Polarization modulation adds little additional information to super-resolution fluorescence microscopy

Frahm, Lars; Keller, Jan

doi:10.1038/nmeth.3687

Cited by 18 publications

(12 citation statements)

References 2 publications

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“…In conclusion, this technique has potential direct applications in single molecule co-localization experiments to discriminate between molecules at a distance below the optical resolution of biological sensors, increase contrast at the nanometer scale of polarization images, or explain increased image quality in super-resolution techniques based on PR microscopy [18][19][20]. …”

Section: Discussionmentioning

confidence: 99%

“…The resulting images are analyzed with a statistics-based algorithm that provides the best set of parameters (fluorophore orientation and emitted intensity) in every region of the sample that fits the actual overall modulation. Although some controversy on the performance of the technique has been addressed, claiming that the PR adds little to the super-resolution capability of the stimulated emission depletion (STED) technique [20], the PR images provide a greater amount of information, which has been used in the past to obtain structural information [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. In addition, it is clear that the extra information can be used to improve an image.…”

Section: Introductionmentioning

confidence: 99%

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Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Artigas

Merino

Polzer

et al. 2017

Optica

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Imaging molecular structures separated by distances of a few nanometers still represents a complex challenge. Moreover, it is normally restricted to observations on thin (few micrometers) samples. In this work, we rotate the polarization of the excitation beam of two-photon excited fluorescence (TPEF) images to show that fluorescent structures at the molecular scale can be discriminated in a living organism. The polarization rotation generates a modulation of the signal intensity in each pixel of the TPEF images that carry information related to the fluorophore orientation. We analyze the signal modulation in every pixel of the polarization-resolved (PR) TPEF images through a Fourier analysis and generate images for the different Fourier components. Doing that, we show that two fluorophores oriented in different directions can be distinguished. Although by imaging the Fourier components the resolution of the optical system restricts the exact localization of two close molecules, discrimination is still possible even when the molecules are located at sub-diffraction distances. We propose a model that predicts this behavior, and demonstrate it experimentally in the neurons of a living Caenorhabditis elegans nematode, where we distinguish the walls of an axon with a diameter below the objective resolution. Since the technique is based in TPEF, the method can be extended to deep tissue imaging and has potential applications in single molecule detection, biological sensors, or super-resolution imaging techniques.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Artigas

Merino

Polzer

et al. 2017

Optica

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“…[27] Later Frahm and Keller showed however that their results were in fact not based on polarization control but due to post processing of the data by a sparsity enhancing deconvolution algorithm. [28] Closer inspection of the data of Hafi and co-workers reveals that they reported very poor modulation depths when changing the polarization (their Figure 2). Two experimental factors might have caused these poor modulation depths, thereby impairing their results, while the conceptual idea is working, as we demonstrate below.…”

Section: Fluorescence Polarization Controlmentioning

confidence: 93%

Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures

Hulleman

Huisman²,

Moerland

et al. 2017

Preprint

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Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells.

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“…There also has been an interesting debate on whether polarization modulation adds super resolution or not. 63,64 Super resolution dipole orientation mapping (SDOM) extended SPoD with measurement of dipole orientations, which adds promptly evidence to the debate. 18 Instead of the SPEED algorithm in SPoD, SDOM establishes a polarization-variant model, in which the intensity determines the super-resolution microscopic image using sparsity-enhanced deconvolution, while the phase determines the e®ective dipole orientation of each super-resolved focal volume using least squares estimation, thus fully exploiting the polarization modulation information.…”

Section: Super-resolution Techniques In Fpmmentioning

confidence: 99%

Super-resolution fluorescence polarization microscopy

Zhanghao

Gao

Jin³

et al. 2017

J. Innov. Opt. Health Sci.

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Fluorescence polarization is related to the dipole orientation of chromophores, making°uores-cence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. Several recent super resolution techniques have been applied to°uorescence polarization microscopy, achieving dipole measurement at nanoscale. In this review, we summarize both di®raction limited and super resolution°uorescence polarization microscopy techniques, as well as their applications in biological imaging.

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Polarization modulation adds little additional information to super-resolution fluorescence microscopy

Cited by 18 publications

References 2 publications

Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures

Super-resolution fluorescence polarization microscopy

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