Spherical nanosized focal spot unravels the interior of cells

  title={Spherical nanosized focal spot unravels the interior of cells},
  author={Roman Schmidt and Christian A Wurm and Stefan Jakobs and Johann Engelhardt and Alexander Egner and Stefan W. Hell},
  journal={Nature Methods},
The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light (λ = 400–700 nm) in the focal plane and >λ along the optical axis (z). Although concepts have been developed to… 
Pi-RESOLFT nanoscopy
By enlarging the aperture along the optic axis, the coherent utilization of opposing objective lenses (4Pi arrangement) has the potential to offer the sharpest and most light-efficient
4Pi-RESOLFT nanoscopy
The 4Pi scheme is applied to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins and it is shown that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible.
STED Microscopy
was considered to be the resolution limit, with λ being the wavelength, n the refractive index of the medium, and α the half aperture angle of the objective lens. Equation (10.1) represents the full
22. Fluorescence Microscopy with Nanometer Resolution
Throughout the twentieth century, it was widely accepted that a light microscope relying on propagating light waves and conventional optical lenses could not discern details that were much finer than
Super‐Resolution Microscopy: A Comparative Treatment
The basic principles of the various super‐resolution imaging modalities are outlined, paying particular attention to the technical considerations for biological imaging, and their various applications in the imaging of both fixed and live biological samples are discussed.
Fluorescence Microscopy with Nanometer Resolution
Throughout the twentieth century, it was widely accepted that a light microscope relying on propagating light waves and conventional optical lenses could not discern details that were much finer than
Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures
A dSTORM system has been constructed and calibrated using a commercially available inverted florescence microscope and total internal reflection florescence (TIRF) imaging and the point spatial distribution of EEA1 is investigated by using the linearised form of Ripley's K-function H(r) and the null hypothesis of complete spatial randomness tested.
Overcoming diffraction limit: From microscopy to nanoscopy
ABSTRACT The advancement of super-resolution imaging technologies has significantly extended microscopy to nanoscopy, making it possible for the study of physical, chemical, and biological processes
A new wave of cellular imaging.
This work examines the optical principles and design of these super-resolution nanoscopy techniques and their ability to see more detail with greater sensitivity--down to single molecules with tens of nanometers resolution.
Axial localization and tracking of self-interference nanoparticles by lateral point spread functions
It is reported that single upconversion nanoparticles, containing multiple emission centres with random orientations, can generate a series of unique, bright and position-sensitive patterns in the spatial domain when placed on top of a mirror.


Photochromic rhodamines provide nanoscopy with optical sectioning.
Fluorescence microscopy concepts emerged that demonstrated that the limiting role of diffraction could be fundamentally overcome, and the main hallmark of these concepts was to use the states of the fluorescent marker not just for generating the signal, but also for breaking the diffraction barrier.
Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.
It is shown that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support.
Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy
3D stochastic optical reconstruction microscopy (STORM) is demonstrated by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy, allowing the 3D morphology of nanoscopic cellular structures to be resolved.
Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.
The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited
Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement
This study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time and map and describe the vesicle mobility within the highly confined space of synaptic boutons.
Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution.
  • M. Gustafsson
  • Physics
    Proceedings of the National Academy of Sciences of the United States of America
  • 2005
Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples, and a recently proposed method in which the nonlinearity arises from saturation of the excited state is experimentally demonstrated.
Lateral resolution of 28 nm (λ /25) in far-field fluorescence microscopy
We demonstrate sub-diffraction lateral resolution of 28±2 nm in far-field fluorescence microscopy through stimulated emission depletion effected by an amplified laser diode. Measurement of the
Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution.
The sub-diffraction spots created with focused light of lambda = 760 nm wavelength and conventional optics enable for the first time far-field fluorescence microscopy with resolution at the tens of nanometer scale, as demonstrated in images of membranes of bacillus megaterium.
Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast
Three-dimensional imaging of live eukaryotic cells at an equilateral resolution of ≈100 nm is demonstrated for the first time, and 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.