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Abstract

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

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

M. Gustafsson (2005)

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.

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Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.

Ahmet Yildiz, Joseph Forkey, Sean McKinney … (2003)

Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.

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Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.

Michael Hofmann, Christian Eggeling, Stefan Jakobs … (2005)

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.

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PubMed ID:: 16896339

PMC ID:: 2700296

DOI:: 10.1038/nmeth929

ScienceOpen disciplines: Chemistry

Keywords: DNA,chemistry,Fluorescent Dyes,Image Interpretation, Computer-Assisted,methods,Microscopy, Fluorescence,Nanotechnology,Stochastic Processes

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ScienceOpen disciplines: Chemistry

Keywords: DNA, chemistry, Fluorescent Dyes, Image Interpretation, Computer-Assisted, methods, Microscopy, Fluorescence, Nanotechnology, Stochastic Processes

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

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Most cited references 10

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

Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.

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