Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.     

Residence Times of Receptors in Dendritic Spines Analyzed by Stochastic Simulations in Empirical Domains

Analysis of high-density superresolution imaging of receptors reveals the organization of dendrites at nanoscale resolution. We present here an apparently novel method that uses local statistics extracted from short-range trajectories for the simulations of long-range trajectories in empirical live cell images. Based on these empirical simulations, we compute the residence time of a receptor in dendritic spines that accounts for receptors’ local interactions and geometrical membrane organization. We report here that depending on the type of the spine, the residence time varies from 1 to 5 min. Moreover, we show that there exists transient organized structures, previously described as potential wells that can regulate the trafficking of receptors to dendritic spine: the simulation results suggest that receptor trafficking is regulated by transient structures.     

A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.     

Two-color STED microscopy of living synapses using a single laser-beam pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.     

Nanoscopy of Filamentous Actin in Cortical Dendrites of a Living Mouse

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43–70 nm resolution reveal dynamical changes in spine morphology.The postsynaptic part of most excitatory synapses in the brain is formed by dendritic spines, which are small protrusions along the dendrites that are highly dynamic during development, but also undergo morphological changes in adulthood (1,2). A prime candidate for regulating these dynamics is the neuronal actin network (3). Filamentous (F-) actin is also important for anchoring postsynaptic receptors and modulating synaptic activities, e.g., through the organization of the postsynaptic density (3). Clearly, the actin dynamics of dendritic spines is best studied in vivo, e.g., in a living mouse, and with confocal and multiphoton microscopy because these techniques can provide three-dimensional optical sectioning several 100 μm inside brain tissue (4). However, because necks of dendritic spines are on the 50–150-nm scale, their details are beyond the 250–400-nm resolution afforded by these diffraction-limited techniques. Fortunately, the diffraction resolution barrier of lens-based fluorescence microscopy has recently been overcome by causing the fluorophores of nearby features to emit sequentially (5). One of the techniques relying on this principle, stimulated emission depletion (STED) microscopy, has recently resolved dendritic spines in the cortex of a living mouse (6). In that initial, in vivo superresolution study, the dendrites were only volume-labeled, and consequently, the spatial arrangements of specific cytoskeletal proteins could not be imaged. On the other hand, F-actin has actually been imaged in living brain slices (7), but in vivo imaging of these structures has not yet been attained.Compared to other superresolution or nanoscopy techniques, STED microscopy bears a number of advantages for imaging spines in the living brain. Implemented as a beam scanning confocal microscope, STED nanoscopy offers optical sectioning and measurements at greater depth. In addition, motion artifacts of the dynamic structures can be minimized by fast scanning. And last but not least, STED can be performed with standard fluorescent proteins. Therefore, we here apply STED nanoscopy to noninvasively uncover the actin cytoskeleton in the living mouse brain. In particular, we show that the 43–70-nm resolution obtained by STED visualizes rearrangements of the dendritic spines in vivo.We took on the challenge of labeling the actin cytoskeleton in the living mouse cortex. We utilized Lifeact-EYFP, a fusion protein consisting of a small peptide and the yellow fluorescent protein EYFP, which directly binds to F-actin without disturbing its polymerization (8). The labeling itself was accomplished by viral infection. To this end, adeno-associated viral particles (AAV) of serotype 2, facilitated by the neuron specific human synapsin promoter hSYN (9) and Semliki Forest viruses (SFV), were created to express Lifeact-EYFP in neurons. For virus injection, the mouse was anesthetized and the head was fixed in a model No. SG-4N head holder (Narishige International USA, East Meadow, NY). A 5-mm incision of the skin of the head enabled drilling a 0.5-mm-diameter hole into the skull. The hole was positioned 0.5 mm outside the prospective imaging center in the visual cortex. The AAVs were injected with a micropipette connected to a pressure generator (Tooheyspritzer; Toohey Company, Fairfield, NJ). Thus, we were able to inject ∼750 nL of concentrated AAV at an angle of 30° over a time of ∼5 min to the layer of pyramidal cells in the prospective imaging center. After injection and 5-min pause, the pipette was retracted with a 5-min break at the half-way point to allow the virus to diffuse into the tissue. The skin was closed with three stitches and the mouse kept on a heating plate in an anesthetic recovery box until wake-up.After 10 days the mouse was prepared for in vivo STED nanoscopy, according to Berning et al. (6) (see also the Supporting Material). At this point, the skin had completely healed and the mouse showed no sign of obvious behavioral abnormality. Optical access was provided by a glass-sealed hole of ∼2 mm in diameter, exposing the visual cortex (Fig. 1 a). STED nanoscopy was performed with an upright beam-scanning microscope similar to that described by Berning et al. (6), with short optical paths and good vibration-damping (Fig. 1 b and see the Supporting Material). The coaligned excitation and STED beams were focused onto the mouse brain using a 1.3 numerical-aperture glycerol immersion lens. The correction collar of the lens allowed compensation of spherical aberrations arising from focusing beneath the brain surface (7).Open in a separate windowFigure 1STED nanoscopy of the dendritic filamentous (F-) actin cytoskeleton in the visual cortex of a living mouse. (a) Clear view of the visual cortex through an optical window. (b) Upright STED imaging of the anesthetized mouse. (c) Dendritic F-actin in the molecular layer of the visual cortex at 4, 25, and 40-μm depths. Maximum intensity projection of a stack of five (xy) images taken in 500-nm axial (z) distances. (Right) Line profile at the marked positions; average of five lines of the raw data and Lorentz fit with full width at half-maximum (FWHM); all image data are raw.Fig. 1 c shows representative parts of dendrites in the molecular layer of the visual cortex. The combination of Lifeact-EYFP labeling and superresolution displayed the dendritic actin of the living mouse neuron in unprecedented detail. Most spines have an actin-rich bulbous end, i.e., a spine head. Sometimes, the dendrite shows small areas with high actin enrichment, which presumably constitute the beginning of filopodia outgrowths (see Fig. S1 in the Supporting Material). The STED image quality was maintained down to a depth of 40 μm below the cover glass. The actin filaments in the spine neck were 43–70-nm thin (see Fig. S2), which can also be interpreted as an upper estimate (poorest value) for the resolution obtained by STED. Note that the images were not processed after recording. All dendrites appeared normal, i.e., in comparison with the morphology of volume-labeled pyramidal cells of transgenic mice (6). The STED beam average laser power was 34 mW. For somewhat greater laser power, we occasionally saw swelling of the dendrites but they were never destroyed. The maximum applicable power depends on the thickness of the dendrite and most likely on the presence of mitochondria as well.Next, we raised the expression level of Lifeact-EYFP by replacing AAV with SFV infection (7,10). We injected 750 nL of SFV (see the Supporting Material) analog to the AAV protocol and allowed the mouse to wake up and recover. After one day, we recorded in vivo STED nanoscopy images of the visual cortex. The labeling was sparser than with the AAV, i.e., fewer cells expressed Lifeact-EYFP, but the signal was brighter and highly specific to neurons. Fig. 2 shows a STED image of a part of a dendrite in the visual cortex at depth <10 μm. The actin label is brighter in the spine heads than in the body of the dendrite, showing that Lifeact-EYFP is primarily attached to F-actin. STED recording over 12 min revealed morphological changes in the actin cytoskeleton. No changes were observed after fixation (see Fig. S4). Bleaching-corrected brightness changes in the spine head inherently reflect density changes in the actin network. In contrast to AAV, SFV shuts down host cell protein synthesis, which leads to cell death after >24 h (11,12); this was not improved by the less cytotoxic SFV(PD) variant (11). Therefore, we recorded in vivo nanoscopy images one day after viral transduction where most dendrites looked healthy. To confirm the viral transduction and verify the subtype of the infected neurons, we perfused the mouse with paraformaldehyde and imaged the brain slices of the region of interest (see Fig. S5). Whereas the AAV labeled mainly neurons of the pyramidal layer, the SFV infected sparsely neurons from all layers of the cortex.Open in a separate windowFigure 2Actin rearrangement in dendritic spines at 60-nm subdiffraction spatial resolution. Image stacks reveal dynamic changes of actin in the spines. (Arrows) Shape changes of spine heads. Maximum intensity projection of five slices of 500-nm axial (z) separation; all data are raw. Average power at back-aperture of objective lens: 2.4 μW excitation and 38-mW STED.The 4–5-fold lateral resolution improvement of STED over standard confocal and multiphoton microscopy is not sufficient to resolve single actin fibers, as with platinum replica electron microscopy (13). Future refinements of both labeling and STED imaging should make this goal achievable. The resolution along the optical (z) axis was kept diffraction-limited (∼500 nm) so that the total illumination dose remained small. At depth >40 μm, scattering and aberrations compromise the image quality. Nonetheless, the molecular layer of the sensory cortex is a highly interesting target for functional optical nanoscopy, because it is the site of the first stage of cortical sensory processing.In summary, STED microscopy can be applied to study subcellular protein structures at 43–70-nm resolution down to 40 μm in the brain of a living mammal. Specifically, we showed that the dynamic actin network responsible for the morphologic plasticity in the brain can be superresolved in the living mouse. Extending in vivo STED microscopy to other protein assemblies as well as to other cell types should provide basic insights into the working principles of the brain.     

STED nanoscopy of actin dynamics in synapses deep inside living brain slices

It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.     

A Highlights from MBoC Selection: Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics

Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro­tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30–100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging.     

Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.     

Time-lapse imaging of dendritic spines in vitro

Dendritic spines are small protrusions present postsynaptically at approximately 90% of excitatory synapses in the brain. Spines undergo rapid spontaneous changes in shape that are thought to be important for alterations in synaptic connectivity underlying learning and memory. Visualization of these dynamic changes in spine morphology are especially challenging because of the small size of spines (approximately 1 microm). Here we describe a microscope system, based on a spinning-disk confocal microscope, suitable for imaging mature dendritic spines in brain slice preparations, with a time resolution of seconds. We discuss two commonly used in vitro brain slice preparations and methods for transfecting them. Preparation and transfection require approximately 1 d, after which slices must be cultured for at least 21 d to obtain spines of mature morphology. We also describe imaging and computer analysis routines for studying spine motility. These procedures require in the order of 2 to 4 h.     

Two-photon imaging of dendritic spine development in the mouse cortex

Dendritic spines are the postsynaptic sites of most excitatory synapses in the mammalian brain. With the advent of two-photon microscopy and transgenic mice expressing fluorescent proteins, dendritic spines can now be imaged in the living cerebral cortex over time scales ranging from seconds to years. Recent studies with this in vivo imaging approach have begun to provide important insights into the development and plasticity of dendritic spines in the intact brain. Here, we review these studies and discuss technical requirements for image acquisition. We envision that intravital two-photon imaging at the level of individual synapses will greatly expand our current understandings of how neuronal networks are assembled and modified throughout life.     

Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy

Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.      

Nanoscopy of Filamentous Actin in Cortical Dendrites of a Living Mouse

We demonstrate superresolution fluorescence microscopy (nanoscopy) of protein distributions in a mammalian brain in vivo. Stimulated emission depletion microscopy reveals the morphology of the filamentous actin in dendritic spines down to 40 μm in the molecular layer of the visual cortex of an anesthetized mouse. Consecutive recordings at 43–70 nm resolution reveal dynamical changes in spine morphology.     

Rapid neurotransmitter uncaging in spatially defined patterns

Light-sensitive 'caged' molecules provide a means of rapidly and noninvasively manipulating biochemical signals with submicron spatial resolution. Here we describe a new optical system for rapid uncaging in arbitrary patterns to emulate complex neural activity. This system uses TeO(2) acousto-optical deflectors to steer an ultraviolet beam rapidly and can uncage at over 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing us to combine patterned uncaging with imaging and electrophysiology. By photolyzing caged neurotransmitter in brain slices we can generate precise, complex activity patterns for dendritic integration. The method can also be used to activate many presynaptic neurons at once. Patterned uncaging opens new vistas in the study of signal integration and plasticity in neuronal circuits and other biological systems.     

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.     

Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses

Activity-induced modification of neuronal connections is essential for the development of the nervous system and may also underlie learning and memory functions of mature brain. Previous studies have shown an increase in dendritic spine density and/or enlargement of spines after the induction of long-term potentiation (LTP). Using two-photon time-lapse imaging of dendritic spines in acute hippocampal slices from neonatal rats, we found that the induction of long-term depression (LTD) by low-frequency stimulation is accompanied by a marked shrinkage of spines, which can be reversed by subsequent high-frequency stimulation that induces LTP. The spine shrinkage requires activation of NMDA receptors and calcineurin, similar to that for LTD. However, spine shrinkage is mediated by cofilin, but not by protein phosphatase 1 (PP1), which is essential for LTD, suggesting that different downstream pathways are involved in spine shrinkage and LTD. This activity-induced spine shrinkage may contribute to activity-dependent elimination of synaptic connections.     

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.     

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.     

LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.     

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.