1P347 Development of reversible protein highlighting techniques

Ando, R.; Mizuno, H.; Miyawaki, Atsushi

doi:10.2142/biophys.45.s118_3

Cited by 3 publications

(4 citation statements)

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“…Several nonlinear effects exist, however, that do hold promise for very high resolution, including stimulated emission depletion (1-3), energy transfer in specially designed fluorophores (6), and reversible photoactivation of molecules (4,(31)(32)(33)(34)(35). The last category is particularly intriguing in that some promising molecules are proteins of the GFP family (32)(33)(34)(35), suggesting the exciting possibility that the extreme resolution of nonlinear structured-illumination microscopy could be combined with the in vivo labeling power of GFP-like genetically encoded protein tags.…”

Section: Discussionmentioning

confidence: 99%

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Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution

Gustafsson

2005

Proc. Natl. Acad. Sci. U.S.A.

1,968

1,385

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Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.super resolution ͉ moiré ͉ resolution extension ͉ saturation T he fluorescence microscope has become a ubiquitous imaging tool in cell biology through its unique ability to image the 3D interior of a living specimen with multicolor molecular labels of extreme specificity, a combination of strengths not shared by higher-resolution techniques such as electron microscopy and scanned-probe methods. It is therefore unfortunate that its spatial resolution is subject to a hard limit caused by diffraction.Recently, ways have been found to bypass the diffraction limit. 2D resolution in the 30-nm range has been realized by using stimulated emission depletion (STED) (1). STED is based on saturated stimulated emission using two synchronized ultrafast laser sources (2, 3); the underlying concept has been generalized to encompass a class of reversible saturable phenomena (4). STED and other proposed methods (5-7) were conceived in the context of laser-scanning microscopy and are designed to directly minimize the size of a scanned focal point. This article demonstrates an alternative approach that brings theoretically unlimited resolution to a wide-field (nonscanning) microscope by using a nonlinear fluorescence response together with a periodic illumination pattern that fills the field of view.Both structured illumination light and optical nonlinearity, of course, are established ideas. Patterned light, for example, has been used for measuring surface shapes (8) and deformations (9) and for enhancing the sensitivity of fluorescence-recovery-afterphotobleaching experiments (10). Axially structured light has been used to enhance axial resolution in standing-wave fluorescence microscopy (11), 4Pi microscopy (12), and I 5 M (13). Lukosz and Marchand (14) suggested in 1963 that lateral light patterns could be used to enhance resolution, and such patterns have been used for both axial (15) and lateral (16-19) resolution enhancement. They can be more effective than point scanning at retrieving high-resolution information (17,19). Nonlinear fluorescence is the basis for multiphoton fluorescence microscopy (20), and several other c...

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Section: Discussionmentioning

confidence: 99%

“…The last category is particularly intriguing in that some promising molecules are proteins of the GFP family (32)(33)(34)(35), suggesting the exciting possibility that the extreme resolution of nonlinear structured-illumination microscopy could be combined with the in vivo labeling power of GFP-like genetically encoded protein tags.…”

Section: Discussionmentioning

confidence: 99%

Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution

Gustafsson

2005

Proc. Natl. Acad. Sci. U.S.A.

1,968

1,385

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“…There is likely to be increasing use for sophisticated photochromic dyes for both security purposes (e.g. for marking banknotes or personal identification documents) and also in erasable electric storage media, as exemplified by certain engineered fluorescent proteins such as Dronpa [103], which is changed from a fluorescent to a non‐fluoresecent form by radiation at 450 nm, but can then be changed back to the original ‘unwritten’ (emissive) form by minimal irradiation at 405 nm [104]. The use of wild‐type GFP, which has similar properties, can also be used as the basis for optical recordings which can be written and later erased for reuse [105].…”

Section: Conclusion and Future Developmentsmentioning

confidence: 99%

Changing colours: now you see them, now you don’t

Dawson¹

2010

Coloration Technology

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Nature offers a range of colour displays which are relatively short‐lived and which were for many centuries little understood. Today we can explain most which arise from diffraction and reflection effects on rays of sunlight, various atmospheric ionisation phenomena and many colour effects resulting from biochemical reactions, although less is understood about some of the strange psychedelic patterns and colours which our brains can sometimes produce. The practical purposes to which certain fluorescent, thermochromic and photochromic dyes can be put, including their presently limited textile applications, are illustrated.

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“… 53 A more extreme perturbation of chromophore structure by the host protein is in the generation of the less common trans form, which is then often significantly distorted from a planar structure, and exhibits both weak fluorescence and photochromism. 10 , 16 , 54 The latter point is central to the application of photoconvertible proteins such as dronpa in super-resolution fluorescence bioimaging. These factors all suggest that an investigation of the photophysics of a nonplanar chromophore outside the protein matrix may be important in assessing the role of nonplanar geometries in the photophysics of GFP-like proteins.…”

Section: Introductionmentioning

confidence: 99%