Mariana Potcoava

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca ²⁺ channels and Ca ²⁺ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca ²⁺ dynamics at the soma is often performed, the imaging of Ca ²⁺ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca ²⁺ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca ²⁺ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca ²⁺ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

We present quantitative 3D live nerve cell imaging with subcellular resolution and extended FOV using incoherent holographic lattice light-sheet system with an optimized SLM phase mask modulation for shaping the light collected from the sample.

We present quantitative 3D live nerve cell imaging using Gaussian-beam light-sheet modalities with conventional lattice light-sheet and interferometric optical detection approaches. The interferometric detection approach offers maximum FOV scanning area than the conventional one.

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.

The purpose of the article is to explore the need and advantages of using the incoherent color holography lattice light-sheet (ICHLLS) to provide multiwavelength quantitative monitoring of 3D cellular dynamics in live tissue to further understand complex functions of cells and cellular compartments. We have explored the use of incoherent color holography lattice light-sheet to investigate colocalization of fluorescent markers in live cells in intact tissue. Neuronal structures provide an attractive target for incoherent color holography lattice light-sheet. The cells show a complex architecture in 3D space in which signaling both between cells and within subcellular structures requires colocalization of proteins and lipids to function. During activity and over long periods it is important in understanding these signaling functions in Parkinson’s, Alzheimer’s and motoneuron diseases within live cells in intact tissue. As a proof of concept this article recalls the key aspects in lattice light-sheet imaging and provides a description of the incoherent detection system configuration to actively control dual diffractive lenses phase-shifting at multiple excitation wavelengths sequentially, and per each z-galvo scanning level, with extended field-of-view. The incoherent color holography lattice light-sheet system will allow simultaneous recording of multidimensional object waves that contain intensity in 3D space, phase, and wavelength information. We measure colocalization of fluorescence indicators introduced into live cells in intact neural tissue.

Recently, lattice light-sheet instruments and imaging technology have shown a great improvement in exploring the dynamics of life at subcellular resolution. However, to reconstruct 3-dimensional structures the objective lens or the sample stage are invariably moved. Invention of incoherent holography lattice light-sheet (IHLLS) using FINCH provides quantitative information on the state and size of subcellular changes of neurons over the visible spectrum. The technique allows both faster three-dimensional amplitude as well as phase imaging without moving either sample stage or the detection objective. Amplitude and phase measurements provide intrinsic instrumental simplicity, larger scanning area, and higher resolution when compared to the original LLS schemes. IHLLS is built as an additional detection arm of an original lattice light-sheet (LLS) system. In this chapter, we review the evolution of IHLLS to address its feasibility and limitations.

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.

Raman spectroscopy using feature selection schemes has considerable advantages over gas chromatography for the analysis of fatty acids’ composition changes. Here, we introduce an educational methodology to demonstrate the potential of micro-Raman spectroscopy to determine with high accuracy the unsaturation or saturation degrees and composition changes of the fatty acids found in the lipid droplets of the LNCaP prostate cancer cells that were treated with various fatty acids. The methodology uses highly discriminatory wavenumbers among fatty acids present in the sample selected by using the Support Vector Machine algorithm. Key words Raman spectroscopy Support vector machine (SVM) Lipids Fatty acids Prostate cancer Near-infrared Noninvasive