TAG-SPARK: Empowering High-Speed Volumetric Imaging With Deep Learning and Spatial Redundancy.

Two-photon high-speed fluorescence calcium imaging stands as a mainstream technique in neuroscience for capturing neural activities with high spatiotemporal resolution. However, challenges arise from the inherent tradeoff between acquisition speed and image quality, grappling with a low signal-to-noise ratio (SNR) due to limited signal photon flux. Here, a contrast-enhanced video-rate volumetric system, integrating a tunable acoustic gradient (TAG) lens-based high-speed microscopy with a TAG-SPARK denoising algorithm is demonstrated.

Roadmap on Label-Free Super-Resolution Imaging.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency.

Electronic Preresonance Stimulated Raman Scattering Spectromicroscopy Using Multiple-Plate Continuum.

Stimulated Raman scattering (SRS) spectromicroscopy is a powerful technique that enables label-free detection of chemical bonds with high specificity. However, the low Raman cross section due to typical far-electronic resonance excitation seriously restricts the sensitivity and undermines its application to bio-imaging. To address this bottleneck, the electronic preresonance (EPR) SRS technique has been developed to enhance the Raman signals by shifting the excitation frequency toward the molecular absorption.

Towards stimulated Raman scattering spectro-microscopy across the entire Raman active region using a multiple-plate continuum.

Stimulated Raman scattering (SRS) has attracted increasing attention in bio-imaging because of the ability toward background-free molecular-specific acquisitions without fluorescence labeling. Nevertheless, the corresponding sensitivity and specificity remain far behind those of fluorescence techniques. Here, we demonstrate SRS spectro-microscopy driven by a multiple-plate continuum (MPC), whose octave-spanning bandwidth (600-1300 nm) and high spectral energy density (∼1 nJ/cm) enable spectroscopic interrogation across the entire Raman active region (0-4000 cm), SRS imaging of a Drosophila brain, and electronic pre-resonance (EPR) detection of a fluorescent dye.