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.

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.

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca²⁺ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca²⁺ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca²⁺ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

We previously developed an incoherent holography technique for use in lattice light sheet (LLS) microscopes that represents a specialized adaptation of light sheet microscopy. Light sheet instruments resolve 3D information by illuminating the sample at 90° to the imaging plane with a sheet of laser light that excites fluorophores in the sample only in a narrow plane. Imaging this plane and then moving it in the imaging z-axis allows construction of the sample volume. Among these types of instruments, LLS microscopy gives higher z-axis resolution and tissue depth penetration. It has a similar working principle to light sheet fluorescence microscopy but uses a lattice configuration of Bessel beams instead of Gaussian beams. Our incoherent light detection technique replaces the glass tube lens of the original LLS with a dual diffractive lens system to retrieve the axial depth of the sample. Here, we show that the system is applicable to all light sheet instruments. To make a direct comparison in the same emission light path, we can imitate the nature of non-Bessel light sheet systems by altering the mask annuli used to create Bessel beams in the LLS system. We change the diffractive mask annuli from a higher NA anulus to a smaller NA anulus. This generates a Gaussian excitation beam similar to conventional light sheet systems. Using this approach, we propose an incoherent light detection system for light sheet 3D imaging by choosing a variable NA and moving only the light sheet while keeping the sample stage and detection microscope objective stationary.