Single molecule light field microscopy

Sims, Ruth; Rehman, Sohaib Abdul; Lenz, Martin; Benaissa, Sarah I.; Bruggeman, Ezra; Clark, Adam J.; Sanders, Edward W.; Ponjavic, Aleks; Mureşan, Leila; Lee, Steven F.; O’Holleran, Kevin

doi:10.1117/12.2584562

Cited by 7 publications

(11 citation statements)

References 40 publications

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“…We imaged fixed Jurkat T cells using the tetrapod PSF (10 μm DOF, 57 Fig. 3d) and the recently developed SMLFM (5 μm DOF, 5 Fig. 3e).…”

Section: Resultsmentioning

confidence: 99%

“…A bespoke lightfield microscope was used as in previous work. 5 Jurkat T cell membranes were imaged using WGA-SiR or HMSiR with continuous excitation at 638 nm (~1 kW cm -2 ). HILO illumination configuration was used to minimise fluorescence background to image a plane near the apical surface of Jurkat T cells.…”

Section: R a F T D R A F Tmentioning

confidence: 99%

“…This makes it suitable for capturing large variations in structure such as the complex morphology of the cell surface, which we illustrate by imaging the entire membrane of a T cell. Furthermore, we show that resPAINT is a generalisable principle that works across a variety of probe control mechanisms (photoactivation or pH-tuned spontaneously blinking probes), as well as large DOF volumetric imaging modalities (DHPSF, tetrapod PSF 4 and SMLFM 5 ). Finally, we use the greatly improved contrast to achieve 3D-PAINT imaging of a T-cell membrane protein, CD45, using an antibody fragment, (Fab).…”

Section: Introductionmentioning

confidence: 97%

“…This has motivated the development of large depth-of-field (DOF) single-molecule imaging techniques, such as the doublehelix point-spread function (DHPSF), 2,3 the tetrapod pointspread function (PSF) 4 and single-molecule light field microscopy (SMLFM). 5 In SMLM, it is becoming necessary to collect substantial numbers of localisations (>100k µm -3 ) to accurately represent increasingly large structures. Obtaining appropriate (i.e.…”

Section: Introductionmentioning

confidence: 99%

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resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Sanders

Carr

Bruggeman

et al. 2022

Preprint

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Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a "reservoir" of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix point-spread function, the tetrapod point-spread function and single-molecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

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“…We imaged fixed Jurkat T cells using the tetrapod PSF (10 μm DOF, 57 Fig. 3d) and the recently developed SMLFM (5 μm DOF, 5 Fig. 3e).…”

Section: Resultsmentioning

confidence: 99%

Section: R a F T D R A F Tmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Sanders

Carr

Bruggeman

et al. 2022

Preprint

Self Cite

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“…Moreover, in the reconstruction, the lateral resolution is constant along the entire depth of field, and there is no artifact near the native image plane. Therefore, FiMic is establishing itself as the standard setup for lightfield microscopy [ 15 , 16 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

The Lightfield Microscope Eyepiece

Incardona

Tolosa

Scrofani

et al. 2021

Sensors

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Lightfield microscopy has raised growing interest in the last few years. Its ability to get three-dimensional information about the sample in a single shot makes it suitable for many applications in which time resolution is fundamental. In this paper we present a novel device, which is capable of converting any conventional microscope into a lightfield microscope. Based on the Fourier integral microscope concept, we designed the lightfield microscope eyepiece. This is coupled to the eyepiece port, to let the user exploit all the host microscope’s components (objective turret, illumination systems, translation stage, etc.) and get a 3D reconstruction of the sample. After the optical design, a proof-of-concept device was built with off-the-shelf optomechanical components. Here, its optical performances are demonstrated, which show good matching with the theoretical ones. Then, the pictures of different samples taken with the lightfield eyepiece are shown, along with the corresponding reconstructions. We demonstrated the functioning of the lightfield eyepiece and lay the foundation for the development of a commercial device that works with any microscope.

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Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5

Hunter

Payne-Dwyer

Shaw

et al. 2022

Preprint

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SummaryThe ability of tumors to establish a pro-tumorigenic microenvironment is becoming an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the “education” of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug-resistance and metastasis, has suggested CCR5 as a target for therapeutic inhibition. However, with several conformational ‘pools’ being reported, phenotypic investigations must be capable of unveiling heterogeneity. Addressing this challenge, we performed structured illumination (SIM) and Partially TIRF coupled HILO (PaTCH) microscopy for super-resolution imaging and single-molecule imaging of CCR5 in fixed cells. Determining the positions of super-resolved CCR5 assemblies revealed a non-random spatial orientation. Further, intensity-tracking of assemblies revealed a distribution of molecular stoichiometries indicative of dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and a plethora of other biomolecules.HighlightsWe use SIM and novel PaTCH microscopy for precise bioimaging and single-molecule tracking of receptor protein CCR5 in model cell linesBy tracking the position of CCR5 assemblies we conclude that they are clustered in the plasma membrane beyond a level expected from a random distributionWe use these high precision data to determine molecular stoichiometries of CCR5 assemblies

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Single molecule light field microscopy

Cited by 7 publications

References 40 publications

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

The Lightfield Microscope Eyepiece

Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5

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