In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.

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Far-Field Optical Nanoscopy

A sensor system is adapted for use with an article of footwear and includes an insert member including a first layer and a second layer, a port connected to the insert and configured for communication with an electronic module, a plurality of force and/or pressure sensors on the insert member, and a plurality of leads connecting the sensors to the port.

Footwear having sensor system

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 (lambda = 400-700 nm) in the focal plane and >lambda along the optical axis (z). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40-45 nm (lambda/16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-lambda-sized organelles.

/pdf/spherical-nanosized-focal-spot-unravels-the-interior-of-11w9l7qhqj.pdf

Spherical nanosized focal spot unravels the interior of cells

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

Two-photon fluorescence excitation and related techniques in biological microscopy.

Fluorescence imaging methods that push or break the diffraction limit of resolution (approximately 200 nm) have grown explosively. These super-resolution nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resolution, speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity—down to single molecules with tens of nanometers resolution. Nanoscopes have revealed transient intermediate states of organelles and molecules in living cells and have led to new discoveries but also biological controversies. We highlight common unifying principles behind nanosc...

A new wave of cellular imaging.

https://pure.mpg.de/pubman/item/item_598391_5/component/file_598390/218581.pdf

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.

The invention discloses an illuminating device ( 1 ) having a laser ( 3 ) that emits a light beam ( 7 ), which is directed onto a microstructured optical element ( 13 ) that spectrally broadens the light from the laser. The laser ( 3 ) and the microstructured optical element ( 13 ) are arranged within the casing.

Illuminating device and microscope

The present invention concerns an apparatus for combining light from at least two laser light sources, preferably in the context of confocal scanning microscopy, and in order to make laser light sources of low output power usable as light sources, in particular for confocal scanning microscopy, is characterized in that the light from the laser light sources has at least approximately the same wavelength; and that at least one beam combining unit that combines the light beams in at least largely lossless fashion is provided.

Apparatus for combining light and confocal scanning microscope

The arrangement has a scanning microscope and contains a laser (10 and an optical arrangement (12) that forms an image of the light from the laser onto the specimen (13) under investigation. An optical component (3) is arranged between the laser and the optical arrangement to spectrally expand the laser light during a single pass through. Independent claims are also included for the following: a scanning microscope and an illumination arrangement for a scanning microscope.

Arrangement for investigating microscopic preparations, has optical component between scanning laser and imaging optical arrangement to spectrally expand laser light during single pass

The invention discloses an entangled-photon microscope ( 1 ) having a light source ( 3 ) and an objective ( 31 ). The entangled-photon microscope ( 1 ) has a microstructured optical element ( 11 ), in which entangled photons can be produced, between the light source ( 3 ) and the objective ( 31 ), the entangled photons propagating in a beam ( 15 ) inside and outside the microstructured optical element.

Rafael Storz

Papers

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.

Illuminating device and microscope

Apparatus for combining light and confocal scanning microscope

Arrangement for investigating microscopic preparations, has optical component between scanning laser and imaging optical arrangement to spectrally expand laser light during single pass

Entangled-photon microscope and confocal microscope