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The cerebellum has been implicated in the regulation of social behavior. Its influence is thought to arise from communication, via the thalamus, to forebrain regions integral in the expression of social interactions, including the anterior cingulate cortex (ACC). However, the signals encoded or the nature of the communication between the cerebellum and these brain regions remains poorly understood. Here, we describe an approach that overcomes technical challenges in exploring the coordination of distant brain regions at high temporal and spatial resolution during social behavior. We developed the E-Scope, an electrophysiology-integrated miniature microscope, to synchronously measure extracellular electrical activity in the cerebellum along with calcium imaging of the ACC. This single coaxial cable device combined these data streams to provide a powerful tool to monitor the activity of distant brain regions in freely behaving animals. During social behavior, we recorded the spike timing of multiple single units in cerebellar right Crus I (RCrus I) Purkinje cells (PCs) or dentate nucleus (DN) neurons while synchronously imaging calcium transients in contralateral ACC neurons. We find that during social interactions, a significant subpopulation of cerebellar PCs were robustly inhibited, while most modulated neurons in the DN were activated. As expected, we find that there are higher correlations in the activity of cerebellar and ACC neurons that are similarly excited or inhibited by social interaction than in the activity of those modulated in an opposing manner. Surprisingly, these distinctions in correlations largely disappear when only non-social bouts were analyzed, suggesting that cerebellar-cortical interactions were social behavior specific. Our work provides new insights into the complexity of cerebellar activation and co-modulation of the ACC during social behavior, and a valuable open-source tool for simultaneous, multimodal recordings in freely behaving mice.

Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm×2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm±100 μm, incorporates an absolute head orientation sensor, and weighs only 13.9 g.The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

Miniaturized calcium imaging is an emerging neural recording technique that has been widely used for monitoring neural activity on a large scale at a specific brain region of rats or mice. Most existing calcium-image analysis pipelines operate offline. This results in long processing latency, making it difficult to realize closed-loop feedback stimulation for brain research. In recent work, we have proposed an FPGA-based real-time calcium image processing pipeline for closed-loop feedback applications. It can perform real-time calcium image motion correction, enhancement, fast trace extraction, and real-time decoding from extracted traces. Here, we extend this work by proposing a variety of neural network based methods for real-time decoding and evaluate the tradeoff among these decoding methods and accelerator designs. We introduced the implementation of the neural network based decoders on the FPGA, and showed their speedup against the implementation on the ARM processor. Our FPGA implementation enables the real-time calcium image decoding with sub-ms processing latency for closed-loop feedback applications., 11 pages, 15 figures

Aversive stimuli can cause hippocampal place cells to remap their firing fields, but it is not known whether remapping plays a role in storing memories of aversive experiences. Here we addressed this question by performingin-vivocalcium imaging of CA1 place cells in freely behaving rats (n=14). Rats were first trained to prefer a short path over a long path for obtaining food reward, then trained to avoid the short path by delivering a mild footshock. Remapping was assessed by comparing place cell population vector similarity before acquisition versus after extinction of avoidance. Some rats received shock after systemic injections of the amnestic drug scopolamine at a dose (1 mg/kg) that impaired avoidance learning but spared spatial tuning and shock-evoked responses of CA1 neurons. Place cells remapped significantly more following remembered than forgotten shocks (drug-free versus scopolamine conditions); shock-induced remapping did not cause place fields to migrate toward or away from the shocked location and was similarly prevalent in cells that were responsive versus non-responsive to shocks. When rats were exposed to a neutral barrier rather than aversive shock, place cells remapped significantly less in response to the barrier. We conclude that place cell remapping occurs in response to motivationally significant (rather than neutral) stimuli that are remembered rather than merely perceived and forgotten, suggesting that reorganization of hippocampal population codes may store memories for aversive events.

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing.

Epifluorescence miniature microscopes (“miniscopes”) are widely used forin vivocalcium imaging of neural population activity. Imaging data is usually collected while subjects are engaged in a task and stored for later offline analysis, but emerging techniques for online imaging offer potential for novel real-time experiments in which closed-loop interventions (such as neurostimulation or sensory feedback) are triggered at short latencies in response to neural population activity. Here we introduceDeCalciOn, a plug-and-play hardware device for online population decoding ofin vivocalcium signals that can trigger closed-loop feedback at millisecond latencies, and is compatible with miniscopes that use the UCLA Data Acquisition (DAQ) interface. In performance tests, the position of rats (n=13) on a linear track was decoded in real time from hippocampal CA1 population activity by 24 linear classifiers. DeCalciOn required

We present a large field of view (FOV) open-source miniature microscope (MiniLFOV) designed to extend the capabilities of the UCLA Miniscope platform to large-scale, single cell resolution neural imaging in freely behaving large rodents and head-fixed mice. This system is capable of multiple imaging configurations, including deep brain imaging using implanted optical probes and cortical imaging through cranial windows. The MiniLFOV interfaces with existing open-source UCLA Miniscope DAQ hardware and software, can achieve single cell resolution imaging across a 3.6 x 2.7 mm field of view at 23 frames per second, has an electrically adjustable working distance of up to 3.5 mm{+/-}150 {micro}m using an onboard electrowetting lens, incorporates an absolute head-orientation sensor, and weighs under 14 grams. The MiniLFOV provides a 30-fold larger FOV and yields 20-fold better sensitivity than Miniscope V3, and a 12-fold larger FOV with 2-fold better sensitivity than Miniscope V4. Power and data transmission are handled through a single, flexible coaxial cable down to 0.3 mm in diameter facilitating naturalistic behavior. We validated the MiniLFOV in freely behaving rats by simultaneously imaging >1000 GCaMP7s expressing neurons in the CA1 layer of the hippocampus and in head-fixed mice by simultaneously imaging ~2000 neurons in the mouse dorsal cortex through a 4 x 4 mm cranial window. For freely behaving experiments, the MiniLFOV supports optional wire-free operation using a 3.5 g wire-free data acquisition expansion board which enables close to 1-hour of wire-free recording with a 400 mAh (7.5 g) on-board single-cell lithium-polymer battery and expands wire-free imaging techniques to larger animal models. We expect this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

Quantitative descriptions of animal behavior are essential to understand the underlying neural substrates. Many behavioral analyses are performed by hand or with expensive and inflexible commercial software that often fail on animals with attached head implants, such as those used for in vivo optogenetics and calcium imaging. With the development of machine learning algorithms that can estimate animal positions across time and space, it is becoming easier for users with no prior coding experience to perform automated animal tracking in behavioral video recordings. Yet classifying discrete behaviors based on positional tracking data remains a significant challenge. To achieve this, we must start with reliable ground truth definitions of behavior, a process that is hindered by unreliable human annotations. To overcome these barriers, we developed BehaviorDEPOT (DEcoding behavior based on POsitional Tracking), a MATLAB-based application comprising six independent modules and a graphical user interface. In the Analysis Module we provide hard-coded classifiers for freezing and rearing. Optionally applied spatiotemporal filters allow users to analyze behaviors in varied experimental designs (e.g. cued tasks or optogenetic manipulations). Even inexperienced users can generate organized behavioral data arrays that can be seamlessly aligned with neurophysiological recordings for detailed analyses of the neural substrates. Four additional modules create an easy-to-use pipeline for establishing reliable ground-truth definitions of behaviors as well as custom behavioral classifiers. Finally, our Experiment Module runs fear conditioning experiments using an Arduino-based design that interfaces with commercialhardware and significantly reduces associated costs. We demonstrate the utility and flexibility of BehaviorDEPOT in widely used behavioral assays including fear conditioning, avoidance, and decision-making tasks. We also demonstrate the robustness of the BehaviorDEPOT freezing classifier across multiple camera types and in mice and rats wearing optogenetic patch cables and head-mounted Miniscopes. BehaviorDEPOT provides a simple, flexible, automated pipeline to move from pose tracking to reliably quantifying a wide variety of task-relevant behaviors.

Miniature microscopes have gained considerable traction for in vivo calcium imaging in freely behaving animals. However, extracting calcium signals from raw videos is a computationally complex problem and remains a bottleneck for many researchers utilizing single-photon in vivo calcium imaging. Despite the existence of many powerful analysis packages designed to detect and extract calcium dynamics, most have either key parameters that are hard-coded or insufficient step-by-step guidance and validations to help the users choose the best parameters. This makes it difficult to know whether the output is reliable and meets the assumptions necessary for proper analysis. Moreover, large memory demand is often a constraint for setting up these pipelines since it limits the choice of hardware to specialized computers. Given these difficulties, there is a need for a low memory demand, user-friendly tool offering interactive visualizations of how altering parameters at each step of the analysis affects data output. Our open-source analysis pipeline, Minian (Miniscope Analysis), facilitates the transparency and accessibility of single-photon calcium imaging analysis, permitting users with little computational experience to extract the location of cells and their corresponding calcium traces and deconvolved neural activities. Minian contains interactive visualization tools for every step of the analysis, as well as detailed documentation and tips on parameter exploration. Furthermore, Minian has relatively small memory demands and can be run on a laptop, making it available to labs that do not have access to specialized computational hardware. Minian has been validated to reliably and robustly extract calcium events across different brain regions and from different cell types. In practice, Minian provides an open-source calcium imaging analysis pipeline with user-friendly interactive visualizations to explore parameters and validate results.

Observing the activity of large populations of neuronsin vivois critical for understanding brain function and dysfunction. The use of fluorescent genetically-encoded calcium indicators (GECIs) in conjunction with miniaturized microscopes is an exciting emerging toolset for recording neural activity in unrestrained animals. Despite their potential, current miniaturized microscope designs are limited by using image sensors with low frame rates, sensitivity, and resolution. Beyond GECIs, there are many neuroscience applications which would benefit from the use of other emerging neural indicators, such as fluorescent genetically-encoded voltage indicators (GEVIs) that have faster temporal resolution to match neuron spiking, yet, require imaging at high speeds to properly sample the activity-dependent signals. We integrated an advanced CMOS image sensor into a popular open-source miniaturized microscope platform. MiniFAST is a fast and sensitive miniaturized microscope capable of 1080p video, 1.5 µm resolution, frame rates up to 500 Hz and high gain ability (up to 70 dB) to image in extremely low light conditions. We report results of high speed 500 Hzin vitroimaging of a GEVI and ∼300 Hzin vivoimaging of transgenic Thy1-GCaMP6f mice. Finally, we show the potential for a reduction in photobleaching by using high gain imaging with ultra-low excitation light power (0.05 mW) at 60 Hz frame rates while still resolving Ca2+spiking activity. Our results extend miniaturized microscope capabilities in high-speed imaging, high sensitivity and increased resolution opening the door for the open-source community to use fast and dim neural indicators.

Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.

One major challenge in neuroscience is to uncover how defined neural circuits in the brain encode, store, modify, and retrieve information. Meeting this challenge comprehensively requires tools capable of recording and manipulating the activity of intact neural networks in naturally behaving animals. Head-mounted miniature microscopes are emerging as a key tool to address this challenge. Here we discuss recent work leading to the miniaturization of neural imaging tools, the current state of the art in this field, and the importance and necessity of open-source options. We finish with a discussion on what the future may hold for miniature microscopy.

Recent studies suggest the hypothesis that a shared neural ensemble may link distinct memories encoded close in time1–13. According to the memory allocation hypothesis1,2, learning triggers a temporary increase in neuronal excitability14–16 that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Accordingly, we report that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Multiple convergent findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability16,17, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged animals, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by aging could affect the temporal structure of memories, thus impairing efficient recall of related information.

Temporal lobe epilepsy causes significant cognitive deficits in both human patients and rodent models, yet the specific circuit mechanisms that alter cognitive processes remain unknown. There is dramatic and selective interneuron death and axonal reorganization within the hippocampus of both humans and animal models, but the functional consequences of these changes on information processing at the neuronal population level have not been well characterized. To examine spatial representations of epileptic and control mice, we developed a novel wire-free miniature microscope to allow for unconstrained behavior during in vivo calcium imaging of neuronal activity. We found that epileptic mice running on a linear track had severely impaired spatial processing in CA1 within a single session, as place cells were less precise and less stable, and population coding was impaired. Long-term stability of place cells was also compromised as place cells in epileptic mice were highly unstable across short time intervals and completely remapped across a week. Because of the large-scale reorganization of inhibitory circuits in epilepsy, we hypothesized that degraded spatial representations were caused by dysfunctional inhibition. To test this hypothesis, we examined the temporal dynamics of hippocampal interneurons using silicon probes to simultaneously record from CA1 and dentate gyrus during head-fixed virtual navigation. We found that epileptic mice had a profound reduction in theta coherence between the dentate gyrus and CA1 regions and altered interneuron synchronization. In particular, dentate interneurons of epileptic mice had altered phase preferences to ongoing theta oscillations, which decorrelated inhibitory population firing between CA1 and dentate gyrus. To assess the specific contribution of desynchronization on spatial coding, we built a CA1 network model to simulate hippocampal desynchronization. Critically, we found that desynchronized inputs reduced the information content and stability of CA1 neurons, consistent with the experimental data. Together, these results demonstrate that temporally precise intra-hippocampal communication is critical for forming the spatial code and that desynchronized firing of hippocampal neuronal populations contributes to poor spatial processing in epileptic mice.

Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 μ m(3) at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 μm and at depths up to 360 μ m can be discriminated.

The discovery potential of experiments searching for rare events, such as dark matter interaction, relies heavily upon achieving a very low background environment. The current generation of noble liquid dark matter detectors is limited by the radioactivity in the detector materials, mostly from the photomultiplier tubes. Quartz Photon Intensifying Detector (Q upid ) is a novel photosensor based upon hybrid APD technology and with intrinsic radioactivity at least an order of magnitude lower than the presently employed phototubes. The basic concept as well as the status and the prospect of the Q upid are reviewed. The performance of the Q upid as photosensor for the ultraviolet scintillation light of liquid xenon is presented.

A multi-target detection system XAX, comprising concentric 10 ton targets of 136Xe and 129/131Xe, together with a geometrically similar or larger target of liquid Ar, is described. Each is configured as a two-phase scintillation/ionization TPC detector, enhanced by a full 4pi array of ultra-low radioactivity Quartz Photon Intensifying Detectors (QUPIDs) replacing the conventional photomultipliers for detection of scintillation light. It is shown that background levels in XAX can be reduced to the level required for dark matter particle (WIMP) mass measurement at a 10^-10 pb WIMP-nucleon cross section, with single-event sensitivity below 10^-11 pb. The use of multiple target elements allows for confirmation of the A^2 dependence of a coherent cross section, and the different Xe isotopes provide information on the spin-dependence of the dark matter interaction. The event rates observed by Xe and Ar would modulate annually with opposite phases from each other for WIMP mass >~100 GeV/c^2. The large target mass of 136Xe and high degree of background reduction allow neutrinoless double beta decay to be observed with lifetimes of 10^27-10^28 years, corresponding to the Majorana neutrino mass range 0.01-0.1 eV, the most likely range from observed neutrino mass differences. The use of a 136Xe-depleted 129/131Xe target will also allow measurement of the pp solar neutrino spectrum to a precision of 1-2%., 16 pages with 17 figures

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera’s native frame rate. We demonstrate that this approach is capable of recording Ca2+ transients resulting from APs in neurons labeled with the Ca2+ sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations.

Understanding of adaptive behavior requires the precisely controlled presentation of multisensory stimuli combined with simultaneous measurement of multiple behavioral modalities. Hence, we developed a virtual reality apparatus that allows for simultaneous measurement of reward checking, a commonly used measure in associative learning paradigms, and navigational behavior, along with precisely controlled presentation of visual, auditory and reward stimuli. Rats performed a virtual spatial navigation task analogous to the Morris maze where only distal visual or auditory cues provided spatial information. Spatial navigation and reward checking maps showed experience-dependent learning and were in register for distal visual cues. However, they showed a dissociation, whereby distal auditory cues failed to support spatial navigation but did support spatially localized reward checking. These findings indicate that rats can navigate in virtual space with only distal visual cues, without significant vestibular or other sensory inputs. Furthermore, they reveal the simultaneous dissociation between two reward-driven behaviors. © 2013 Cushman et al.

The hippocampal cognitive map is thought to be driven by distal visual cues and self-motion cues. However, other sensory cues also influence place cells. Hence, we measured rat hippocampal activity in virtual reality (VR), where only distal visual and nonvestibular self-motion cues provided spatial information, and in the real world (RW). In VR, place cells showed robust spatial selectivity; however, only 20% were track active, compared with 45% in the RW. This indicates that distal visual and nonvestibular self-motion cues are sufficient to provide selectivity, but vestibular and other sensory cues present in RW are necessary to fully activate the place-cell population. In addition, bidirectional cells preferentially encoded distance along the track in VR, while encoding absolute position in RW. Taken together, these results suggest the differential contributions of these sensory cues in shaping the hippocampal population code. Theta frequency was reduced, and its speed dependence was abolished in VR, but phase precession was unaffected, constraining mechanisms governing both hippocampal theta oscillations and temporal coding. These results reveal cooperative and competitive interactions between sensory cues for control over hippocampal spatiotemporal selectivity and theta rhythm.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Dark Matter and Double Beta Decay experiments require extremely low radioactivity within the detector materials. For this purpose, the University of California, Los Angeles and Hamamatsu Photonics have developed the QUartz Photon Intensifying Detector (QUPID), an ultra-low background photodetector based on the Hybrid Avalanche Photo Diode (HAPD) and entirely made of ultraclean synthetic fused silica. In this work we present the basic concept of the QUPID and the testing measurements on QUPIDs from the first production line. Screening of radioactivity at the Gator facility in the Laboratori Nazionali del Gran Sasso has shown that the QUPIDs safely fulfill the low radioactive contamination requirements for the next generation zero background experiments set by Monte Carlo simulations. The quantum efficiency of the QUPID at room temperature is > 30% at the xenon scintillation wavelength. At low temperatures, the QUPID shows a leakage current less than 1 nA and a global gain of 10^5. In these conditions, the photocathode and the anode show > 95% linearity up to 1 uA for the cathode and 3 mA for the anode. The photocathode and collection efficiency are uniform to 80% over the entire surface. In parallel with single photon counting capabilities, the QUPIDs have a good timing response: 1.8 +/- 0.1 ns rise time, 2.5 +/- 0.2 ns fall time, 4.20 +/- 0.05 ns pulse width, and 160 +/- 30 ps transit time spread. The QUPIDs have also been tested in a liquid xenon environment, and scintillation light from 57Co and 210Po radioactive sources were observed., 15 pages, 22 figures

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors.

Single-molecule spectroscopy is a set of powerful techniques for detailed analysis of molecular interactions and motion, but due to the requirement of low concentrations, long acquisition times are required to achieve sufficient statistics. Newly developed detectors permit acquiring data in parallel, resulting in faster acquisition and giving access to shorter time scales for the observation of dynamic phenomena. We present several new single-photon counting detectors and acquisition systems capable of high throughput single-molecule spectroscopy including multi-pixel CMOS detectors and a multi-pixel hybrid photodetector. To take advantage of these detectors, we developed a novel approach for multi-spot excitation utilizing a liquid crystal on silicon spatial light modulator (LCOS), which allows dynamic excitation spot generation corresponding to the detector geometry. We also developed a high throughput field programmable gate array (FPGA)-based parallel acquisition system. We present examples of these approaches for single-molecule applications such as Fluorescence Correlation Spectroscopy (FCS), and demonstrate the feasibility for high throughput single-molecule Forster resonance energy transfer (smFRET) measurements. We also discuss the ways in which our approaches permit measurements of faster dynamic processes. These high throughput developments will significantly expand the power of single-molecule spectroscopy for biophysical and other applications.