Biophysics

Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD⁺ ratio and the intracellular Ca²⁺ levels in individual dentate granule cells from acute hippocampal slices.

Graphic abstract:

Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.

Visualizing the function of pancreatic β-cells in vivo has been a long-sought goal for β-cell researchers. Unlike imaging of β-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of β-cells in their native physiological microenvironment in vivo. Here, we detail a protocol for real-time visualization of individual β-cell function in vivo in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg (ins:Rcamp1.07) with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous β-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote β-cell function or maturation using a living whole organism system.

Elevations in cytosolic calcium (Ca²⁺) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca²⁺ indicators remains the gold standard for visualization and quantification of intracellular Ca²⁺ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca²⁺ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca²⁺ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability – an essential requirement for recording dynamic changes in cytosolic Ca²⁺ levels – and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca²⁺ signals over time using Fiji (ImageJ) software.

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in absolute cross sections must be characterized. Excitation at optimum wavelength will make it possible to reduce the laser power and therefore minimize photodamage. Obtaining 2PA spectra and cross sections requires correcting the two-photon excited fluorescence signals for a combination of laser properties, including the beam spatial profile, pulse duration, and absolute power, at each wavelength of the tuning range. To avoid such tedious day-to-day laser characterization required in the absolute measurement method, a relative method based on independently characterized 2PA reference standards is often used. By carefully analyzing the available literature data, we selected the most reliable standards for both the 2PA spectral shape and cross section measurements. Here we describe a protocol for measuring the 2PA spectral shapes and cross sections of fluorescent proteins and other fluorophores with the relative fluorescence method using these reference standards. Our protocol first describes how to build an optical system and then how to perform the measurements. In our protocol, we use Coumarin 540A in dimethyl sulfoxide and LDS 798 in chloroform for the spectral shape measurements to cover the range from 680 to 1300 nm, and Rhodamine 590 in methanol and Fluorescein in alkaline water (pH 11) for the absolute two-photon cross section measurements.