In near-field scanning optical microscopy, a light source or detector with dimensions less than the wavelength (lambda) is placed in close proximity (lambda/50) to a sample to generate images with resolution better than the diffraction limit. A near-field probe has been developed that yields a resolution of approximately 12 nm ( approximately lambda/43) and signals approximately 10(4)- to 10(6)-fold larger than those reported previously. In addition, image contrast is demonstrated to be highly polarization dependent. With these probes, near-field microscopy appears poised to fulfill its promise by combining the power of optical characterization methods with nanometric spatial resolution.

Commentary: Introduced the adiabatically tapered single mode fiber probe to near-field scanning optical microscopy which, together with shear force feedback, made the technique a practical reality. Although earlier claims of superresolution via near-field microscopy existed for nearly a decade, this paper was the first to convincingly break Abbe’s limit with visible light, as demonstrated by reproducibly resolving known, complex nanoscale patterns having features separated by much less than the wavelength. Whereas our fiber probe and shear force technologies were soon widely adopted and key to many novel applications (see above), the earlier methods proved to be technological dead ends, never achieving the results of their original claims. This experience taught me the most valuable lesson of my career: while it’s bad to bullshit others, it’s even worse to bullshit yourself. It’s a lesson sadly unheeded by many current practitioners of superresolution microscopy.

The performance of mass spectrometers with limited pumping capacity is shown to be improved through use of a discontinuous atmospheric pressure interface (DAPI). A proof-of-concept DAPI interface was designed and characterized using a miniature rectilinear ion trap mass spectrometer. The interface consists of a simple capillary directly connecting the atmospheric pressure ion source to the vacuum mass analyzer region; it has no ion optical elements and no differential pumping stages. Gases carrying ionized analytes were pulsed into the mass analyzer for short periods at high flow rates rather than being continuously introduced at lower flow rates; this procedure maximized ion transfer. The use of DAPI provides a simple solution to the problem of coupling an atmospheric pressure ionization source to a miniature instrument with limited pumping capacity. Data were recorded using various atmospheric pressure ionization sources, including electrospray ionization (ESI), nano-ESI, atmospheric pressure chemical ionization (APCI), and desorption electrospray ionization (DESI) sources. The interface was opened briefly for ion introduction during each scan. With the use of the 18 W pumping system of the Mini 10, limits of detection in the low part-per-billion levels were achieved and unit resolution mass spectra were recorded.

Optical microscopy has so far been restricted to superficial layers, leaving many important biological questions unanswered. Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth. Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves. Deep inside highly scattering media, the sound focus dimensions restrict the imaging resolutions. Here we show that by iteratively focusing light into an ultrasound focus via phase conjugation, we can fundamentally overcome this resolution barrier in deep tissues and at the same time increase the focus to background ratio. We demonstrate fluorescence microscopy beyond the ballistic regime of light with a threefold improved resolution and a fivefold increase in contrast. This development opens up practical high resolution fluorescence imaging in deep tissues.

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Many unexpected discoveries in developmental biology have depended on advancement of imaging technologies to visualize developmental processes as they unfold across multiple spatial and temporal scales. This essay surveys the recent advances in imaging, highlighting emerging capabilities with an eye toward those poised to have the greatest impact on developmental biology.

Genetically encodable calcium ion (Ca) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

Small-molecule fluorophores manifest the ability of chemistry to solve problems in biology. As we noted in a previous review (Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142-155), the extant collection of fluorescent probes is built on a modest set of "core" scaffolds that evolved during a century of academic and industrial research. Here, we survey traditional and modern synthetic routes to small-molecule fluorophores and highlight recent biological insights attained with customized fluorescent probes. Our intent is to inspire the design and creation of new high-precision tools that empower chemical biologists.

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular "core" dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.