Fresnel incoherent correlation holography (FINCH) was a milestone in incoherent holography. In this roadmap, two pathways, namely the development of FINCH and applications of FINCH explored by many prominent research groups, are discussed. The current state-of-the-art FINCH technology, challenges, and future perspectives of FINCH technology as recognized by a diverse group of researchers contributing to different facets of research in FINCH have been presented.

We propose an Incoherent holography detection technique for lattice light-sheet (IHLLS) systems for 3D imaging without moving either the sample stage or the detection microscope objective, providing intrinsic instrumental simplicity and high accuracy when compared to the original LLS schemes. The approach is based on a modified dual-lens Fresnel Incoherent Correlation Holography technique to produce a complex hologram and to provide the focal distance needed for the hologram reconstruction. We report such an IHLLS microscope, including characterization of the sensor performance, and demonstrate a significant contrast improvement on beads and neuronal structures within a biological test sample as well as quantitative phase imaging. The IHLLS has similar or better transverse performances when compared to the LLS technique. In addition, the IHLLS allows for volume reconstruction from fewer z-galvo displacements, thus facilitating faster volume acquisition.

Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca²⁺entry and neurotransmitter release. However, Ca²⁺requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca²⁺entry may occur through small numbers or even single open Ca²⁺channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca²⁺channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca²⁺entry, using cell-attached patch clamp and lattice light-sheet microscopy (LLSM), resolving small channel numbers evoking Ca²⁺entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca²⁺channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca²⁺transients exhibiting quantal amplitude variations of 0–6 event sizes between individual APs and stochastic variation of precise locations of Ca²⁺entry within the AZ. Further, total Ca²⁺channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca²⁺entry via as few as one channel may trigger neurotransmitter release.SIGNIFICANCE STATEMENTPresynaptic Ca²⁺entry through voltage-gated calcium channels (VGCCs) causes neurotransmitter release. To understand neurotransmission, its modulation, and plasticity, we must quantify Ca²⁺entry and its relationship to vesicle fusion. This requires direct recordings from active zones (AZs), previously possible only at calyceal terminals containing many AZs, where few channels open following action potentials (APs; Sheng et al., 2012), and even single channel openings may trigger release (Stanley, 1991, 1993). However, recording from more conventional terminals with single AZs commonly found centrally has thus far been impossible. We addressed this by cell-attached recordings from acutely dissociated single lamprey giant axon AZs, and by lattice light sheet microscopy of presynaptic Ca²⁺entry. We demonstrate nanodomains of presynaptic VGCCs coupling with primed vesicles with 1:1 stoichiometry.

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective.