fastSIM: a practical implementation of fast structured illumination microscopy

Lu-Walther, Hui-Wen; Kielhorn, Martin; Förster, Ronny; Jost, Aurélie; Wicker, Kai; Heintzmann, Rainer

doi:10.1088/2050-6120/3/1/014001

Cited by 80 publications

(86 citation statements)

References 17 publications

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“…For example, a period of 9 pixels for 488 nm illumination and 12 pixels for 640 nm illumination. For a comprehensive discussion of SLM pattern design, including sub-pixel optimization of pattern spacing using sheared gratings, see the previous work of Kner et al 16 and Lu-Walther et al 20 The position of the two excitation foci must be inside the TIR ring for all wavelengths, however the diffraction angle of the ±1 orders from the SLM is wavelength dependent. For standard SIM, multicolor imaging can be achieved by optimizing the grating period for the longest wavelength, and tolerating a loss in performance for the shorter channels.…”

Section: Total Internal Reflection (Tir)mentioning

confidence: 99%

“…The first in vivo images obtained with TIRF-SIM were reported in 2009 16 achieving frame rates of 11 Hz to visualize tubulin and kinesin dynamics, and two color TIRF-SIM systems have been presented 17,18 . Most recently, a guide for the construction and use of a single color two-beam SIM system was presented featuring frame-rates of up to 18 Hz 19,20 .…”

Section: Introductionmentioning

confidence: 99%

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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Young

Ströhl

Kaminski

2016

JoVE

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Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

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Section: Total Internal Reflection (Tir)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Young

Ströhl

Kaminski

2016

JoVE

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“…Our NL-SIM design therefore protected the SLM from the potentially destructive 405nm. A benefit is that our data acquisition method is exactly as in linear SIM (LSIM) configurations [6]. Although Padron also offers positive photoswitchability, a disadvantage of Padron is that violet light also excites fluorescence emission which is problematic in our acquisition scheme [17, 20].…”

Section: Acquisition Methods In Nl-simmentioning

confidence: 99%

“…Our NL-SIM system was adopted from the two-beam fastSIM setup, described in [6] as depicted in Fig 4. A small mirror (see Fig 4, M 1 ) was used to integrate the 405 nm laser into the system in the Fourier plane of the SLM.…”

Section: Principle Of Nl-simmentioning

confidence: 99%

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

et al. 2016

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Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.

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“…73 The further improvement of raw data acquisition rate is achieved at 162 fps. 74 Recent spatial light modulator–based 3D SIM has been applied for live-cell imaging. The recording speed can be up to 5 seconds/volume with a 120-nm lateral and 360-nm axial resolution.…”

Section: Available Super-resolution Microscopy Techniquesmentioning

confidence: 99%

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

Zhi

Wang

Yao

2015

Crit Rev Biomed Eng

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Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina.

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fastSIM: a practical implementation of fast structured illumination microscopy

Cited by 80 publications

References 17 publications

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

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

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

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