We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation.
Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To address this issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-photon light-sheet microscopy the maximum achievable frame rate is limited by the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarisation, and we implemented remote scanning of the focal plane to record without disturbing the sample. Using this setup, as a preliminary result, we characterized the intensity spectra of neuronal calcium traces of 4 days post fertilisation larvae during the day/night phases. We aim to extend these results to multiple brain regions and frequency bands.
The three-dimensional reconstruction of large volumes of the human neural networks at cellular resolution is one of the biggest challenges of our days. Commonly, fine slices of samples marked with colorimetric techniques are individually imaged. This approach in addition to being time-consuming does not consider space cell organization, leading to loss of information. The aim of this work was to develop a methodology that allows analyzing the cytoarchitecture of the human brain in three dimensions at high resolution. In particular, we exploit the possibility of combining high-resolution 3D imaging techniques with clearing methodologies. We successfully integrate the SWITCH immunohistochemistry technique with the TDE clearing method to image a large volume of human brain tissue with two-photon fluorescence microscopy. In conclusion, this new approach enables to characterize large human brain specimens with high-resolution optical techniques, giving the possibility to expand the histological studies to the third dimension.
Recently, optogenetics has provided interesting insights into cardiovascular research, leading to cardiac pacing, re-synchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac pathway manipulation, optical stimulation has not been directly driven by the cardiac electrical dynamics, limiting the full potential of such a new technology. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control whole mouse heart electrical activity. The system combines an ultrafast wide-field mesoscope with a digital micro-mirror device (Texas Instruments Lightcrafter), capable of drawing arbitrarily-chosen patterns, thus allowing optogenetic activation. Cardiac functionality can be manipulated either in free-run mode with sub-millisecond temporal resolution or in a closed-loop fashion: an ad hoc hardware and software platform allows real-time intervention capable of reacting to threatening anomalous electrical conditions within 2 ms. The methodology has been applied to restore atrioventricular block, by triggering the optical stimulation of the ventricle according to optically mapped atrial activity. Furthermore, real-time intra-ventricular manipulation of the propagating electrical wave-front has been demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. The development of this innovative optical methodology provides the first proof-of-concept that a real-time, self-sustaining, optical-based stimulation can efficiently control cardiac rhythm in normal and abnormal conditions, promising a new approach to the investigation of (patho)physiology of the heart.
Remodeling processes associated with genetic and non-genetic cardiac diseases can cause alterations of electrical conduction and electro-mechanical dysfunction, eventually leading to arrhythmias. These alterations consist mainly in collagen deposition (fibrosis) and cellular disorganization (myofilament alignment), and their predictive models are often based on non-integrated and low-resolution information. Here, we combine advances in tissue clearing, immunostaining and high-resolution optical microscopy to reconstruct the three-dimensional organization of cardiac conduction system in the whole mouse heart. We developed a passive 2’2-thiodiethanol - Clarity protocol for clearing the heart and for achieving antibody penetration into the whole tissue. We simultaneously reconstructed the cellular organization stained by immunostaining and imaged the collagen distribution by second-harmonic generation deep in the cardiac tissue. A cytoarchitectonic analysis was applied to identify cells and to map myofilaments alignment in three dimensions, defining the conduction pathway of action potential propagation at intercellular level. We investigated the three-dimensional cytoarchitectonic remodeling in a transgenic mouse model of hypertrophic cardiomyopathy characterized by a severe degree of left ventricle hypertrophy and interstitial fibrosis. First, using a recently-developed ultra-fast optical system we mapped the propagation of electrical activity in whole diseased hearts. Then, we correlated the propagation maps with the pathological disorganization of myofilaments and collagen deposition using the three-dimensional high-resolution optical reconstruction. This innovative experimental approach will allow to dissect the morphological causes leading to alterations of electrical conduction and to electro-mechanical dysfunction, and, more generally, will represent a whole new paradigm for diagnostic and therapeutic investigations.
Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.
Light-sheet microscopy enables whole mouse brain imaging in association with clearing methodologies. Here, we present a pipeline for optimal investigation of the vascular component, which offers improved image quality for morphological analysis.
KEYWORDS: Robotics, In vivo imaging, Brain, Two photon excitation microscopy, Spine, Neurons, Cortical activation, Optogenetics, Functional imaging, Current controlled current source
Neurorehabilitation protocols based on the use of robotic devices provide a highly repeatable therapy and have recently shown promising clinical results. Little is known about how rehabilitation molds the brain to promote motor recovery of the affected limb. We used a custom-made robotic platform that provides quantitative assessment of forelimb function in a retraction test. Complementary imaging techniques allowed us to access to the multiple facets of robotic rehabilitation-induced cortical plasticity after unilateral photothrombotic stroke in mice Primary Motor Cortex (Caudal Forelimb Area - CFA). First, we analyzed structural features of vasculature and dendritic reshaping in the peri-infarct area with two-photon fluorescence microscopy. Longitudinal analysis of dendritic branches and spines of pyramidal neurons suggests that robotic rehabilitation promotes the stabilization of peri-infarct cortical excitatory circuits, which is not accompanied by consistent vascular reorganization towards pre-stroke conditions. To investigate if this structural stabilization was linked to functional remapping, we performed mesoscale wide-field imaging on GCaMP6 mice while performing the motor task on the robotic platform. We revealed temporal and spatial features of the motor-triggered cortical activation, shining new light on rehabilitation-induced functional remapping of the ipsilesional cortex. Finally, by using an all-optical approach that combines optogenetic activation of the contralesional hemisphere and wide-field functional imaging of peri-infarct area, we dissected the effect of robotic rehabilitation on inter-hemispheric cortico-cortical connectivity.
Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6.
Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity.
We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.
Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.
Fatal cardiac arrhythmias are a major medical and social issue in Western countries. Current implantable pacemaker/defibrillators have limited effectiveness and are plagued by frequent malfunctions and complications. Here, we aim at setting up a new method to map and control the electrical activity of whole isolated mouse hearts. We employ a transgenic mouse model expressing Channel Rhodopsin-2 (ChR2) in the heart coupled with voltage optical mapping to monitor and control action potential propagation. The whole heart is loaded with the fluorinated red-shifted voltage sensitive dye (di-4-ANBDQPQ) and imaged with the central portion (128 x 128 pixel) of sCMOS camera operating at frame rate of 1.6 kHz. The wide-field imaging system is implemented with a random access ChR2 activation developed using two orthogonally-mounted acousto-optical deflectors (AODs). AODs rapidly scan different sites of the sample with a commutation time of 4 μs, allowing us to design ad hoc ChR2-stimulation pattern. First, we demonstrate the capability of our system in manipulating the conduction system of the whole mouse heart by changing the electrical propagation features. Then, we explore the efficacy of the random access ChR2 stimulation in inducing arrhythmias as well as to restore the cardiac sinus rhythm during an arrhythmic event. This work shows the potentiality of this new method for studying the mechanisms of arrhythmias and reentry in healthy and diseased hearts, as well as the basis of intra-ventricular dyssynchrony.
Ludovico Silvestri, Nikita Rudinskiy, Marco Paciscopi, Marie Caroline Müllenbroich, Irene Costantini, Leonardo Sacconi, Paolo Frasconi, Bradley Hyman, Francesco Pavone
Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information.
Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution.
The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.
Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples. The strong entanglement between clearing methods and imaging technology is often overlooked, as typical classification of the former is based only on the chemicals used. Here, we review the recent literature in the field, proposing a taxonomy of clearing techniques based on their mating with the major high-throughput microscopies. We hope that this application-oriented classification can help researchers to find the protocol best suited to their experiment among the many present in the literature.
Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.
M. Caroline Müllenbroich, Ludovico Silvestri, Leonardo Onofri, Irene Costantini, Marcel van’t Hoff, Leonardo Sacconi, Giulio Iannello, Francesco S. Pavone
Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.
Light scattering inside biological tissue is a limitation for large volumes imaging with microscopic resolution. Based on
refractive index matching, different approaches have been developed to reduce scattering in fixed tissue. High refractive
index organic solvents and water-based optical clearing agents, such as Sca/e, SeeDB and CUBIC have been used for
optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the
fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high
transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a
highly expensive refractive index matching solution limiting practical applicability to large volumes.
In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear
mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not
introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform
brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue
slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for
CLARITY. The potential of this method has been explored by imaging blocks of dysplastic human brain transformed
with CLARITY, immunostained and cleared with the TDE. This clearing approach significantly expands the application
of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in
mouse and human brain.
Nowadays, there are several imaging techniques offering a complementary approach to visualize intact neural networks on large areas. Each of those offers a different strategy and furnish complementary information on the role of neural components. We will describe different approaches enabling to move from single neuron details to whole brain imaging, connecting short range structural information to long range one. In particular, some examples of correlative microscopies, combining linear and non linear techniques will also be described.
KEYWORDS: Brain, Microscopy, Neuroimaging, In vivo imaging, Functional magnetic resonance imaging, Neurons, Axons, Calcium, Electron microscopy, Brain mapping
Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation.
Brain imaging is becoming an important field in the frame of the neurophotonics in correlations with other medical ones in neuroscience studying functional and morphological aspects. In this presentation an overview on multi photon imaging of the brain will be presented, together with innovative aspects related to big area imaging and correlative microscopy approaches. Multiphoton imaging applications will be described together with methods to improve the penetration depth and obtain large area detection, or correlating functional aspects in vivo on single neuron with large area, even on whole brain, morphological aspects. Connecting super resolution features at the nanometer level with micro, meso and macroscopic architectures is in fact one of the challenging aspects to understand brain functioning.
T-tubules (TT) are invaginations of the surface sarcolemma (SS) that mediate the rapid propagation of the action potential (AP) to the cardiomyocyte core. We employed the advantages of an ultrafast random access multi-photon (RAMP) microscope (Sacconi et al., PNAS 2012) with a double staining approach to optically record t-tubular AP and, simultaneously, the corresponding local Ca2+-release in different positions across the cardiomyocytes. Despite a uniform AP between SS and TT at steady-state stimulation, in control cardiomyocytes we observed a non-negligible be variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced by applying 0.1μM Isoproterenol, which increases the opening probability of Ca2+-release units. In the rat heart failure model (HF), we previously demonstrated that some tubular elements fail to propagate AP. We found that the tubules unable to propagate AP, displayed a reduced correspondent Ca2+-transient amplitude as well as a slower Ca2+ rise compared to electrically coupled tubules. Moreover variability of Ca2+-transient kinetics were increased in HF. Finally, TT that did not show AP, occasionally exhibited spontaneous depolarizations that were never accompanied by local Ca2+-release in the absence of any pro-arrhythmogenic stimulation. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodeled tubules. This work was funded by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162).
KEYWORDS: In vivo imaging, Axons, Neurons, Californium, Electron microscopy, Dendrites, Optical fibers, Cranial windows, Two photon imaging, Microscopy
Multi-photon imaging provides valuable insights into the continuous reshaping of neuronal connectivity in live brain. We
previously showed that single neuron or even single spine ablation can be achieved by laser-mediated dissection.
Furthermore, single axonal branches can be dissected avoiding collateral damage to the adjacent dendrite and the
formation of a persistent glial scar. Here, we describe the procedure to address the structural plasticity of cerebellar
climbing fibers by combining two-photon in vivo imaging with laser axotomy in a mouse model. This method is a
powerful tool to study the basic mechanisms of axonal rewiring after single branch axotomy in vivo. In fact, despite the
denervated area being very small, the injured axons consistently reshape the connectivity with surrounding neurons, as
indicated by the increase in the turnover of synaptic boutons. In addition, time-lapse imaging reveals the sprouting of
new branches from the injured axon. Newly formed branches with varicosities suggest the possible formation of synaptic
contacts. Correlative light and electron microscopy revealed that the sprouted branch contains large numbers of vesicles,
with varicosities in the close vicinity of Purkinje dendrites.
One of the unique features of the brain is that its activity cannot be framed in a single spatio-temporal scale, but rather spans many orders of magnitude both in space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.
We combined the advantage of an ultrafast random access microscope with novel labelling technologies to study the intra- and inter-cellular action potential propagation in neurons and cardiac myocytes with sub-millisecond time resolution. The random accesses microscopy was used in combination with a new fluorinated voltage sensitive dye with improved photostability to record membrane potential from multiple Purkinje cells with near simultaneous sampling. The RAMP system rapidly scanned between lines drawn in the membranes of neurons to perform multiplex measurements of the TPF signal. This recording was achieved by rapidly positioning the laser excitation with the AOD to sample a patch of membrane from each cell in <100 μs; for recording from five cells, multiplexing permits a temporal resolution of 400 μs sufficient to capture every spike. The system is capable to record spontaneous activity over 800 ms from five neighbouring cells simultaneously, showing that spiking is not temporally correlated. The system was also used to investigate the electrical properties of tubular system (TATS) in isolated rat ventricular myocytes.
KEYWORDS: Californium, In vivo imaging, Injuries, Two photon imaging, Optical fibers, Axons, Green fluorescent protein, Nervous system, Information operations, Luminescence
In the adult nervous system, different neuronal classes show different regenerative behavior. Although previous studies demonstrated that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Here we describe the approach followed to characterize the reactive plasticity after injury.
Fluorescence light sheet microscopy has known a true renaissance in the last years. In fact, since optical sectioning is achieved in a wide-field detection scheme, this technique allows high resolution three-dimensional imaging with high frame rate. Light sheet microscopy is therefore an ideal candidate for reconstructing macroscopic specimens with micron resolution: coupled with clearing protocols based on refractive index matching it has been exploited to image entire mouse brains without physical sectioning. Use of clearing protocols poses several challenges to light sheet microscopy. First of all, residual light scattering inside the tissue expands the excitation light sheet, leading to the excitation of out-of-focus planes, and thus frustrating the very principle of light sheet illumination. To reject out-of-focus contributions we recently coupled light sheet illumination with confocal detection, achieving significant contrast enhancement in real time. Another issue which often arises when working with clearing agents is the refractive index mismatch between the clearing and the medium objective design medium. This introduces severe spherical aberration, which leads to broadening of the point spread function and to a strong reduction in its peak value: When imaging deep (several mm) inside macroscopic specimens, the signal can be reduced by more than an order of magnitude. We investigated the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path.
KEYWORDS: Microscopy, In vivo imaging, Neurons, Brain, Confocal microscopy, Neuroimaging, Electron microscopy, Axons, Two photon excitation microscopy, Luminescence
Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy,
combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can
be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron
imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting
from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.
In the adult nervous system, different populations of neurons correspond to different regenerative behavior. Although previous works showed that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury1, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Time lapse two-photon imaging has been combined to laser nanosurgery2, 3 to define a temporal pattern of the degenerative event and to follow the structural rearrangement after injury. To characterize the damage and to elucidate the possible formation of new synaptic contacts on the sprouted branches of the lesioned CF, we combined two-photon in vivo imaging with block face scanning electron microscopy (FIB-SEM). Here we describe the approach followed to characterize the reactive plasticity after injury.
Action potential, via the transverse axial tubular system (TATS), synchronously triggers uniform Ca2+ release throughout
the cardiomyocyte. Cardiac diseases associated with TATS structural remodeling preclude a uniform Ca2+ release across
the myocyte, contributing to contractile dysfunction. A simultaneous recording of intracellular local Ca2+ release and
action potential in tubular network can be useful to unravel the link between TATS abnormality and dysfunctional EC coupling. Here we combine the advantage of an ultrafast random access multi-photon (RAMP) microscope with a double staining approach to optically record AP in several TATS elements and, simultaneously, the corresponding local Ca2+
transient. Isolated rat cardiomyocytes were labeled with a novel voltage sensitive dye (VSD) and a calcium indicator.
RAMP microscope rapidly scans between lines drawn across the TATS of the cardiomyocyte to perform a multiplexed
measurement of the two fluorescence signals. Although the calcium and voltage indicators can be excited at the same wavelength, the large Stokes shift of the VSD emission allows us to use spectral unmixing to resolve the voltage and
calcium responses. In healthy cardiomyocytes, we found uniform AP propagation within the TATS and homogeneous Ca2+ release throughout the whole cell. The capability of our technique in probing spatiotemporal relationship between
Ca2+ and electrical activity was then explored in a model of acute detubulation in which failure to conduct AP in disconnected TATS may cause local delay of Ca2+ transient rise leading to non-homogenous Ca2+ release.
Second-harmonic-generation (SHG) microscopy has emerged as a powerful tool to image unstained living tissues and probe their molecular and supramolecular organization. In this article, we review the physical basis of SHG, highlighting how coherent summation of second-harmonic response leads to the sensitivity of polarized SHG to the three-dimensional distribution of emitters within the focal volume. Based on the physical description of the process, we examine experimental applications for probing the molecular organization within a tissue and its alterations in response to different biomedically relevant conditions. We also describe the approach for obtaining information on molecular conformation based on SHG polarization anisotropy measurements and its application to the study of myosin conformation in different physiological states of muscle. The capability of coupling the advantages of nonlinear microscopy (micrometer-scale resolution in deep tissue) with tools for probing molecular structure in vivo renders SHG microscopy an extremely powerful tool for the advancement of biomedical optics, with particular regard to novel technologies for molecular diagnostic in vivo.
In the adult nervous system, different population of neurons corresponds to different regenerative behavior. Although
previous works show that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a
response to injury1, we have hitherto no details about the real dynamics of fiber regeneration. We coupled two photon
imaging to laser-induced lesions to perform in vivo multiphoton nanosurgery in the CNS of living mice expressing
fluorescent proteins to investigate the reparative properties of Climbing Fibers (CFs) in the adult CNS, following the
time evolution of this plastic process in vivo. Here we show that a regenerative event may take place in a murine model
in the days that follow a sub-micrometric lesion on the distal portion of the climbing fiber. Furthermore this unique
model could allow, through manipulation of the viral vector, to explore in detail the biochemical mechanisms underlying
the reparative process. The great potential of long-term two photon imaging, coupled to genetic manipulation, opens
great opportunities to further investigate the dynamic properties of neurons and their rearrangement following an injury.
In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an
indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon
absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without
causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is
applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are
then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the
procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the
fiber itself over a period of days. These results show the possibility of this innovative technique to investigate
regenerative processes in adult brain.
In parallel with imaging and manipulation technique, non-linear microscopy offers the opportunity to optically record
electrical activity in intact neuronal networks. In this work, we combined the advantages of second-harmonic generation
(SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording
fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk
loading of tissue
with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar
slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given
population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where
action potentials were recorded without averaging across trials. These results show the strength of this technique in
describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the
computations of neuronal networks.
Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored
with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of
proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries
and provides selective information on the structural order and orientation of the emitting proteins and the
dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We
characterise the polarization-dependence of SHG intensity in three different physiological states: resting,
rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The
orientation of motor domains of the myosin molecules is dependent on their physiological states and
modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different
molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor.
We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from
its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the
fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We
estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the
dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an
isometric contraction.
In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation
scheme to realize a new microscope (RA-SHG) capable of optically recording fast membrane potential events occurring
in a wide-field configuration. The RA-SHG microscope in combination with a bulk staining method with FM4-64 was
used to simultaneously record electrical activity from clusters of Purkinje cells (PCs) in acute cerebellar slices.
Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where APs were recorded in a
single trial without averaging. These results show the strength of this technique to describe the temporal dynamics of
neuronal assemblies.
The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic
Generation (SHG). As previously found (Vanzi et al., J. Muscle Cell Res. Motil. 2006) by fractional
extraction of proteins, myosin is the source of SHG signal. A full characterization of the polarization-dependence
of the SHG signal can provide very selective information on the orientation of the emitting
proteins and their dynamics during contraction. We developed a line scan polarization method, allowing
measurements of a full polarization curve in intact muscle fibers from skeletal muscle of the frog to
characterize the SHG polarization dependence on different physiological states (resting, rigor and isometric
tetanic contraction). The polarization data have been interpreted by means of a model in terms of the
average orientation of SHG emitters.The different physiological states are characterized by distinct patterns
of SHG polarization. The variation of the orientation of emitting molecules in relation to the physiological
state of the muscle demonstrates that one part of SHG signal arises from the globular head of the myosin
molecule that cross-links actin and myosin filaments. The dependence of the SHG modulation on the
degree of overlap between actin and myosin filaments during an isometric contraction, provides the
constraints to estimate the fraction of myosin heads generating the isometric force in the active muscle
fiber.
KEYWORDS: Dendrites, Spine, Neurons, In vivo imaging, Fluorescent proteins, Absorption, Two photon excitation microscopy, Two photon imaging, Green fluorescent protein, Brain
Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact
neocortex of living mice. Multi-photon absorption has also been used as a tool for the selective disruption of cellular
structures in living cells and simple organisms. In this work we exploit the spatial localization of multi-photon excitation
to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins.
This methodology was applied to dissect single dendrites with sub-micrometric precision without causing any visible
collateral damage to the surrounding neuronal structures. The spatial precision of this method was demonstrated by
ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The
morphological consequences were then characterized with time lapse 3D two-photon imaging over a period of minutes to
days after the procedure. Here we present the results of our systematic study of the morphological response of cortical
pyramidal neurons to nanosurgical perturbations. Dendritic branches were followed after transecting distal segments,
whilst the plasticity and remodeling of individual dendritic spines on a given branch was also followed after removing of
a subset of spines.
KEYWORDS: Dendrites, Spine, Neurons, In vivo imaging, Fluorescent proteins, Two photon excitation microscopy, Absorption, Two photon imaging, Neuroscience, Multiphoton processes
Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.
Recently, two-photon microscopy has been used for high spatial resolution imaging of the intact neocortex in living
rodents. In this work we used near-IR femtosecond laser pulses for a combination of two-photon microscopy and
microdissection on fluorescently-labeled neuronal structures in living mice. Three-dimensional reconstructions of
dendrites expressing the green fluorescence protein were made in the cortex of GFP-M and YFP-H transgenic mice.
Afterwards, single dendrites were laser-dissected irradiating the structure with a high femtosecond laser energy dose. We
report that laser dissection can be performed with micrometric precision and without any visible collateral damage of the
surrounding neuronal structures. After laser irradiation, one part of the severed dendrite underwent degeneration and
disappeared within 5 hours.
Using a chronically implanted glass window, we performed long-term imaging in the area of the dissected dendrite.
Images of the long-term morphological changes in the neuronal network after dendritic lesioning will be provided. Laser
microdissection of selected structures of the neuronal branching in vivo represents a promising tool for neurobiological
research.
V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, C. Tesi, N. Piroddi, C. Poggesi, C. Castiglioni, A. Milani, M. Linari, G. Piazzesi, V. Lombardi, F. Pavone
The intrinsically ordered arrays of proteins in skeletal muscle allows imaging of this tissue by Second Harmonic
Generation (SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution
of the molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very
powerful tool in the investigation of structural dynamics occurring in muscle during active production of force. A full
characterization of the polarization-dependence of the SHG signal represents a very selective information on the
orientation of the emitting proteins and their dynamics during contraction, provided that different physiological states of
muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here polarization data are
obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by means of a model, in
terms of an average orientation of the SHG emitters which are structured with a cylindrical symmetry about the fiber
axis. Optimizing the setup for accurate polarization measurements with SHG, we developed a line scan imaging method
allowing measurement of SHG polarization curves in different physiological states. We demonstrate that muscle fiber
displays a measurable variation of the orientation of SHG emitters with the transition from rest to isometric contraction.
Valentina Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, C. Tesi, N. Pirrodi, C. Poggesi, C. Castiglioni, A. Milani, M. Linari, G. Piazzesi, V. Lombardi, F. Pavone
The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation
(SHG). Biochemical and colocalization studies have gathered an increasing wealth of clues for the attribution of the
molecular origin of the muscle SHG signal to the motor protein myosin. Thus, SHG represents a potentially very
powerful tool in the investigation of structural dynamics occurring in muscle during active production of force and/or
shortening. A full characterization of the polarization-dependence of the SHG signal represents a very selective
information on the orientation of the emitting proteins and their dynamics during contraction, provided that different
physiological states of muscle (relaxed, rigor and active) exhibit distinct patterns of SHG polarization dependence. Here
polarization data are obtained from single frog muscle fibers at rest and during isometric contraction and interpreted, by
means of a model, in terms of an average orientation of the SHG emitters which are structured with a cylindrical
symmetry about the fiber axis. The setup is optimized for accurate polarization measurements with SHG, combined with
a line scan imaging method allowing acquisition of SHG polarization curves in different physiological states. We
demonstrate that muscle fiber displays a measurable variation of the orientation of SHG emitters with the transition from
rest to isometric contraction.
Recently, two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the
intact neocortex in living mice. In this work we study the in vivo spine rearrangements after an acute and selective
damage. For this purpose, we have used a near-IR femtosecond pulsed laser to combine two-photon microscopy imaging
with microdissection operation on fluorescently-labeled neurons. Three-dimensional reconstructions of dendrites
expressing fluorescence protein have been performed in the cortex of YFP-H and GFP-M transgenic living mice.
Afterwards, single dendrites have been laser-dissected irradiating the structure with a high femtosecond laser energy
dose. By using a chronically implanted glass window we performed long-term imaging in the area of the dissected dendrite.
We will show that laser ablation can be performed with micrometric precision and without visible collateral damage to
nearby neuronal structures. Also, we will evidence the morphological changes of the dendritic branches and dendritic
spines after this specific perturbation inside the intact neuronal network. Laser microdissection of selected structures of
the neuronal branching in vivo represents a promising tool for neurobiological research.
The intrinsically ordered arrays of proteins (mainly actin and myosin) constituting the myofibrils within muscle cells are at the basis of a strong Second Harmonic Generation (SHG) from muscle fibers and isolated myofibrils. We have characterized the SHG signal with regard to its polarization and potential source within the muscle cell. The lateral resolution that can be achieved through SHG imaging of muscle strongly depends on sample depth. In fact, a comparison between intact muscle fibers and single myofibrils demonstrates that, whereas in both cases the alternation of dark I bands and bright A bands is visible, the contours of these bands are much better resolved in myofibrils than in fibers. Further, imaging of myofibrils revealed the presence of a darker zone in the centre of the A band. These effects of scattering by tissue on the image resolution were also studied with regard to the polarization of the SHG signal. The polarization-dependence of SHG intensity represents a powerful tool for the investigation of the structural dynamics occurring in the emitting proteins during the active cycle of muscle contraction. The prospective to perform functional studies requires a complete characterization of the effects of scattering and possibly multiple emitting populations on the measured SHG signal. Also, SHG is extremely sensitive to the degree of order present in the filament array, offering an interesting potential in the development of non-invasive tools for the diagnosis of degenerative diseases affecting skeletal muscles.
In eukaryotic cells, proper position of the mitotic spindle and the division plane is necessary for successful cell division and development. In this work the nature of forces governing the positioning and elongation of the mitotic spindle and the spatio-temporal regulation of the division plane positioning in fission yeast was studied. By using a mechanical perturbations induced by laser dissection of the spindle and astral microtubules, we found that astral microtubules push on the spindle poles. Further, laser dissection of the spindle midzone induced spindle collapse inward. This suggests that the spindle is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Exploiting a combination of non-linear microscopy and optical trapping, we performed an optical manipulation procedure designed to displace the cell nucleus away from its normal position in the center of the cell. After the laser-induced displacement, the nucleus typically returned towards the cell center, in a manner correlated with the extension of a microtubule from the nucleus to the closer tip of the cell. This observation suggests that the centering of the nucleus is provided by microtubule pushing force. Moreover the cells in which the nucleus was displaced during interphase displayed asymmetric division, whereas when the nucleus was displaced during late prophase or metaphase, the division plane formed at the cell center as in non-manipulated cells. This result suggests that in fission yeast the division plane is selected before pro-metaphase and that the signal is not provided by the mitotic spindle.
We present a combination of nonlinear microscopy, laser nanosurgery and optical trapping applied to the 3D imaging and manipulation of intracellular structures in live cells. We use Titanium-sapphire laser pulses for a combined nonlinear microscopy and nanosurgery on microtubules tagged with green fluorescent protein (GFP) in fission yeast. The same laser source is also used to trap small round lipid droplets naturally present in the cell. The trapped droplets are used as handles to exert a pushing force on the nucleus, allowing for a displacement of the nucleus away from its normal position in the center of the cell. We show that nonlinear nanosurgery and optical manipulation can be performed with sub-micrometer precision and without visible collateral damage to the cell. We present this combination as an important tool in cell biology for the manipulation of specific structures in alternative to genetic methods or chemical agents. This technique can be applied to several fundamental problems in cell biology, including the study of dynamics processes in cell division.
Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology.
We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology.
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