Significance: Light-field microscopy (LFM) enables fast, light-efficient, volumetric imaging of neuronal activity with calcium indicators. Calcium transients differ in temporal signal-to-noise ratio (tSNR) and spatial confinement when extracted from volumes reconstructed by different algorithms.
Aim: We evaluated the capabilities and limitations of two light-field reconstruction algorithms for calcium fluorescence imaging.
Approach: We acquired light-field image series from neurons either bulk-labeled or filled intracellularly with the red-emitting calcium dye CaSiR-1 in acute mouse brain slices. We compared the tSNR and spatial confinement of calcium signals extracted from volumes reconstructed with synthetic refocusing and Richardson–Lucy three-dimensional deconvolution with and without total variation regularization.
Results: Both synthetic refocusing and Richardson–Lucy deconvolution resolved calcium signals from single cells and neuronal dendrites in three dimensions. Increasing deconvolution iteration number improved spatial confinement but reduced tSNR compared with synthetic refocusing. Volumetric light-field imaging did not decrease calcium signal tSNR compared with interleaved, widefield image series acquired in matched planes.
Conclusions: LFM enables high-volume rate, volumetric imaging of calcium transients in single cell somata (bulk-labeled) and dendrites (intracellularly loaded). The trade-offs identified for tSNR, spatial confinement, and computational cost indicate which of synthetic refocusing or deconvolution can better realize the scientific requirements of future LFM calcium imaging applications.
KEYWORDS: Signal to noise ratio, Deconvolution, 3D image processing, Image resolution, Luminescence, Dendrites, Point spread functions, Neurophotonics, Microscopes, Microlens
Significance: Light-field microscopy (LFM) enables high signal-to-noise ratio (SNR) and light efficient volume imaging at fast frame rates. Voltage imaging with genetically encoded voltage indicators (GEVIs) stands to particularly benefit from LFM’s volumetric imaging capability due to high required sampling rates and limited probe brightness and functional sensitivity.
Aim: We demonstrate subcellular resolution GEVI light-field imaging in acute mouse brain slices resolving dendritic voltage signals in three spatial dimensions.
Approach: We imaged action potential-induced fluorescence transients in mouse brain slices sparsely expressing the GEVI VSFP-Butterfly 1.2 in wide-field microscopy (WFM) and LFM modes. We compared functional signal SNR and localization between different LFM reconstruction approaches and between LFM and WFM.
Results: LFM enabled three-dimensional (3-D) localization of action potential-induced fluorescence transients in neuronal somata and dendrites. Nonregularized deconvolution decreased SNR with increased iteration number compared to synthetic refocusing but increased axial and lateral signal localization. SNR was unaffected for LFM compared to WFM.
Conclusions: LFM enables 3-D localization of fluorescence transients, therefore eliminating the need for structures to lie in a single focal plane. These results demonstrate LFM’s potential for studying dendritic integration and action potential propagation in three spatial dimensions.
Surface plasmon resonance (SPR) at planar metal/dielectric interfaces and localised SPR (LSPR) for metal nanoparticles have both been extensively studied, but it is less clear what happens to the optical properties of surface plasmon-polaritons (SPPs) at the micrometer scale. This paper characterises the angular responses of microscale gold patterns on a glass substrate using an SPR imaging configuration using a high numerical aperture objective lens. The potential use for biosensing is also discussed.
The propagation length of a SPP equals approximately 10 μm, so the geometries that are investigated are 10 × 16, 10 × 10, 6 × 16, and 6 × 6 μm sized rectangles with 2 μm spacings. The gold patterns are photolithographically produced using an image reversal process. Bright-field microscopy is used to investigate their morphology and stylus profilometry is used to check the gold thickness. The SPR responses were compared to planar gold film to see if they were characteristic and confirm the relation to SPR.
All the gold microstructures present plasmonic properties. SPR dips are observed for all samples, however, they are not as sensitive and are wider than that of planar gold film. This phenomenon becomes more pronounced as the length of the gold structure decreases, because of the spatial constriction of the propagating SPP. The process suggests that the SPR technique can be successfully implemented to detect individual action potentials. Further work is required in order to achieve a reliable process and investigate the capabilities and sensitivity of the proposed technique.
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