Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer

doi:10.1371/journal.pone.0165148

Cited by 7 publications

(7 citation statements)

References 19 publications

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“…The absolute magnitude of Fourier space images to the power of 0.3 is shown. PALM/STORM, photoswitchable fluorescent proteins, such as Dronpa, 49 mEos2, Skylan NS, 50 RS-eGFP, IRIS-FP, and Kohinoor, 51 are also becoming increasingly popular choices to enable NL-SIM with a resolution down to about 50 nm.…”

Section: Labeling Strategies For Sr-simmentioning

confidence: 99%

“…However, it is also possible to determine the individual phases from the weighted autocorrelation at the precise grating frequency of each image. 51 As usually limited volumes are acquired along the axial direction, significant problems can be caused by out-of-focus light in the reconstruction. At illumination frequencies below the detection Abbe limit, 2D-SIM can be equipped with optical sectioning capability, albeit usually at the cost of a slightly reduced spatial resolution, by utilizing a method called OTF attenuation.…”

Section: The Nitty-gritty Details Of Sim Image Reconstructionmentioning

confidence: 99%

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Super-Resolution Structured Illumination Microscopy

2017

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Super-resolved structured illumination microscopy (SR-SIM) is among the most rapidly growing fluorescence microscopy techniques that can surpass the optical diffraction limit. The strength of SR-SIM is that it can be readily applied to samples prepared for conventional fluorescence microscopy, requiring no sophisticated sample preparation protocols. As an extension of wide-field fluorescence microscopy, it is inherently capable of multicolor imaging and optical sectioning and, with sufficiently fast implementations, permits live cell imaging. Image reconstruction, however, currently relies on sophisticated computational procedures, susceptible to reconstruction artifacts, requiring trained users to recognize and avoid them. Here, we review the latest developments in SR-SIM research. Starting from a historical overview of the development of SR-SIM, we review how this method can be implemented in various experimental schemes, we provide an overview of the important parameters involved in successful image reconstruction, we summarize recent biological applications, and we provide a brief outlook of the directions in which we believe SR-SIM is headed in the future.

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Section: Labeling Strategies For Sr-simmentioning

confidence: 99%

Section: The Nitty-gritty Details Of Sim Image Reconstructionmentioning

confidence: 99%

Super-Resolution Structured Illumination Microscopy

2017

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“…We denoted this SR technique as super-resolution polarization demodulation/on-state polarization angle narrowing (SPoD-OnSPAN) [ 9 ]. RSFPs have also been shown to be useful for other SR bioimaging methods, such as reversible saturable/switchable optical fluorescence transitions (RESOLFT) [ 7 , 10–12 ] and nonlinear structured illumination microscopy (NL-SIM) [ 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

“…As Kohinoor shares the same wavelength for fluorescence excitation and on-switching, it is denoted as a positively reversibly photoswitchable fluorescent protein (pRSFP). Although Kohinoor has allowed for SR observation by RESOLFT, NL-SIM and SPoD-OnSPAN with very low doses of light [ 7–9 , 14 ], some photoproperties, such as fluorescence intensity and photoswitching speed, have yet to be improved for time-lapse and high-time-resolution SR imaging.…”

Section: Introductionmentioning

confidence: 99%

A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

et al. 2021

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Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

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“…RSFPs can emit fluorescence multiple times with adequate inactivation and reactivation by excitation and activation lights, respectively, allowing the detection of several signals from a single fluorophore and leading to robust image reconstruction. Skylan-S and Skylan-NS have been applied for super-resolution optical fluctuation imaging (SOFI) (Dertinger et 5 al., 2009;Zhang et al, 2015) and nonlinear-SIM (Lu-Walther et al, 2016;Zhang et al, 2016), respectively. However, the contrast and fluorescence of most of the RSFPs, including Skylans, decreases upon chemical fixation (Shinoda et al, 2019), thus limiting their applications.…”

Section: Introductionmentioning

confidence: 99%

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

Osuga

Nishimura

Suetsugu

2021

MBoC

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Super-resolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Super-resolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photo-convertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and re-activated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

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Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

Cited by 7 publications

References 19 publications

Super-Resolution Structured Illumination Microscopy

Super-Resolution Structured Illumination Microscopy

A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

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