True optical resolution beyond the Rayleigh limit achieved by standing wave illumination

300

253

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it lightefficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.extended resolution | frequency mixing | multicolor | patterned excitation F luorescence microscopy allows noninvasive 3D imaging of the interior of living specimens with molecular specificity, and is therefore an invaluable resource to the biological sciences. Unfortunately, the resolving power of fluorescence microscopy is fundamentally limited by the diffraction of light.Lately, many techniques have been introduced to extend the resolution beyond the classic diffraction limit (1-8). Although the improvement of spatial resolution is impressive, the practical impact of these new techniques will largely depend on whether they can keep the two key advantages of fluorescence microscopy, namely, the capability of imaging living cells in three dimensions and multicolor labeling. Live-cell imaging over many time points has been demonstrated for some resolution-extending techniques (9-15) but, to our knowledge, not for multicolor 3D imaging of whole cells.Stimulated emission depletion (STED) microscopy increases the spatial resolution by suppressing the fluorescence emission on the rim of a focused laser spot, theoretically enabling unlimited resolution (16). STED microscopy has been demonstrated for live imaging at high frame rates (12, 15), but its point-scanning nature makes it unsuitable for fast volumetric imaging over large fields of view. Furthermore, to achieve high spatial resolution, very high power densities (38-540 MW/cm 2 ) (12) are necessary for the STED laser beam, which may cause increased photobleaching and phototoxicity, thus limiting the application of live-cell STED microscopy.In localization-based microscopy, only sparse subsets of photoswitchable fluorophores in a sample are imaged at a time, which allows the isolation and precise localizati...

mentioning

confidence: 99%

Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Fiolka

Shao

Rego

et al. 2012

300

253

“…In real space, the resolution limit is described by the minimal fluorescence spot size, which for a standard highaperture confocal microscope is Ϸ180 and Ϸ500 nm in the transverse and axial directions, respectively (1). The demand for higher resolution in biological imaging has spurred a number of interesting developments, such as scanning 4Pi-confocal microscopy (3), stimulated emission depletion fluorescence microscopy (4, 5), wide-field I 5 M (6), and combinations of structured illumination with image restoration (7,8).…”

mentioning

confidence: 99%

Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast

Egner

Jakobs

Hell

2002

278

216

By introducing beam-scanning multifocal multiphoton 4Pi-confocal microscopy, we have attained fast fluorescence imaging of live cells with axial super resolution. Rapid scanning of up to 64 pairs of interfering high-angle fields and subsequent confocal detection enabled us to perform three to five times finer optical sectioning than confocal microscopy. In conjunction with nonlinear image restoration, we demonstrate, to our knowledge for the first time, three-dimensional imaging of live eukaryotic cells at an equilateral resolution of Ϸ100 nm. This imaging mode allowed us to reveal the morphology and size of the green fluorescent protein-labeled mitochondrial compartment of live Saccharomyces cerevisiae (bakers' yeast) growing on different carbon sources. Our studies show that mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum. We determine the average tubular diameter and find that it increases from 339 ؎ 5 nm to 360 ؎ 4 nm when changing from glucose to glycerol, that is, from a fermentable to a nonfermentable carbon source. Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 ؎ 0.2. F ocused light is the only means by which it is possible to visualize and quantify biochemical processes in a live cell. Catalyzed by the advent of specific exogenous and endogenous markers, such as the green fluorescent protein (GFP) and its mutants, fluorescence-based far-field microscopy has evolved into a powerful tool for elucidating the relationship between cellular structure and function. This fact particularly applies to confocal and multiphoton fluorescence microscopes, which, by providing three-dimensional (3D) images, facilitate quantitative studies in the interior of cells (1). Despite its obvious advantages, the application of far-field microscopy has been seriously restricted. A major reason is the limited spatial resolution that has precluded 3D imaging and the quantitative 3D analysis of submicron-scale cellular compartments.In Fourier space, where the imaging process is expressed in terms of spatial frequencies, the limited resolution of light microscopes is attributed to their inability to grasp and transfer object frequencies higher than about one-half of the reciprocal wavelength (2). In real space, the resolution limit is described by the minimal fluorescence spot size, which for a standard highaperture confocal microscope is Ϸ180 and Ϸ500 nm in the transverse and axial directions, respectively (1). The demand for higher resolution in biological imaging has spurred a number of interesting developments, such as scanning 4Pi-confocal microscopy (3), stimulated emission depletion fluorescence microscopy (4, 5), wide-field I 5 M (6), and combinations of structured illumination with image restoration (7,8).In fluorescence imaging, the mathematical function describing th...

“…In any case, the pattern has to be scanned across the specimen and the camera read out for each position of the pattern. Scanning with line patterns in conjunction with deconvolution has been shown to enhance the lateral resolution by up to 2-fold over conventional microscopy (22,23). However, the fundamental advantage of a (similarly parallelized) RESOLFT type of microscope over the latter is the fact that, in the RESOLFT case, the resolution increase is not limited by diffraction.…”

Section: Discussion Conclusion and Outlookmentioning

confidence: 99%

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

Hofmann

Eggeling

Jakobs

et al. 2005

750

673

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 GW͞cm 2 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.photoswitching ͉ nanoscopy ͉ resolution ͉ saturation ͉ photochromic