Spatio-angular fluorescence microscopy III. Constrained angular diffusion, polarized excitation, and high-NA imaging

Chandler, Talon; Shroff, Hari; Oldenbourg, Rudolf; Rivière, Patrick La

doi:10.1364/josaa.389217

Cited by 16 publications

(18 citation statements)

References 49 publications

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“…This rotational diffusion can be assumed to be uniform within a hardedged symmetric cone, 25,29 or one can directly model rotational diffusion within an orientational potential well. 26,35 If an SM's rotational diffusion possesses sufficient symmetry, one may conveniently consider it as a mixture of two ideal sources: one immobilized dipole at a particular orientation plus one freely rotating molecule (i.e., an isotropic point source), each of a particular brightness. 7,26,27 Here, we adopt a generalized model for rotational diffusion introduced by Backer and Moerner.…”

Section: Modeling the Rotational Diffusion Of A Dipolelike Emittermentioning

confidence: 99%

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Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Ding

Lew

2021

Preprint

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Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule's wobble, simultaneously from a single image. Even when imaging dim emitters (~500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive using molecular positions alone.

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Section: Modeling the Rotational Diffusion Of A Dipolelike Emittermentioning

confidence: 99%

“…Assuming that a molecule's rotational correlation time is faster than its fluorescence lifetime and the temporal resolution of an imaging sensor, 25,26,35 its orientation trajectory can be fully characterized by a second-moment vector 41 m = [ µ 2…”

Section: Figurementioning

confidence: 99%

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Ding

Lew

2021

Preprint

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“…Spatio-angular measurements of biological systems is a rapidly growing field. Spatio-angular measurements akin to uPTI are being developed with fluorescence polarization imaging (35,69,70). We anticipate that the models and deconvolution approaches we have reported will be of value to spatio-angular fluorescence imaging as well.…”

Section: Discussionmentioning

confidence: 99%

uPTI: uniaxial permittivity tensor imaging of intrinsic density and anisotropy

Yeh

Ivanov

Guo

et al. 2020

Preprint

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Architecture of biological systems is important for their homeostatic functions and is predictive of pathology. Volumetric imaging of intrinsic density, anisotropy, and 3D orientation of cell and tissue components is informative, but still challenging. These physical properties can be described succinctly by the volumetric distribution of the specimen’s permittivity tensor (PT). We report uniaxial permittivity tensor imaging (uPTI), a novel approach for label-free volumetric imaging with diffraction-limited resolution. uPTI combines the oblique illumination and the polarization imaging with high numerical apertures to encode the specimen’s permittivity tensor into intensity modulations, which are decoded with a novel vector diffraction model and a multi-channel convex optimization. The uPTI volumes of polystyrene beads and laser-fabricated anisotropic glass targets show that 3D uPT measurements are quantitative, with diffraction-limited spatial resolution of 0.23 × 0.23 × 0.8 μm3. Automated 2D and 3D imaging of a mouse brain section reveals anatomy at multiple scales (cm – 250 nm) and demonstrates that uPTI enables analysis of the density, anisotropy, and orientation of axon bundles and single axons. We multiplex uPTI with fluorescence de-convolution microscopy to enable correlative analysis of physical and molecular architecture of iPSC-derived cardiomyocytes. uPTI can be added to an existing widefield microscope as a low cost module. We share our implementation of the forward model and optimization algorithms via open source repository. Collectively, the reported advances in optical design, image formation, reconstruction algorithms, and biological interpretation open multiple avenues to study architecture of organelles, cells and tissue sections, including human cells and tissues that are challenging to label.

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“…Beyond FLIM, the all-optical processing of resonant EO imaging will allow new capabilities for widefield lock-in detection at high modulation frequencies. This may prove useful for polarization-modulated microscopy techniques to image molecular orientations [53][54][55] and also for label-free contrast that relies on weak signals detected by modulation transfer such as in stimulated Raman microscopy 30 and photothermal microscopy. 27,28 High frequency lock-in detection improves rejection of low frequency 1/f noise sources for optical metrology.…”

Section: Discussionmentioning

confidence: 99%

Resonant Electro-optic Imaging for Microscopy at Nanosecond Resolution

Bowman,

Kasevich

2021

Preprint

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We present an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently capture nanosecond image dynamics on standard camera sensors. Images are gated at 39 MHz with 16 dB extinction and 86% modulation depth, allowing lifetimes to be estimated with sensitivity 1.9 times the shot-noise limit and without restricting photon throughput. High throughput is shown by acquiring FLIM images with millisecond exposure and > 10 8 photons per image. High sensitivity is shown through wide-field lifetime imaging of single-molecule populations and dynamics. Using both the throughput and sensitivity of the technique, we enable the first combination of wide-field lifetime imaging with single-molecule localization microscopy. We further demonstrate lifetime imaging of single-molecule FRET and multi-parameter imaging of molecular binding events combining intensity, lifetime, and polarization. More generally, resonant electro-optic imaging can provide lifetime contrast in any wide-field method and will enable nanosecond time-domain measurements in microscopy.

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Spatio-angular fluorescence microscopy III. Constrained angular diffusion, polarized excitation, and high-NA imaging

Cited by 16 publications

References 49 publications

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

uPTI: uniaxial permittivity tensor imaging of intrinsic density and anisotropy

Resonant Electro-optic Imaging for Microscopy at Nanosecond Resolution

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