Confocal microscopes have been the workhorses of 3-D biological imaging, but they are slow, offer limited depth penetration and collect only ballistic photons. With their inefficient use of excitation photons they expose biological samples to an often intolerably high light burden. The speed limitation and photo-bleaching risk can be somewhat relaxed in a spinning-disk geometry, due to shorter pixel dwell times and rapid re-scans during image capture. Alternatively, light-sheet microscopes rapidly image large volumes of transparent or chemically cleared samples. Finally, with infrared excitation and efficient scattered-light collection, 2-photon microscopy allows deep-tissue imaging, but it remains slow. Here, we describe a new optical scheme that borrows the best from three different worlds: the speed and direct-view from a spinning-disk confocal, deep tissue-penetration and intrinsic optical sectioning from 2-photon excitation, and a large field of view and a low invasiveness of a selective-plane illumination microscope – all with a single objective lens. We validate the performance of our 2-photon spinning-disk microscope in various applications that have in common to simultaneously require a large depth penetration, high speed and larger fields of view. Beyond biological fluorescence, we demonstrate an application in material science, imaging coherent non-linear scattering from a 3-D nano-porous network.

Human inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies (EBs) enlarge the scope of investigations to multi-cellular phenomena. The highest level of complexity, brain organoids that—in many aspects—recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that uses planar two-photon (2P) excitation. Its particularity is that—unlike two- or three-lens light-sheet microscopes—it uses a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petráň spinning-disk scheme for achieving wide-field excitation. However, unlike the Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This new design, advantageous for 2P excitation, circumvents problems arising with the tandem disk from the large wavelength difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited by the light sheet is collected with the same objective and imaged onto a fast sCMOS camera. We demonstrate 3-D imaging of TO-PRO3-stained EBs and of brain organoids, uncleared and after rapid partial transparisation with triethanolamine formamide (RTF) and we compare the performance of our instrument to that of a confocal laser-scanning microscope (CLSM) having a similar numerical aperture. Our large-field 2P-spinning disk microscope permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

Here we report the successful first operation of FLUMIAS-DEA, a miniaturized high-resolution 3D fluorescence microscope on the International Space Station (ISS) by imaging two scientific samples in a temperature-constant system, one sample with fixed cells and one sample with living human cells. The FLUMIAS-DEA microscope combines features of a high-resolution 3D fluorescence microscope based on structured illumination microscope (SIM) technology with hardware designs to meet the requirements of a space instrument. We successfully demonstrated that the FLUMIAS technology was able to acquire, transmit, and store high-resolution 3D fluorescence images from fixed and living cells, allowing quantitative and dynamic analysis of subcellular structures, e.g., the cytoskeleton. The capability of real-time analysis methods on ISS will dramatically extend our knowledge about the dynamics of cellular reactions and adaptations to the space environment, which is not only an option, but a requirement of evidence-based medical risk assessment, monitoring and countermeasure development for exploration class missions.

The FLUMIAS (Fluorescence-Microscopic Analyses System for Life-Cell-Imaging in Space) confocal laser spinning disk fluorescence microscope represents a new imaging capability for live cell imaging experiments on suborbital ballistic rocket missions. During the second pioneer mission of this microscope system on the TEXUS-54 suborbital rocket flight, we developed and performed a live imaging experiment with primary human macrophages. We simultaneously imaged four different cellular structures (nucleus, cytoplasm, lysosomes, actin cytoskeleton) by using four different live cell dyes (Nuclear Violet, Calcein, LysoBrite, SiR-actin) and laser wavelengths (405, 488, 561, and 642 nm), and investigated the cellular morphology in microgravity (10−4 to 10−5 g) over a period of about six minutes compared to 1 g controls. For live imaging of the cytoskeleton during spaceflight, we combined confocal laser microscopy with the SiR-actin probe, a fluorogenic silicon-rhodamine (SiR) conjugated jasplakinolide probe that binds to F-actin and displays minimal toxicity. We determined changes in 3D cell volume and surface, nuclear volume and in the actin cytoskeleton, which responded rapidly to the microgravity environment with a significant reduction of SiR-actin fluorescence after 4–19 s microgravity, and adapted subsequently until 126–151 s microgravity. We conclude that microgravity induces geometric cellular changes and rapid response and adaptation of the potential gravity-transducing cytoskeleton in primary human macrophages.

Of all 3D-super resolution techniques, structured illumination microscopy (SIM) provides the best compromise with respect to resolution, signal-to-noise ratio (S/N), speed and cell viability. Its ability to achieve double resolution in all three dimensions enables resolving 3D-volumes almost 10× smaller than with a normal light microscope. Its major drawback is noise contained in the out-of-focus-signal, which—unlike the out-of-focus signal itself—cannot be removed mathematically. The resulting “noise-pollution” grows bigger the more light is removed, thus rendering thicker biological samples unsuitable for SIM. By using a slit confocal pattern, we employ optical means to suppress out-of-focus light before its noise can spoil SIM mathematics. This not only increases tissue penetration considerably, but also provides a better S/N performance and an improved confocality. The SIM pattern we employ is no line grid, but a two-dimensional hexagonal structure, which makes pattern rotation between image acquisitions obsolete and thus simplifies image acquisition and yields more robust fit parameters for SIM.

Optical visualization of neural network activity is limited by imaging system‐dependent technical tradeoffs. To overcome these constraints, we have developed a powerful low‐cost and flexible imaging system with high spectral variability and unique spatio‐temporal precision for simultaneous optical recording and manipulation of neural activity of large cell groups. The system comprises eight high‐power light‐emitting diodes, a camera with a large metal‐oxide‐semiconductor sensor and a high numerical aperture water‐dipping objective. It allows fast and precise control of excitation and simultaneous low noise imaging at high resolution. Adjustable apertures generated two independent areas of variable size and position for simultaneous optical activation and image capture. The experimental applicability of this system was explored in semi‐isolated preparations of larval axolotl ( Ambystoma mexicanum ) with intact inner ear organs and central nervous circuits. Cyclic galvanic stimulation of semicircular canals together with glutamate‐ and γ‐aminobutyric acid ( GABA )‐uncaging caused a corresponding modulation of Ca 2+ transients in central vestibular neurons. These experiments revealed specific cellular properties as well as synaptic interactions between excitatory and inhibitory inputs, responsible for spatio‐temporal‐specific sensory signal processing. Location‐specific GABA ‐uncaging revealed a potent inhibitory shunt of vestibular nerve afferent input in the predominating population of tonic vestibular neurons, indicating a considerable impact of local and commissural inhibitory circuits on the processing of head/body motion‐related signals. The discovery of these previously unknown properties of vestibular computations demonstrates the merits of our novel microscope system for experimental applications in the field of neurobiology.

Here we introduce a novel fluorescence microscope concept which expands the application of functional fluorescence imaging and further enables optical manipulation of biological samples. Taking advantage of the LED technique allows a space saving housing of multiple light sources and illumination at various wavelengths. Furthermore, the microscope provides an optical separation into two independently controllable excitation pathways enabling a simultaneous illumination of different regions which are adjustable in size. Image detection is realized via a super resolution high speed camera that allows high resolution image capturing (2560x2160) at rates up to 100 Hz. Moreover, we implemented a voice-coil driven high NA objective to ensure z-movement at maximum speed and precision. The multiple LED arrangement and the separation of excitation pathways therefore facilitate the usage of multiple imaging approaches at the same time. To test the performance of the microscope we used in vitro whole head preparations of Ambystoma mexicanum tadpoles. This preparation allows studying neuronal systems with all sensory pathways intact and can be maintained up to one week. Thus, we were able to use calcium imaging to record sensory evoked neuronal responses of central vestibular neurons elicited by electric stimulation of specific semicircular canals. Further, to quantify the glutamate uncaging efficiency we calculated the required light intensity and duration to optically evoke action potentials by patching those neurons. Finally, we could show that by means of spatially separating calcium imaging and glutamate uncaging we were able to manipulate ipsilateral semicircular canal evoked calcium responses by optically activating contralateral inhibitory/excitatory pathways.

In contrast to previously presented methods [1, 2] our technique does not use predefined scan trajectories or scans restricted in the axial dimension. Rather our trajectory mode connects user-defined points in space not limited by number and scans them either in order of input or in the fastest calculated way. This technique was shown to work in the GCaMP-stained antennal lobe of drosophila. It will be used to identify olfactory glomeruli matching specific odors and to visualize the time-resolved reactions of the glomeruli distributed in 3D. The image mode allows scanning user-defined two-dimensional images lying in different depths repeatedly with high frequency. This was used to image a group of neurons within a chick dorsal root ganglion bulkstained with Fluo-3 upon stimulation of a neighboring neuron. A rectangular region of interest was fit into each neuron in order to increase the signal to noise ratio compared to a single line going through the cell. 2D scans were repeatedly imaged to record the temporal behavior of the calcium signal.

2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small deviations in the sample orientation, however, make comparison of three-dimensional image stacks taken at different time-points challenging. When analysing changes of three-dimensional structures over time (4D imaging) this fundamental problem is one of the main limitations when complex structures are studied repetitively. We used an upright two-photon microscope complemented with a software-controlled stage-rotation instead of a conventional stage for chronic in vivo imaging in the brain of transgenic mouse models of Alzheimer's disease. Before every session an optimal imaging condition was successfully created by aligning the surface of the cranial window perfectly perpendicular to the laser beam. Deviations in the sample orientation between consecutive imaging sessions could be eliminated which improves conditions for chronic in vivo studies.

Low-magnification high-numerical aperture objectives maximize the collection efficiency for scattered two-photon excited fluorescence (2PEF), but non-descanned detection schemes for such objectives demand optical components much bigger than standard microscope optics. Fiber coupling offers the possibility of removing bulky multi-channel detectors from the collection site, but coupling and transmission losses are generally believed to outweigh the benefits of optical fibers. We present here two new developments based on large-core fiber-optic fluorescence detection that illustrate clear advantages over conventional air-coupled 2PEF detection schemes. First, with minimal modifications of a commercial microscope, we efficiently couple the output of a 20×/NA0.95 objective to a large-core liquid light guide and we obtain a 7-fold collection gain when imaging astrocytes at 100 μm depth in acute brain slices of adult ALDH1L1-GFP mice. Second, combining 2PEF microscopy and 4-color detection on a custom microscope, mode scrambling inside a 2-mm plastic optical fiber is shown to cancel out the spatially non-uniform spectral sensitivity observed with air-coupled detectors. Spectral unmixing of images of brainbow mice taken with a fiber-coupled detector revealed a uniform color distribution of hippocampal neurons across a large field of view. Thus, fiber coupling improves both the efficiency and the homogeneity of 2PEF collection.

DOI of original article:10.1016/j.jneumeth.2011.03.015. ∗ Corresponding author at: Laboratorie de Neurophysiologie et Nouvelles Microscopies Universite Paris Descartes, Paris F-75006, France. Tel.: +33 1 42 86 42 21; fax: +33 42 86 42 41 51. E-mail address: martin.oheim@parisdescartes.fr (M. Oheim). 1 Present address: Dept. of Neurophysiology, Brain Research Institute, University of Zurich, Winterhurestrasse 190, CH-8057 Zurich, Switzerland.

The goal was to develop a light-microscope platform concept, which allows characterization of live cells in microtiter plates or in live-cell slide chambers with a speed, sensitivity and versatility unattainable so far.The goal was achieved by combining several novel technological concepts: a model-based digital control for a voice coil focus drive; scanner technology to follow a continuously moving sample during image acquisition, thus avoiding the usual stop-and-go; fast sectioning capabilities by using slit-scan confocal concepts; motorized dual emission image registration; and integrated environmental control.

The goal was to develop an upright microscope platform for the screening of slides employing one- and two-photon laser scanning techniques. A highly compact, vibration damping unit was created, which combines novel concepts for moving a slide in three dimensions, keeping it focused while doing so, scanning a laser-focus over the sample using novel galvanometer-control concepts, combining and separating excitation and emission beam and spectrally dispersing the emitted light by a linearized prism-spectrograph. Spectral detection is achieved by turning a 128 x 128 back-thinned EMCCD detector in a continuously reading spectral point-detector. To make the unit even ore versatile, it can be turned into a conventional wide field fluorescence microscope, enabling rapid routine observation to select regions of the sample for a subsequent, more detailed confocal analysis.

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited‐state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild‐type(wt)‐GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.