Random Access Multi-photon Microscope Research Articles

Fast optical recording of neuronal activity by three-dimensional custom-access serial holography.

Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.

Compact and adjustable compensator for AOD spatial and temporal dispersion using off-the-shelf components.

Random access multiphoton microscopy using two orthogonal acousto-optic deflectors (AODs) allows sampling only particular regions of interest within a plane, greatly speeding up the sampling rate. AODs introduce spatial and temporal dispersions, which distort the point spread function and decrease the peak intensity of the pulse. Both of these effects can be compensated for with a single dispersive element placed a distance before the AODs. An additional acousto-optic modulator, a custom cut prism, and a standard prism used with additional cylindrical optics have been demonstrated. All of these introduce additional cost or complexity and require an extended path length to achieve the needed negative group delay dispersion (GDD). By introducing a telescope between a transmission grating and the AODs, we correct for spatial and temporal dispersions in a compact design using only off-the-shelf components, and we show that the GDD can be tuned by translation of the telescope without adjustment of any other elements.

Two-Photon Voltage Imaging of Spontaneous Activity from Multiple Neurons Reveals Network Activity in Brain Tissue.

SummaryRecording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access multiphoton microscopy (RAMP) and the latest two-photon-compatible high-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.

In Vivo Melanoma Imaging on Living Mice Using Dynamic Nuclear Polarization Magnetic Resonance Imaging

Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the function of neuronal circuits. The combination of the fast scanning by random-access multiphoton microscopy (RAMP) and the latest two-photon-compatible high-performance fluorescent genetically encoded voltage indicators (GEVIs) has enabled action potential detection in deep layers in in vivo brain. However, neuron connectivity analysis on optically recorded action potentials from multiple neurons in brain tissue has yet to be achieved. With high expression of a two-photon-compatible GEVI, ASAP3, via in utero electroporation and RAMP, we achieved voltage recording of spontaneous activities from multiple neurons in brain slice. We provide evidence for the developmental changes in intralaminar horizontal connections in somatosensory cortex layer 2/3 with a greater sensitivity than calcium imaging. This method thus enables investigation of neuronal network connectivity at the cellular resolution in brain tissue.

Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

Electrical defects of the transverse-axial tubular system in cardiac diseases.

Electrical excitability is an essential feature of cardiomyocytes and the homogenous propagation of the action potential is guaranteed by a complex network of membrane invaginations called the transverse‐axial tubular system (TATS). TATS structural remodelling is a hallmark of cardiac diseases and we demonstrated that this can be accompanied by electrical defects at single T‐tubular level. Using a random‐access multi‐photon (RAMP) microscope, we found that pathological T‐tubules can fail to conduct action potentials, which delays local Ca2+ release. Although the underlying causes for T‐tubular electrical failure are still unknown, our findings suggest that they are likely to be related to local ultrastructural alterations. Here, we first review the experimental approach that allowed us to observe and dissect the consequences of TATS electrical dysfunction and then propose two different strategies to unveil the reasons for T‐tubular electrical failures. The first strategy consists in a correlative approach, in which the failing T‐tubule identified with the RAMP microscope is then imaged with electron microscopy. The second approach exploits the diffusion of molecules within TATS to gain insights into the local TATS structure, even without a thorough reconstruction of the tubular network. Although challenging, the local electrical failure occurring at single T‐tubules is a fundamental question that needs to be addressed and could provide novel insights in cardiac pathophysiology.

Impact of detubulation on force and kinetics of cardiac muscle contraction.

Action potential-driven Ca(2+) currents from the transverse tubules (t-tubules) trigger synchronous Ca(2+) release from the sarcoplasmic reticulum of cardiomyocytes. Loss of t-tubules has been reported in cardiac diseases, including heart failure, but the effect of uncoupling t-tubules from the sarcolemma on cardiac muscle mechanics remains largely unknown. We dissected intact rat right ventricular trabeculae and compared force, sarcomere length, and intracellular Ca(2+) in control trabeculae with trabeculae in which the t-tubules were uncoupled from the plasma membrane by formamide-induced osmotic shock (detubulation). We verified disconnection of a consistent fraction of t-tubules from the sarcolemma by two-photon fluorescence imaging of FM4-64-labeled membranes and by the absence of tubular action potential, which was recorded by random access multiphoton microscopy in combination with a voltage-sensitive dye (Di-4-AN(F)EPPTEA). Detubulation reduced the amplitude and prolonged the duration of Ca(2+) transients, leading to slower kinetics of force generation and relaxation and reduced twitch tension (1 Hz, 30°C, 1.5 mM [Ca(2+)]o). No mechanical changes were observed in rat left atrial trabeculae after formamide shock, consistent with the lack of t-tubules in rodent atrial myocytes. Detubulation diminished the rate-dependent increase of Ca(2+)-transient amplitude and twitch force. However, maximal twitch tension at high [Ca(2+)]o or in post-rest potentiated beats was unaffected, although contraction kinetics were slower. The ryanodine receptor (RyR)2 Ca-sensitizing agent caffeine (200 µM), which increases the velocity of transverse Ca(2+) release propagation in detubulated cardiomyocytes, rescued the depressed contractile force and the slower twitch kinetics of detubulated trabeculae, with negligible effects in controls. We conclude that partial loss of t-tubules leads to myocardial contractile abnormalities that can be rescued by enhancing and accelerating the propagation of Ca(2+)-induced Ca(2+) release to orphan RyR2 clusters.

Determinants of Mechanical Dysfunction in Myocardium with Reduced Density of T-Tubules

We have previously shown (Ferrantini et al 2010, Biophysical Journal98(3) pp. 5a) that formamide-induced osmotic shock is effective in achieving acute detachment of t-tubules from the surface in thin (< 200 µm thickness) rodent ventricular trabeculae, in which mechanical measurements can be performed in isometric conditions. Here, disconnection of t-tubules from the sarcolemma is verified by two-photon fluorescence imaging of labelled membrane and by directly probing the absence of tubular action potential by Random Access Multi-Photon (RAMP) microscopy. Formamide-induced detubulation in intact myocardium is incomplete and variable between different myocytes. This resembles t-tubule remodelling observed in disease settings. Simultaneous measurements of Force and Ca2+ transients in Fura-2 loaded trabeculae show that acute detachment of t-tubules leads to reduced twitch force and Ca2+ transient amplitude, impairment of the positive inotropic effect of high stimulus-rate, slower kinetics of contraction and prolonged Ca2+ transients that exhibit asynchronous time-course between different regions of the muscle. The DHPR blocker (Lacidipine) and the NCX blocker (SN-6) are used to prove that: (i) loss of tubular ICa-L accounts for the reduction of force and Ca2+-transient amplitude at both low and high pacing rates; (ii) loss of INCX contributes to the reduction in amplitude at high stimulation rates and plays a role in the prolongation of force relaxation and Ca2+ transient decay. Caffeine, applied at low doses (200 µM) to detubulated myocardium to increase RyR2 open probability, is able to re-synchronize Ca2+ transients within the muscle and partially reverse the loss of contractility at high stimulation rates. Simultaneous recordings of local Ca2+ release and action potential in the tubular network of single detubulated cardiomyocytes by RAMP microscopy indicate that non-uniform Ca2+release plays a major role in detubulation-induced mechanical dysfunction.

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity

The dynamic ability of neuronal dendrites to shape and integrate synaptic responses is the hallmark of information processing in the brain. Effectively studying this phenomenon requires concurrent measurements at multiple sites on live neurons. Substantial progress has been made by optical imaging systems that combine confocal and multiphoton microscopy with inertia-free laser scanning. However, all of the systems developed so far restrict fast imaging to two dimensions. This severely limits the extent to which neurons can be studied, as they represent complex three-dimensional structures. Here we present a new imaging system that utilizes a unique arrangement of acousto-optic deflectors to steer a focused, ultra-fast laser beam to arbitrary locations in three-dimensional space without moving the objective lens. As we demonstrate, this highly versatile random-access multiphoton microscope supports functional imaging of complex three-dimensional cellular structures such as neuronal dendrites or neural populations at acquisition rates on the order of tens of kilohertz.