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By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

 

Introduction Using data collected from hearing aid users’ own hearing aids could improve the customization of hearing aid processing for different users based on the auditory environments they encounter in daily life. Prior studies characterizing hearing aid users’ auditory environments have focused on mean sound pressure levels and proportions of environments based on classifications. In this study, we extend these approaches by introducing entropy to quantify the diversity of auditory environments hearing aid users encounter. Materials and Methods Participants from 4 groups (younger listeners with normal hearing and older listeners with hearing loss from an urban or rural area) wore research hearing aids and completed ecological momentary assessments on a smartphone for 1 week. The smartphone was programmed to sample the processing state (input sound pressure level and environment classification) of the hearing aids every 10 min and deliver an ecological momentary assessment every 40 min. Entropy values for sound pressure levels, environment classifications, and ecological momentary assessment responses were calculated for each participant to quantify the diversity of auditory environments encountered over the course of the week. Entropy values between groups were compared. Group differences in entropy were compared to prior work reporting differences in mean sound pressure levels and proportions of environment classifications. Group differences in entropy measured objectively from the hearing aid data were also compared to differences in entropy measured from the self-report ecological momentary assessment data. Results Auditory environment diversity, quantified using entropy from the hearing aid data, was significantly higher for younger listeners than older listeners. Entropy measured using ecological momentary assessment was also significantly higher for younger listeners than older listeners. Discussion Using entropy, we show that younger listeners experience a greater diversity of auditory environments than older listeners. Alignment of group entropy differences with differences in sound pressure levels and hearing aid feature activation previously reported, along with alignment with ecological momentary response entropy, suggests that entropy is a valid and useful metric. We conclude that entropy is a simple and intuitive way to measure auditory environment diversity using hearing aid data. 

Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.