Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Hell, Stefan W.; Schrader, Martin; Voort, H. T. M. van der

doi:10.1046/j.1365-2818.1997.2410797.x

Cited by 114 publications

(73 citation statements)

References 23 publications

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“…Phase artifacts leading to noteworthy misrepresentations of the objects can be ruled out. Nonlinear image restoration further increases the resolution by 50% (13), so that we applied nonlinear image processing to all 3D sets shown further in the paper. The final transverse resolution of Ϸ140 and Ϸ100 nm along the optic axis is not yet sufficient to discriminate single cristae; however, the 3D representation of the tubular structure of the mitochondria is fundamentally improved.…”

Section: Multifocal Multiphoton 4pimentioning

confidence: 99%

“…This concept is particularly powerful in connection with multiphoton excitation, because it substantially reduces undesired interference side lobes and provides a higher contrast of the interference pattern. Thus multiphoton 4Pi-confocal microscopy leads to a reliable axial resolution improvement (3,13) of the order of 90-150 nm in both aqueously mounted (14) and glycerol-mounted fixed cells (13). Unfortunately, relying on stage scanning, whereby the sample is scanned slowly through the focus of the lenses, 4Pi-confocal microscopy has been hampered by slow data acquisition.…”

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

Egner

Jakobs

Hell

2002

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

281

216

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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...

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Section: Multifocal Multiphoton 4pimentioning

confidence: 99%

mentioning

confidence: 99%

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

Egner

Jakobs

Hell

2002

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

281

216

View full text Add to dashboard Cite

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“…2 Two-photon techniques, which break the diffraction resolution limit, are based on point spread function engineering and must be mentioned when giving a general review of imaging techniques for biosciences. 4π microscopy, STED microscopy and triple-state depletion microscopy are just some of these methods, which have been especially developed in the group of Stefan Hell (Max Plank Institute, Goettingen, Germany) [23][24][25] . These techniques are particularly appropriate for optical imaging on the nanometer scale but are completely inadequate for measurements in (usually) thick biological samples.…”

Section: Fluorescence Imaging Techniquesmentioning

confidence: 99%

Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

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“…As the mounting medium of our specimens (glycerol) had a refractive index smaller than that of immersion oil and glass, n Ϸ 1.47 as opposed to n ϭ 1.51, the optical paths' lengths and hence the phase of the two beams changed during the axial translation of the sample. It has been shown that this phase change is a linear function of the scan and can be actively compensated by synchronously moving the piezo mirror with the axial movement of the sample (Egner et al, 1998;Hell et al, 1997). Therefore, in a largely uniform volume such as the cytoplasm of a fixed cell, the 4Pi PSF can be kept constant even in the presence of a significant refractive index mismatch between the mounting medium and the glass cover slip.…”

Section: Pi-confocal Microscopymentioning

confidence: 99%

4Pi-Confocal Microscopy Provides Three-Dimensional Images of the Microtubule Network with 100- to 150-nm Resolution

Nagorni

Hell

1998

Journal of Structural Biology

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We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse fibroblast cell. Comparison with two-photon confocal resolution reveals a fourfold better axial resolution in the 4Pi-confocal case. By combining 4Pi-confocal microscopy with Richardson-Lucy image restoration a further resolution increase is achieved. Featuring a threedimensional resolution in the range 100-150 nm, the 4Pi-confocal (restored) images are intrinsically more detailed than their confocal counterparts. Our images constitute what to our knowledge are the best-resolved three-dimensional images of entangled cellular microtubules obtained with light to date. Academic Press

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Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range

Cited by 114 publications

References 23 publications

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

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

Fluorescence lifetime imaging in biosciences: technologies and applications

4Pi-Confocal Microscopy Provides Three-Dimensional Images of the Microtubule Network with 100- to 150-nm Resolution

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