Acousto-optic systems for advanced microscopy

Acoustic waves in an optical medium cause rapid periodic changes in the refraction index, leading to diffraction effects. Such acoustically controlled diffraction can be used to modulate, deflect, and focus light at microsecond timescales, paving the way for advanced optical microscopy designs that feature unprecedented spatiotemporal resolution. In this article, we review the operational principles, optical properties, and recent applications of acousto-optic (AO) systems for advanced microscopy, including random-access scanning, ultrafast confocal and multiphoton imaging, and fast inertia-free light-sheet microscopy. As AO technology is reaching maturity, designing new microscope architectures that utilize AO elements is more attractive than ever, providing new exciting opportunities in fields as impactful as optical metrology, neuroscience, embryogenesis, and high-content screening

Multiplane Encoded Light-Sheet Microscopy for Enhanced 3D Imaging

Light-sheet microscopes have become the tool of choice for volumetric imaging of large samples. Based on a wide-field acquisition scheme, they are capable of optical sectioning at diffraction-limited resolution and minimal overall photodamage. Unfortunately, traditional architectures are limited in speed because 3D images are collected by either sample translation or synchronized movement of both light-sheet and detection objective lens. A promising solution avoiding slow mechanical movements is to extend the depth-of-field of the microscope and moving only the light-sheet. However, this normally comes at the cost of losing light and contrast, compromising the signal-to-noise ratio of the images. Here, we propose an innovative technique devoted to restoring the quality of the images, while preserving the speed of extended depth-of-field microscopes. It is based on generating a stack of parallel light-sheets using a pair of orthogonal acousto-optic deflectors, enabling the simultaneous illumination of different sample planes. Given the extended depth-of-field, all such planes appear in focus and can be acquired in a superimposed single frame. By applying a single-step inversion algorithm, we can decode a stack of frames into a volumetric image whose signal-to-noise ratio and contrast are greatly enhanced. We provide a detailed theoretical framework of the method and demonstrate its feasibility with volumetric images of kidney cell spheroids

SmartScope2: Simultaneous Imaging and Reconstruction of Neuronal Morphology.

Quantitative analysis of neuronal morphology is critical in cell type classification and for deciphering how structure gives rise to function in the brain. Most current approaches to imaging and tracing neuronal 3D morphology are data intensive. We introduce SmartScope2, the first open source, automated neuron reconstruction machine integrating online image analysis with automated multiphoton imaging. SmartScope2 takes advantage of a neuron\u27s sparse morphology to improve imaging speed and reduce image data stored, transferred and analyzed. We show that SmartScope2 is able to produce the complex 3D morphology of human and mouse cortical neurons with six-fold reduction in image data requirements and three times the imaging speed compared to conventional methods

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Combined voltage and calcium imaging and signal calibration

Voltage imaging using fluorescent voltage-sensitive dyes can be combined with other optical measurements, in particular with Ca2+ imaging, allowing for correlation of membrane potential changes with intracellular Ca2+ signals. Calibration of fluorescence voltage signals permits the comparison of membrane potential changes from different sites allowing spatial mapping of membrane potential changes. These two technical aspects enhance the capability of voltage imaging to address several fundamental problems of neurobiology. Here we discuss how to combine voltage imaging with the optical measurement of intracellular Ca2+ transients and different approaches to calibrate voltage-sensitive dye signals on an absolute scale

Activity-dependent modulation of K+ currents at presynaptic terminals of mammalian central synapses

The activity-dependent regulation of presynaptic K+ currents at the CA3-CA1 synapse in the rat hippocampus was investigated during a train of evoked afferent action potentials. The waveforms of presynaptic compound action potentials (cAPs) and presynaptic Ca2+ transients ([Ca2+]pre,t) were measured with fluorescent voltage-sensitive and Ca2+-sensitive indicators in rat brain slices.Under control conditions, presynaptic cAPs and the accompanying [Ca2+]pre,t displayed similar amplitudes for each stimulus, suggesting that there was no cumulative change of K+ and Ca2+ currents during the test train. However, when a subgroup of presynaptic K+ channels was blocked by a low concentration of 4-aminopyridine (4-AP, 40 μm), a significant facilitation of the [Ca2+]pre,t was observed.This phenomenon was not due to a direct action of 4-AP on presynaptic Ca2+ channels, but to cumulative suppression of the K+ conductance as indicated by the corresponding change in waveforms of the cAP and presynaptic fibre volley. The observed facilitation was not an artifact by virtue of increased fibre recruitment, nor was it related to the accumulation of extracellular K+; rather, it was dependent on Ca2+ influx and stimulation frequency. The time course of recovery from facilitation was closely related to the decay of the intracellular Ca2+ concentration.The facilitation was not blocked by a saturating concentration of 4-AP (8 mM) but was reduced during the application of the K+ channel blocker tetraethylammonium (TEA, 10 mM), implicating the involvement of TEA-sensitive K+ channels. Such activity-dependent suppression of presynaptic K+ conductance could lead to excessive transmitter release and might explain the hippocampal epileptiform activity that can be induced by application of 4-AP