010

Cited by 8 publications

(13 citation statements)

References 55 publications

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“…To correct the flatness of the mirror and later for modulating the PSFs, we implemented REALM (github.com/MSiemons/REALM) (Siemons et al, 2020), which allows for corrections without requiring an additional sensor to monitor the incoming wavefront. We further wrote a plugin for Micromanager to connect REALM to the deformable mirror used in our study (github.com/HohlbeinLab/Thorlabs_DM_Device_Adapter) Martens et al (2020).…”

Section: Methodsmentioning

confidence: 99%

“…CC-BY-NC 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 3, 2021. ; https://doi.org/10.1101/2021.03.03.433739 doi: bioRxiv preprint Martens et al (2020).…”

Section: Introductionmentioning

confidence: 99%

“…Originally a 2D imaging technique, 3D resolution in SMLM can be achieved by breaking the axial symmetry of the imaged point spread function (PSF) using astigmatism (Huang et al, 2008; Holtzer et al, 2007) or further PSF engineering via phase masks or AO enabling saddle point, tetrapod (Aristov et al, 2018) and double helix (Pavani et al, 2009) PSF. Our recent work introduced a method called circular-tangent phasor-based SMLM (ct-pSMLM) that enables fast and accurate localization of emitters after PSFs engineering on standard CPUs (Martens et al, 2020). For our purposes, we will use a sensorless approach based on a deformable mirror to compensate aberrations and introduce engineered PSFs providing access to three-dimensional super-resolution microscopy.…”

Section: Introductionmentioning

confidence: 99%

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Enabling single-molecule localization microscopy in turbid food emulsions

Jabermoradi

Yang

Gobes

et al. 2021

Preprint

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Turbidity poses a major challenge for the microscopic characterization of many food systems. In these systems, local mismatches in refractive indices can cause reflection, absorption and scattering of incoming as well as outgoing light leading to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the achievable optical resolution, we combined adaptive optics (AO) with single-molecule localization microscopy (SMLM). Building on our previously published open hardware microscopy framework, the miCube, we first added a deformable mirror to the detection path. This element enables both the compensation of aberrations directly from single-molecule data and, by further modulating the emission wavefront, the introduction of various point spread functions (PSFs) to enable SMLM in three dimensions. We further added a top hat beam shaper to the excitation path to obtain an even illumination profile across the field of view (FOV). As a model system for a non-transparent food colloid in which imaging in depth is challenging, we designed an oil-in-water emulsion in which phosvitin, a ferric ion binding protein present in from egg yolk, resides at the oil water interface. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain 2D and 3D images of phosvitin covered oil droplets with sub 100 nm resolution. Droplets with radii as low as 200 nm can be discerned, which is beyond the range of conventional confocal light microscopy. Our data indicated that in the model emulsion phosvitin is homogeneously distributed at the oil-water interface. With the possibility to obtain super-resolved images in depth of nontransparent colloids, our work paves the way for localizing biomacromolecules at colloidal interfaces in heterogeneous food emulsions.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enabling single-molecule localization microscopy in turbid food emulsions

Jabermoradi

Yang

Gobes

et al. 2021

Preprint

Self Cite

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“…To correct the flatness of the mirror and later for modulating the PSFs, we implemented REALM (github.com/MSiemons/REALM) [32], which allows for corrections without requiring an additional sensor to monitor the incoming wavefront. We further wrote a plugin for Micromanager to connect REALM to the deformable mirror used in our study (github.com/HohlbeinLab/Thorlabs_DM_Device_Adapter) [21].…”

Section: (D) Rescan-confocal Microscopymentioning

confidence: 99%

“…Originally, a two-dimensional imaging technique, three-dimensional resolution in SMLM can be achieved by breaking the axial symmetry of the imaged point spread function (PSF) using astigmatism [17,18] or further PSF engineering via phase masks or AO enabling saddle point, tetrapod [19] and double helix [20] PSFs. Our recent work introduced a method called circular-tangent phasor-based SMLM (ct-pSMLM) that enables fast and accurate localization of emitters after PSFs engineering on standard CPUs [21].…”

Section: Introductionmentioning

confidence: 99%

Enabling single-molecule localization microscopy in turbid food emulsions

Jabermoradi

Yang

Gobes

et al. 2022

Phil. Trans. R. Soc. A.

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Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field illumination to our previously published open microscopy framework, the miCube. In the detection path, we implemented AO via a deformable mirror to compensate aberrations and to modulate the emission wavefront enabling the engineering of point spread functions (PSFs) for single-molecule localization microscopy (SMLM) in three dimensions. As a model system for a non-transparent food colloid such as mayonnaise, we designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain two- and three-dimensional images of phosvitin covered oil droplets with sub 100 nm resolution. Our data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, our work paves the way for localizing biomacromolecules at heterogeneous colloidal interfaces in food emulsions. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

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Correlative approaches in single-molecule biophysics: A review of the progress in methods and applications

Leake

2021

Methods

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Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

Cited by 8 publications

References 55 publications

Enabling single-molecule localization microscopy in turbid food emulsions

Enabling single-molecule localization microscopy in turbid food emulsions

Enabling single-molecule localization microscopy in turbid food emulsions

Correlative approaches in single-molecule biophysics: A review of the progress in methods and applications

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