Photobleaching imprinting microscopy: seeing clearer and deeper

Gao, Liang; Garcia-Uribe, Alejandro; Liu, Yan; Li, Chiye; Wang, Lihong V.

doi:10.1242/jcs.142943

Cited by 14 publications

(15 citation statements)

References 23 publications

(23 reference statements)

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“…The switching-off rate typically has a power dependence of one on the excitation intensity in one-photon absorption, and at least two in two-photon absorption. 13 Therefore, the fluorescence signal in the center of the excitation volume decays faster than that in the periphery. As a result, the fluorescence signal decay not only reflects the excitation intensity profile, but also incorporates the fluorophore switching-off distribution [ Fig.…”

Section: Principle Of Reversibly Switchablementioning

confidence: 99%

“…Following its first demonstration in photoacoustic imaging, this method has been adopted in fluorescence imaging, including confocal microscopy, two-photon microscopy, wide-field microscopy, and light-sheet microscopy. [11][12][13] However, photobleaching is a nonreversible process and will inevitably perturb the original contrast. Therefore, photobleaching-based subdiffraction imaging is not suitable for biological studies where permanent change is not tolerable.…”

Section: Introductionmentioning

confidence: 99%

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Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Yao

Shcherbakova

et al. 2014

J. Biomed. Opt

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Abstract. Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.

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Section: Principle Of Reversibly Switchablementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Yao

Shcherbakova

et al. 2014

J. Biomed. Opt

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“…If permanent photobleaching is a concern, the dynamics of photoswitchable chromophores can be used instead. Further, the same principle of PI-PAM can be readily transferred to fluorescence microscopy [11,17,18]. Since the resolution enhancement gained by the photobleaching effect is complementary to other super-resolution mechanisms and it does not require modification to the existing systems, the principle of PI-PAM can be incorporated into other super-resolution methods to further improve their resolving powers [8,19,20].…”

Section: Conclusion and Discussionmentioning

confidence: 99%

Photo-imprint super-resolution photoacoustic microscopy

Yao

Wang

et al. 2015

SPIE Proceedings

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Combining the absorption-based photoacoustic effect and intensity-dependent photobleaching effect, we demonstrate a simple method for super-resolution photoacoustic imaging of both fluorescent and non-fluorescent samples. Our method is based on a double-excitation process, where the first excitation pulse partially and inhomogeneously bleaches the molecules in the diffraction-limited excitation volume, thus biasing the signal contributions from a second excitation pulse striking the same region. By scanning the excitation beam, we performed three-dimensional sub-diffraction imaging of varied fluorescent and non-fluorescent species. A lateral resolution of 80 nm and an axial resolution of 370 nm have been demonstrated. This technique has the potential to enable label-free super-resolution imaging, and can be transferred to other optical imaging modalities or combined with other super-resolution methods.

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“…A variety of microscopy techniques rely on photobleaching. For example, photo-imprint microscopy can increase resolution beyond the diffraction limit in both in-plane and axial dimensions 8,9 . Another super-resolution method called bleaching/blinking assisted localization microscopy (BALM) uses discrete photobleaching events to localize single molecules 10 .…”

Section: Introductionmentioning

confidence: 99%

Super-multiplexed fluorescence microscopy via photostability contrast

Orth

Ghosh

Wilson

et al. 2018

Preprint

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Many areas of biological research rely heavily on fluorescence microscopy to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously with spectrally distinct fluorescent labels. A high degree of multiplexing is desirable as it affords the experiment greater information content, speeding up research timelines. Multiplexing can be increased by imaging a larger number of spectral channels, however, the wide emission spectra of most fluorophores limits multiplexing to four or five labels in standard fluorescence microscopes. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins, and show that the latter are particularly well suited to our method as their bleaching is often reversible. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

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Photobleaching imprinting microscopy: seeing clearer and deeper

Cited by 14 publications

References 23 publications

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Photo-imprint super-resolution photoacoustic microscopy

Super-multiplexed fluorescence microscopy via photostability contrast

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