Patent Publication Number: US-2023152561-A1

Title: Techniques for High-Speed Volumetric Sampling

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of priority from PCT Application Serial No. PCT/US21/15957, filed on Jan. 31, 2021, entitled “Techniques for High-Speed Volumetric Sampling,” which claims priority from U.S. Provisional Patent Application Ser. No. 62/968,330, filed on Jan. 31, 2020, and entitled “Massively Axial Multiplexed Mesoscopy,” and from U.S. Provisional Patent Application Ser. No. 63/122,508, filed on Dec. 8, 2020, and entitled “High-Speed, Cortex-Wide Volumetric Recording of Neuroactivity at Cellular Resolution Using Light Beads Microscopy,” and from U.S. Provisional Patent Application Ser. No. 63/106,684, filed on Oct. 28, 2020, and entitled “Mesoscopic Signal Neuron Resolution Volumetric Ca2+ Imaging.” The content of all of these applications is incorporated herein by reference in their entirety. 
    
    
     TECHNICAL FIELD 
     The described subject matter relates to microscopic systems, and methods of use thereof. 
     BACKGROUND 
     Two-photon (2p) scanning microscopy paired with genetically encoded Calcium indicators (GECIs) has become the gold-standard for recording activity, particularly at depth, in scattering brain tissue. However, 2p microscopy systems are still limited in both volumetric field-of-view (FOV) and recording speed by the need to scan a small, focused beam in order to form an image. To begin to understand the underlying cortical circuitry behind emergent, complex behavior in human-analogous models, tools are required that can record the activity of single neurons, across depth, and in multiple cortical areas simultaneously. 
     Current state-of-the-art scanning techniques face a number of technical limitations related to large-scale functional imaging of the mammalian brain. Understanding how sensory information and behavioral states are encoded within the mammalian brain requires the ability to record the activity of large populations of individual neurons distributed across functional and anatomical regions that span the entire cortex in awake and behaving animals. However, the inherent tradeoffs among imaging speed (i.e., voxel acquisition rate), spatial resolution, signal to noise ratio (SNR) and the size of the recording volume, and the finite limits of brain exposure to laser power have prevented the realization of a mesoscopic-scale volumetric imaging of single neuron activity across different layers of the entire cortical surface at adequate physiological volume rates. Solving this engineering challenge within the constrained and highly interrelated parameter space necessitates a principled approach that thus far has been missing in previous realizations of calcium (Ca2+) imaging. Hence, there is a need for improved systems and methods that provide a technical solution for overcoming the inherent tradeoffs a speed, resolution, and acquisition volume-size of current scanning techniques. 
     SUMMARY 
     In one general aspect, the instant application describes a multiplexing module. The multiplexing module is configured to perform operations of: receiving a plurality of laser pulses from a pulsed laser source; splitting each laser pulse into a plurality of beamlets; introducing a delay between each adjacent beamlet of the plurality of beamlets, such that the plurality of beamlets associated with a respective laser pulse of the plurality of laser pulses is distributed equally across a pulse repetition period associated with the pulsed laser source; changing a divergence of each subsequent beamlet of the plurality of beamlets associated with each respective laser pulse to introduce a distinguishing feature between each beamlet of the plurality of beamlet to cause each beamlet to focus on a different axial plane or lateral position of the sample; and outputting the plurality of beamlets associated with each respective laser pulse. 
     In another general aspect, the instant application describes a spatial multiplexing module. The spatial multiplexing module is configured to perform the operations of: receiving a plurality of laser pulses from a pulsed laser source; splitting the plurality of laser pulses into a plurality of spatially separated laser pulses; and outputting the plurality of spatially separated laser pulses. The spatial multiplexing module provides for equal splitting of power while minimizing dispersion and introduces no pathlength difference between the plurality of laser pulses. 
     In another general aspect, the instant application describes an optical modulation module. The optical modulation module is configured to perform the operations of: receiving a plurality of beamlets associated with each laser pulse of a plurality of laser pulses, wherein each a volume of a sample; determining whether an object of interest is present at a respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse; selectively adjust the amount of power associated with a set of beamlets from the plurality of beamlets for which no object of interest is located at the respective location in the volume associated with the respective location in the volume of the sample associated with the respective beamlet to reduce an amount of light associated with the set of beamlets from reaching the sample to generate modified beamlets; and outputting the modified beamlets. 
     This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Furthermore, the claimed subject matter is not limited to implementations that solve any or all disadvantages noted in any part of this disclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements. Furthermore, it should be understood that the drawings are not necessarily to scale. 
         FIG.  1 A  is a diagram showing an example implementation of the spatiotemporal multiplexing module that may be used by Massively Axial Multiplexed Mesoscopy (MAxiMuM). 
         FIG.  1 B  is a temporal schematic of pulses from the two sub-volumes each associated with a separate cavity of the spatiotemporal multiplexing module of the multiplexing module of  FIG.  1 A . 
         FIG.  1 C  is a diagram showing the two sub-volumes of MAxiMuM that are spatially separated along the optical axis and a scanning scheme that may be used in 2p microscopy for scanning those two sub-volumes. 
         FIG.  1 D  is a diagram showing an example implementation of a lateral spatiotemporal multiplexing module. 
         FIG.  2    is a diagram depicting elements of Light Beads Microscopy which may use MAxiMuM shown in  FIG.  1 A . 
         FIG.  3    is a diagram of an example microscopy setup in which MAxiMuM in integrated with a mesoscope. 
         FIGS.  4 A and  4 B  present a set of charts that present information related to post-object calibration. 
         FIG.  5    is a temporal schematic and schematic of the relative pulse energies of pulses from the two sub-volumes of the multiplexing module of  FIG.  1 A . 
         FIG.  6 A  is a diagram showing an example of a Co-linear Many-fold Axial Multiplexing (c-MAM). 
         FIG.  6 B  is a diagram showing an example spatial multiplexing module. 
         FIG.  6 C  is a diagram of an example detection module that may be used with the spatial multiplexing module of  FIG.  6 B . 
         FIG.  7    shows a schematic of an example system that incorporates the c-MAM. 
         FIG.  8    shows examples of simulated system performance data. 
         FIG.  9    is a diagram of a selective switching module enabling Selective Light Beads Mesoscopy (s-LBM). 
         FIG.  10    provides a first chart of simulated sensitivity as a function of the polling frequency F and threshold value and a second chart of simulated sensitivity versus reduction in required imaging power for different threshold values. 
         FIG.  11    is a flow diagram of a process for operating a spatiotemporal multiplexing module to image a sample. 
         FIG.  12    is a flow diagram of a process for operating a spatial multiplexing module to image a sample. 
         FIG.  13    is a flow diagram of a process for operating an optical modulation module to image a sample. 
         FIG.  14    is a block diagram showing an example software architecture, various portions of which may be used in conjunction with various hardware architectures herein described, which may implement any of the described features. 
         FIG.  15    is a block diagram showing components of an example machine configured to read instructions from a machine-readable medium and perform any of the features described herein. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. It will be apparent to persons of ordinary skill, upon reading this description, that various aspects can be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the p[resent teachings. 
     The imaging techniques provided herein provide a technical solution to the technical problem of overcoming the inherent tradeoffs among speed, resolution, sampling density and acquisition volume-size of current scanning techniques discussed above in particular in scattering or other types of samples that require different pulse energies to be delivered at different sample locations. The Massively Axial Multiplexed Mesoscopy (MAXiMuM), Light Beads Microscopy (LBM), and Co-linear Many-fold Axial Multiplexing (c-MAM) techniques presented herein provide technical solutions improving imaging techniques for volumetric recordings. These techniques may be used for volumetric recording of neural activity or for volumetric recordings of other types of activity within a sample. Some implementations of these techniques provide a spatiotemporal multiplexing module that facilitates excitation of the sample across multiple axial planes of the sample which eliminates the need for axial scanning required by current scanning techniques. Other implementations provide a spatiotemporal multiplexing module that facilitates excitation of the sample across multiple laterally separated regions of the sample. The spatiotemporal multiplexing module generates, from a laser pulse, a set of axially or laterally separated and temporally distinct foci referred to herein as “light beads.” These light beads may be used to in axial scanning to rapidly record information throughout the entire depth of the sample at the same time and at a rate that often exceeds the rate at which current microscopes record a single voxel on a single axial plane of a sample. Alternatively, the light beads may be used to laterally scan across multiple sampling locations of a lateral plane of the sample rather than across multiple axial planes of the sample. As a result, the imaging techniques provided herein may scan an entire volume in the same amount of time that current microscopes take to scan a single axial plane of a sample, because the spatiotemporal multiplexing module facilitates simultaneous scanning across multiple axial planes of the sample. Another technical benefit of these approaches is that the individual light beads may be dynamically and individually switched on and off or the power delivered to them individually and arbitrarily tuned during the scanning process based on the location of an object of interest, such as but not limited to a neuron, cell, or other object of interest in sample, to reduce the utilized power level at the sample. These and other technical benefits of the techniques disclosed herein will be evident from the discussion of the example implementations that follow. While these examples discuss applying these techniques to calcium imaging for recording activity of neurons at high speed in a large volume, these techniques may be applied in any context for which volumetric imaging is to be provided. The techniques provided herein may be used with other types of biological and/or non-biological samples. Furthermore, the type of scanning that may be performed is not limited to fluorescence to study the properties of a sample. Other types of optical properties of the samples may be studied, including but not limited to scattering, reflection, and attenuation or absorption of properties of the organic or inorganic samples being studies. 
     MAXiMuM 
     MAXiMuM is a module for volumetric sampling. MAxiMuM optimizes temporal and spatial sampling in combination with spatiotemporal axial or lateral multiplexing, to facilitate fully volumetric imaging through 2D scanning. MAxiMuM may be applied to Calcium (Ca 2+ ) imaging, voltage imaging, and/or other types of imaging. 
     MAXiMuM provides a technical solution to the technical problem of overcoming the inherent tradeoffs between speed, resolution, and acquisition volume-size of current scanning techniques with an optimized spatial and temporal sampling strategy that maximizes neuron extraction fidelity for objects of interest in a sample within a finite power budget and a spatiotemporal multiplexing module for sampling axially without the need for axial scanning. MAxiMuM is a scalable solution that further provides the ability to control the pulse energy of each of a set of beamlets in a relatively lossless manner as will be described in greater detail in the examples which follow. Furthermore, the pulse energy of each beamlet may be arbitrarily set as will be discussed in the examples which follow. 
     Sampling in a scanning 2p microscope may be optimized by determining a minimum number of pixels required to sample a neuronal cell body or other object of interest with sufficient spatial and temporal signal to faithfully extract a time series associated with the neuronal body or other object of interest from surrounding noise for a given shape of the microscope&#39;s point-spread function (PSF). In the case of a Gaussian PSF, the minimum sampling criterion may be investigated by analyzing the receiver operating characteristics of neuron detection as a function of downsampling the data. To that end, multiple single-plane videos of mouse brain tissue have been run through an automated neuronal extraction pipeline based on the CalmAn tools for identifying active neurons in brain images and the statistics of neuronal detection were determined as spatial pixels were removed from the data set. An F-score representing a measure of the accuracy of the analysis may be obtained. The F-score may be defined as the harmonic mean of the true-positive and false-negative detection rates as a function of the effective pixel size. Extraction fidelity may be preserved up to a pixel size of ˜5 μm, as predicted by the Nyquist theorem for a mean neuron size of ˜12 μm. While many of the example implementation discuss imaging of brain tissue, the techniques provided herein are not limited to imaging of neurons within a volume of brain tissue or other types of cells in other types of tissue. Other types of organic and/or inorganic samples may be scanned using these techniques to image objects of interest within a volume of the sample. 
     If extraction fidelity is to be preserved within a plane, the axial resolution and sampling must be designed to detect signal from all neurons or other objects of interest present without a high probability of detecting multiple neurons or other objects of interest within a voxel. Accordingly, some implementations of MAXiMuM have been designed for 15 μm axial pixels and a PSF with axial extent &lt;20 μm full width at half maximum (FWHM). Due to the Gaussian shape of the PSF, the lateral size (FWHM˜1.xx μm) is significantly smaller than the maximum lateral pixel size (5 μm). This resolution disparity can be addressed by using temporal focusing (TeFo) to create a larger lateral PSF diameter without extending axially. However, this can be optional when demonstrating the capability to switch to high resolution (˜1 μm) on the fly. However, TeFo modules are compatible with the MAxiMuM multiplexing module. While the application references specific axial pixel sizes and PSF sizes in the various example implementations provided herein, these examples are intended to illustrate possible configurations of MAXiMuM but do not limit MaXiMuM to these specific example implementations. 
     Conventional volumetric scanning 2p microscopes needs scanning along the optical axis, requiring scanning of each plane in the volume sequentially, and thus severely limiting the obtainable volumetric acquisition rate. MAxiMuM provides a technical solution to this problem by eliminating axial scanning and instead sampling along the axis through a series of 30 spatiotemporal multiplexed beamlets. While the example implementation discussed herein utilize 30 spatiotemporal multiplexed beamlets, other implementations may be configured to utilize a different number of beamlets. Each voxel in a MAxiMuM data set corresponds to a single beamlet or bead, thereby maximizing signal-to-noise ratio while minimizing heat penalty. 
     In conventional volumetric scanning 2p microscopes without MAxiMuM, the laser may have a relatively slow repetition rate, which could result in dead time between voxels. For example, if the laser has a repetition rate of 4.68 MHz, which may result in approximately 214 ns of dead time between voxels. To address this technical problem, MAxiMuM provides a spatiotemporal multiplexing module is provided to split a single pulse from the laser into multiple beamlets which are each delayed such that they may be equally spaced in time across a time window. For example, the spatiotemporal multiplexing module may be configured to split a single pulse of the laser into 30 beamlets and to delay each beamlet such that the beamlets are equally spaced in time across the 214 ns window for the laser having a 4.68 MHz repetition rate. The number of beamlets and the size of time window may vary depending upon the implementation. Additionally, each beamlet is given a different divergence during the splitting process to focus each beamlet to a different axial plane of the sample. A technical benefit of this approach is that, based on the time of arrival to the detector, light resulting from excitation of the sample can be binned and re-assigned to the plane from which the light originated. This light may result from fluorescence of an indicator applied to the sample which is excited by the laser light of a beamlet. Another technical benefit of this approach is that an entire column is sampled within the time that it would have normally taken to record a single pixel laterally. Thus, the volume can be imaged at the planar frame rate of the mesoscope. 
     In some implementations such as the example implementation such as that shown in  FIG.  1 A , the multiplexing module may be based on an 8f re-imaging cavity constructed with concave mirrors and/or with a combination of flat mirrors and lenses. The round-trip time of the cavity provides temporal delay between beamlets, and an offset between the plane where the beam is re-imaged by the concave mirror pairs and the partially reflective mirror which re-injects beams back into the cavity results in an increase in divergence for each beam exiting the cavity. In one implementation, the axial offset of the cavity is configured such that the 30 beams cover an axial extent of 450 μm in tissue, corresponding to ˜15 μm axial sampling. An example implementation of the multiplexing module is discussed below with respect to  FIG.  1 A . 
       FIG.  1 A  is a diagram of an example implementation of a spatiotemporal multiplexing module  100  that may be used to implement MAxiMuM. In  FIG.  1 A , ‘Ms’ denote mirrors, Is&#39; denote lenses, and ‘HWP’ denotes a half-wave plate. 
     The cavity of the spatiotemporal multiplexing module  100  reimages the focused beam from a pulsed laser source using four concave mirrors. A partially reflective mirror (PRM) may be used before the cavity to reflect the majority of light back into the cavity for the subsequent round trips while the chosen fraction of the light (also referred to herein as a “beamlet”) is coupled out of the cavity and sent towards the microscope. Each roundtrip introduces a lateral offset Δx and an axial offset Δz, and a temporal offset Δt for each successive beam. The relative pulse energy for each beamlet may be set by adjusting the splitting ratio of the PRM. 
     The beam propagates a distance Δz from the nominal focal plane before encountering a partially reflective mirror. The reflected portion is reintroduced into the cavity and for each successive round-trip experiences an extra axial shift Δz and a relative temporal delay τ. Accordingly, the beams transmitted out of the cavity have distinct axial focal points (z 1 , z 2 , . . . z 30 ) and distinct temporal delays (t 1 , t 2 , . . . t 30 ). To be incident on the partially reflective mirror after the first roundtrip, spatiotemporal multiplexing module  100  also imparts a small lateral shift as well, resulting in slight tilt to the light column and a total lateral separation of &lt;200 μm between the top and bottom beads in the sample. 
     The introduced focal offset between each beam allows their focusing to depths in the sample with a relative decrease in optical power for the i th  given by P i =T(1−T) i , with T the transmission of the PRM while the size and geometry of the cavity allows for the temporal separation of sub-pulses to be adjusted to fulfill the fluorescence lifetime limited principle. The above equation shows that T can be chosen to match the exponential power change to the specific scattering length of the imaged tissue. A technical benefit of this approach is that it provides a flexible means for adjusting the power such that the power increases in subsequent light beads as a function of sample depth and independently the axial separation of the light beads in the scattering sample. An example implementation of MAxiMuM using a mirror with T˜8% allows the generation of 30 temporally multiplexed beams, such as those shown in the example implementation of  FIG.  1 A . 
     One of the technical benefits of the MAxiMuM techniques provided herein, is that it allows for a decoupling of the number of axially multiplexed beams and the sample-specific needs to adjust the power as a function of depth in order to maintain a constant SNR. A technical benefit of this versatility is that MAxiMuM enables the realization of sampling conditions at different densities and axial imaging ranges within the same sample which in turn enables the realization of imaging modalities with different applications. More specifically, this decoupling allows the axial separation between the foci of two sequential beams in the sample, δz, to be freely chosen by fulfilling δz=−l s  ln(1−T) with δz=Δz/M 2  where M is the magnification of the microscope and Δz the axial separation of beams exiting the cavity. This degree of freedom together with the lateral voxel spacing given by the laser repetition rate, the resonant scanner frequency, the optical design of the system and the flexibility to choose size of the PSF allows the effective realization of different imaging modalities aimed at large-scale cellular-resolution volumetric recording, synaptic resolution volumetric recording of dendrites and axonal processes and volumetric cellular resolution imaging at up to ˜400 Hz and beyond, which opens up applications of our approach to volumetric voltage imaging of genetically encoded voltage indicators (GEVIs). 
     The spatiotemporal multiplexing module  100  may be implemented as a standalone module that is disposed between a laser source and a microscope where the spatiotemporal multiplexing module  100  performs sequential re-imaging of the beam waist at the entrance of the spatiotemporal multiplexing module  100 . As a result, the spatiotemporal multiplexing module  100  may be combined with existing 2 pM systems. By using appropriately selected cavity parameters and telescopes in addition to the demultiplexed detection, existing 2 pMs can be converted to fast volumetric Ca2+ imaging platforms with desired spatiotemporal resolution for different neurobiological applications provided that the laser source can produce sufficient pulse energies. Thereby, the spatiotemporal multiplexing module  100  addresses the inherent tradeoffs between volume acquisition rate, voxel spacing, and resolution within the limits of sample exposure to laser light in the most efficient manner irrespective of which parameter is optimized. 
     In the example implementation of the spatiotemporal multiplexing module  100  includes a cavity  110  that includes concave mirrors configured in an 8f, non-inverting, re-imaging scheme. However, other implementations may utilize a combination of flat mirrors and lenses to replace some or all of the concave mirrors. An input beam  120  is focused by lens L 1 , which is disposed above the aperture of the output coupler M 1  and in front focal plane of the mirror M 2 . The mirrors M 2 , M 3 , M 4 , and M 5  are concave mirrors with custom low-dispersion dielectric coatings. The mirrors M 2 , M 3 , M 4 , and M 5  reimage the initial spot of the laser pulse onto the turning mirror M 6 . The mirror M 6  provides a slight vertical tilt to the beam such that it intersects the output coupler M 1 . The output coupler M 1  is a low dispersion ultrafast beam splitter. The majority of the light incident on the output coupler M 1  undergoes another round-trip through the cavity  110 , and the rest of the light is output by the cavity  110 . In implementations that include the optional cavity  115 , the light output by cavity  110  is transmitted to cavity  115 . Thus, a “beamlet” is split off from the laser light incident on the output coupler M 1  and output from the cavity  110 , while the remainder of the light incident on the output coupler M 1  makes another round trip of the cavity  110 . 
     Each round trip through the cavity  110  provides a temporal delay as well as an offset in the focal plane of the beam dictated by the distance between the mirror M 6  and the output coupler M 1 . In the example shown in  FIG.  1 A , the temporal delay τ may be represented by the equation τ=8f/c. The angle of the mirror M 6  ensures that the beam  120  intersects the aperture of the output coupler M 1  and causes a small lateral offset between subsequent round trips of the cavity  110 . This offset is minimized during alignment to reduce offset between axial planes in the sample.  FIG.  1 A  shows an example beam profile  125  that represents a simulation of a beam profile exiting the cavity  110 . 
     The non-zero transmission of the output coupler M 1  also causes the beams emitted from the cavity  110  to fall off in optical power exponentially according to the splitting ratio of M 1 . It is well known that scattering in brain tissue requires an increase in imaging power to preserve signal to noise ratio at increasing tissue depths. Accordingly, the spatiotemporal multiplexing module  100  can finely tune the rate of decrease in power between subsequent beams focused to different axial depths in the sample by manipulating the splitting ratio of the mirror M 1  in order to match the expected scattering properties of the tissue or other sample. Therefore, by orienting the least-powerful beams towards the shallowest depths in the sample to be scanned, the spatiotemporal multiplexing module  100  facilitates volumetric imaging without the need for active adjustment of the imaging power as shown in the chart  140  of  FIG.  1 B . Chart  140  is a temporal schematic of pulses from cavity  110  and cavity  115 . The pulses from cavity  110  are represented by z 1 a-z 5 a and the pulses from cavity  110  are represented by z 1   b -z 5   b . Due to the shorter delay of cavity  115  relative to cavity  110 , the pulse trains from cavity  110  and cavity  115  are interleaved. The pulse energies for each beam decreases exponentially according to the transmission/reflection ratio of mirror M 1 , a partially transmissive mirror (PRM) in cavity  110 . The transmission/reflection ratio may be adjusted to control the pulse energy drop off of the individual beamlets. For brain tissues, exponential decrease may be matched to the expected scattering length (is) for brain tissue (˜200 μm). The exponential decrease may be matched to the expected scattering length for other types of samples. Power of the pulses from cavity  115  is lower than those from cavity  110  since cavity  115  pulses are sent to more superficial layers in the sample. The offset can be controlled by the HWP in cavity  110 . 
     In example implementation of the spatiotemporal multiplexing module  100  used for 2 pM where the sample comprises brain tissue, excitation power must increase exponentially with depth in order to preserve signal-to-raise ratio in the presence of tissue scattering. The spatiotemporal multiplexing module  100  is configured such that the pulse energy for beams exiting the cavity fall off according to an exponential decay chosen by optimizing the partial reflectivity of the output coupler M 1 . Using a reflectivity of R=10% allows for a fall-off of pulse energy that matches the scattering length of brain tissue (l s ˜200 μm), such that the signal-to-noise ratio from each light bead is conserved and maximized across all depths within the column for the total delivered pulse energy. A series of relay telescopes may be used to couple the light beads into a mesoscopy platform such that the center of the light bead column is conjugated to the nominal focal plane of the objective. 
       FIG.  5    is a temporal schematic of pulses from cavity  110  and cavity  115 . Due to the shorter delay of cavity  115  relative to  110 , the pulse trains are interleaved. The pulse energies for each beam decreases exponentially due to the partially transmissive mirror in cavity  110 . Exponential decrease is matched to the expected scattering length (l s ) for brain tissue (˜200 μm). Power for cavity  115  pulses is lower than those from cavity  110  since cavity  115  pulses are sent to more superficial layers in the brain. The offset may be controlled by the HWP in cavity  110 . 
     The light exiting cavity  110  is recollimated by the lens L 2 . L 2  may be implemented using an achromatic doublet. The lenses L 3  and L 4  form a unitary magnification telescope which may ensure that the lowest power beams were directed to the shallowest depths in a sample. In the example implementation shown in  FIG.  1 A , the lenses F 3  and F 4  have a focal length of 100 mm and may be implemented using an achromatic doublet. In other implementations, the lenses F 3  and F 4  may have a different focal length or could be realized by using curved mirrors. 
     The beams are transmitted through a half-wave plate (HWP) and onto a polarizing beam splitter (PBS) of cavity  115 . The reflected portion of the beam undergoes a single round-trip through another custom-mirror-based  8   f  re-imaging cavity  115  before being re-combined with the transmitted portion of the beam as shown in  FIG.  1 A . In the example implementation shown in  FIG.  1 A , the mirrors M 9 , M 10 , M 11 , and M 12  may have a focal length of 250 mm and 2-inch diameter. Other realizations using flat mirrors and lenses is also possible. Cavity  115  includes mirrors having a shorter focal length than the mirrors in cavity  110  mirrors to create a copy of the 15 pulses from cavity  110  and shift them in time and axial location to achieve the full 30 beams and 465 μm axial range of the MAxiMuM system. 
     The beams coupled to the secondary cavity  115  are delayed an additional 6.67 ns in the example shown in  FIG.  1 A , interleaving the beams in time with the beams transmitted by the PBS. The focal planes of these delayed beams can be globally shifted by adjusting the position of the mirrors M 9  and mirrors M 11 , forming two sub-volumes  150  and  155  that are spatially separated along the optical axis as shown in  FIG.  1 C . The cavities  110  and  115  together form the two sub-volumes  150  and  155  such that the planes from cavity  110  are below those of cavity  115  so that together the two sub-volumes  150  and  155  can sample the entire axial range of a volume of the sample. Manipulation of the HWP can be used to adjust the relative optical power of the sub-volumes in order to preserve matching to the scattering properties of the tissue. In the example implementation shown in  FIG.  1 A , thirty spatiotemporally multiplexed beams exit the secondary cavity  115 , and the axial separation between imaging planes is ˜15 μm in this example. 
     MAxiMuM provides a significant technical benefit through its arbitrarily large and scalable degree of multiplexing that results in eliminating the need for any axial scanning in the volume. The degree of multiplexing provided is scalable through careful design of the primary and secondary cavities  110  and  115  and through careful tuning of the inter-cavity dispersion and output leakage. The example implementation showing in  FIG.  1 A  samples a 450 μm axial range with 15 μm axial width voxels using 30×multiplexing, with the number of beams limited primarily by the temporal response of the photo-multiplier tubes (PMTs) and by tissue heating. Outside these practical limitations, the design form could be applied to even higher degrees of multiplexing, to increase the axial range or sample with higher resolution. 
       FIG.  1 D  is a diagram showing an example implementation of a lateral spatiotemporal multiplexing module  170 . In contrast with the examples shown in  FIGS.  1 A and  1 C , the lateral spatiotemporal multiplexing module  170  is configured to provide lateral spatiotemporal multiplexing rather than the axial multiplexing provided by the spatiotemporal multiplexing module  100 . In  FIG.  1 D , ‘Ms’ denote mirrors, Is&#39; denote lenses, ‘DOE’ denotes a diffractive optical element, and ‘KEs’ denote knife edge mirrors. The lateral multiplexing module  170  includes mirrors M 1 , M 2 , M 3 , and M 4  which are curved mirrors. KE 1  is a horizontal blade knife edge mirror and KE 2  is a vertical blade knife edge mirror. 
     A laser beam input  170  may be received from a pulsed laser source as in the preceding examples of the axial multiplexing module. The DOE  172  diffracts the laser beam input  170  onto the lens  173 , which focuses the laser beam input into a plurality of beamlets  174 . The lateral spatiotemporal multiplexing module  170  shown in  FIG.  1 D  includes the DOE  172  for splitting the laser beam input  174  into a plurality of beamlets. However, other implementations may include other beam splitting elements for separating the laser beam input  170  into the plurality of beamlets  174 . 
     The beamlets  174  move horizontally by Δx and vertically by Δy for each round trip around the re-imaging cavity of the lateral multiplexing module  170 . KE 2  picks off a new beamlet to be output from the lateral multiplexing module  170  for each round trip around the cavity. The values of Δy is configurable to cause the beamlets to be distributed laterally. The beamlets output by the lateral spatiotemporal multiplexing module  170  may be used to sample laterally along the same axial plane of the sample in contrast with the examples shown in FIG.  1 C in which the spatiotemporal multiplexing module  100  produces axially separated beamlets across multiple axial planes. The specific delay associated with each beamlet may be configured in a similar manner as the delay associated with the beamlets in the spatiotemporal multiplexing module  100 . The delay associated with each beamlet is generated based on a number of round trips that the beamlet makes around the re-imaging cavity of the lateral multiplexing module  170 . 
     Light Beads Microscopy 
     Light Beads Microscopy (LBM) may use a spatiotemporal multiplexer, such as but not limited to the MAxiMuM spatiotemporal multiplexing module  100  or the c-MAM  601 , to facilitate volumetric recording of a sample. The examples which follow apply LBM to recording neuroactivity in brain tissue. LBM enables near-simultaneous in-vivo volumetric recording of &gt;807,748 neurons at single-cell resolution in the mouse cortex. The LBM techniques presented herein provide a technical solution to the technical problem of recording neuroactivity in a volumetric fashion at physiological time scales and at a cellular resolution. However, the LBM techniques disclosed herein are not limited to studying neuroactivity in brain tissue. The LBM techniques may be applied to study objects of interest and/or activity of interest within volumes of organic and/or inorganic samples. 
     The LBM techniques described herein provide a technical solution in which information is acquired from sample voxels at the maximum possible rate, limited only by fluorescence lifetime, while minimizing the laser-induced heat penalty per unit generated signal, and spatially sampling features of interest, such as cell bodies, as efficiently as possible. The LBM techniques are not limited to calcium image and may be applied to other imaging techniques such as but not limited to volumetric voltage imaging of genetically encoded voltage indicators (GEVIs). 
       FIG.  2    is a diagram showing various aspects of LBM which uses MAxiMuM to generate light beads. LBM is a scalable high-speed optical acquisition technique that provides a voxel acquisition rate of more than 140 MHz. In LBM, the microscope scans a set of axially separated and temporally distinct foci (also referred to herein as “light beads”  215 ) as opposed to a single spot  205  as shown in  FIG.  2   . The beads record information throughout the entire depth of the sample at the same, if not a higher, rate than a typical microscope records a single voxel. Thus, LBM can obtain the entire volume at the same rate as a conventional microscope records a single plane, which eliminates the need for additional axial scanning. The LBM techniques may use various spatiotemporal multiplexing techniques to form the light beads, such as but not limited to the MAxiMuM spatiotemporal multiplexing module  100  or the c-MAM  601 . 
     In the example shown in  FIG.  2 A , MAxiMuM  210  may be implemented by the spatiotemporal multiplexing module  100  shown in  FIG.  1 A . MAxiMuM  210  may be configured to generate ˜500 μm long columns composed of 30 axially and temporally distinct focused beams in this example implementation. Other implementations may be configured to generate columns having a different length and/or that include a different number of temporally distinct focused beams. LBM has been realized on a mesoscopy platform that allows access to a lateral field of view (FOV) of ˜6×6 mm2 at subcellular resolution (NA=0.6), demonstrating volumetric and single-cell resolution recording from volumes of ˜3×5×0.5 mm3, encompassing portions of the primary visual (VISp), primary somatosensory (SSp), posterior parietal (PTLp), and retrosplenial (RSP) areas of GCaMP6s-labeled mouse neocortex at ˜5 Hz volume rate. The versatility of LBM on this mesoscopy platform has been demonstrated by recording in a variety of configurations ranging from moderately sized FOVs (600×600×500 μm3) with voxel resolution capable of resolving subcellular features, to FOVs (5.4×6×0.5 mm3) encompassing both hemispheres of the mouse cortex and capturing the dynamics of populations exceeding 800,000 neurons. 
     LBM provides an optical recording system that offers an acquisition bandwidth limited only by the properties of the molecular sensors, while inducing minimal heating of the tissue or other sample per unit generated fluorescence. To achieve this in densely labeled brain tissue without a priori information about the sample or any post-imaging reconstruction, LBM employs a high degree of spatiotemporal multiplexing along the axial dimension. Multiplexing is facilitated by splitting a single highly energetic pump pulse into many sub-pulses. Each sub-pulse undergoes a differing amount of free-space propagation prior to entering the microscope, causing relative temporal delays between adjacent pulses that exceed the fluorescence lifetime of the indicator. The sub-pulses are arranged into a column of light beads  215 , and focused to different planes  225  in the sample, such that each voxel in the volume is sampled by a single laser pulse. This approach maximizes signal-to-noise ratio and increases the extracted information rate to the limit posed by the fluorescence lifetime and the detector response time. By scanning the column of light beads  215  over the transverse plane, a volumetric image can be formed at the nominal frame rate of the microscope. As the light beads  215  are both temporally and spatially distinct, the fluorescence emitted from each bead can be recorded with the same single-point detector, and the detection time of the fluorescence can be used to determine the location of each voxel in three-dimensional sample space. 
     The column of light beads is formed using a spatiotemporal multiplexing module, such as but not limited to the spatiotemporal multiplexing module  100  or the c-MAM  601 . The spatiotemporal multiplexing module output a set of beamlets having distinct axial or lateral focal points. The relative pulse energy for each beamlet may be set by adjusting the splitting ratio of the PRM. 
     When 2 pM is used in scattering media, excitation power must increase exponentially with depth in order to preserve signal-to-raise ratio in the presence of tissue scattering. The spatiotemporal multiplexing module  100  is configured such that the pulse energy for beams exiting the cavity fall off according to an exponential decay chosen by optimizing the partial reflectivity of the output coupler M 1 . Using a reflectivity of R=10% allows for a fall-off of pulse energy that matches the scattering length of brain tissue (l s ˜200 μm), such that the signal-to-noise ratio from each light bead is conserved and maximized across all depths within the column for the total delivered pulse energy. Lastly, a series of relay telescopes are used to couple the light beads into our mesoscopy platform such that the center of the light bead column is conjugated to the nominal focal plane of the objective. 
     Integration with mesoscope: The output of the multiplexing module was interfaced with a commercial mesoscope. An example mesoscope layout and accompanying electronics are shown in  FIG.  3   .  FIG.  3    is a diagram providing an example mesoscopic system  300 . The mesoscopic system  300  includes a fiber chirped-pulse amplifier (FCPA)  301  which emits the pulsed laser beam, which passes through the optical parametric chirped-pulse amplifier (OPCPA)  302  followed by the electro-optic modulator (EOM)  303 , the dispersion compensation path  304 , MAxiMuM  305 , and into the microscope. Is&#39; denote lenses, ‘Rs’ denote relay lens pairs, ‘PMT’ denotes photo-multiplier tube, ‘ADC’ denotes analog to digital converter, and ‘PLL’ denotes phase-locked loop. The channel plot  310  shows channel allocation for demultiplexing on the Field Programmable Gate Array (FPGA). Data points are the measured impulse response for fluorescence from GCaMP6f measured with our PMT and associated electronics, captured with 1614 MHz (0.62 ps) resolution. Shaded regions denote the integration boundaries for each demultiplexed channel. 
     MAxiMuM post-objective calibration:  FIG.  4    presents a set of charts that present information related to post-object calibration. Charts  405 ,  410 , and  415  are charts relating to axial light bead positions, relative light bead pulse energy, and transverse position of the light beads, respectively, calibrated by translating a pollen grain through the focus of the microscope. Chart  420  presents lateral point-spread function full-width and half-maximum diameters for each light beam. The horizontal line across chart  420  represents the mean value. Error bars denote the 95% confidence interval values for the Gaussian fits used to determine PSF widths. Chart  425  presents axial point-spread function full-width at half-maximum diameters for each light bead. The horizontal line across chart  425  represents the mean value. Error bars denote the 95% confidence interval values for the Lorentzian fits used to determine PSF widths. Chart  430  presents pulse duration measurements of each beam from MAxiMuM, post-objective. Chart  435  presents measurements of crosstalk between demultiplexed channels. The black horizontal line on chart  435  shows the mean value. 
     Chart  405  shows a calibration of the relative axial focal position for each beam post-objective, achieved by recording fluorescence signal from a grain of pollen (˜20 μm diameter) translated through the focus of the objective by a piezo stage. The offset between axial foci of the beams is linear (r 2 =0.98) over the 30 beams. Additionally, by tracking the maximum time-averaged signal from a pollen grain measured at the nominal focal plane of each beam, the power in each plane was calibrated (chart  410 ). The power measurements were fit with an exponential curve, finding that the effective scattering length (ls=220 μm) matched the expectation for the sample (ls˜200 μm) reasonably well. Pollen grain measurements were also used to determine the lateral offset between the beams (chart  415 ) which corresponded to less than 200 μm total shift across the axial range. 
     The point spread function (PSF) for each beam was also recorded using a 1 μm diameter fluorescent bead. The measured full-width-at-half-maximum (FWHM) diameter for the PSF of each beam is shown for the lateral and axial dimensions in charts  420  and  425 , respectively. The measured axial widths ensure that for the given separation between axial planes (˜15 μm) and typical neuron sizes (10-20 μm), the probability of detecting any neurons present is high, while the likelihood of the PSF intersecting two neurons simultaneously (thus rendering them indistinguishable) is negligibly low. The average lateral FWHM is 1.3 μm, which when deconvolved (assuming a step function with 1 μm width as a model for the fluorescent bead) yields an average PSF diameter of 1.1 μm. 
     Care was taken to minimize the inter-cavity dispersion for MAxiMuM. While dispersion accumulation throughout the microscope is inevitable and can be compensated, any dispersion within the cavity would lead to variation of the individual pulse duration of each beam exiting MAxiMuM. Accordingly, the cavity was designed with entirely reflective components and used low-dispersion dielectric coatings for as many components as possible. Chart  430  shows the post-objective pulse durations measured by auto-correlation for each of the 30 beams, measured using APE pulseCheck autocorrelator. While the pulse duration (120-160 fs) is not transform-limited (˜90 fs) for each beam after the objective, the degradation is acceptably small and can be accounted for by adjusting the power of each beam through optimization of the output coupling from the cavity. 
     Crosstalk analysis: A crucial element of the spatiotemporal multiplexing scheme employed here is that signal from the beams is distinguishable in time in order to effectively demultiplex and assign signal to the proper axial location. Any leakage of the signal from one beam to the time slot of another will lead to distortion of the volume, typically referred to as crosstalk. Crosstalk in the system estimation was measured by recording mouse brain tissue in vivo with 30× multiplexing enabled, but with the beams in cavity  115  ( FIG.  1 A ) blocked. As shown in the channel plots  310  inset in  FIG.  3   , the fluorescence response from a single voxel has an exponential tail that inevitably extends to some degree into the signal from the subsequent voxel. The fraction of residual signal generated by the beams from cavity  110 , recorded at time values allocated for beams from cavity  115 , indicates the crosstalk for the system. As shown in chart  435  of  FIG.  4   , the crosstalk is fairly channel-invariant, and the average value (7%) is largely negligible. Crosstalk is a linear mixing phenomenon and can be removed post-recording if the contributions from each channel are known a priori. 
     Data acquisition: Data was acquired using the commercial mesoscope-compatible version of the ScanImage software platform (Vidrio, Inc.) with necessary additional customizations, as well as upgraded digitization hardware ( FIG.  4   , chart  405 ). An evaluation board was used to multiply a trigger signal from the OPCPA laser to 1614 MHz, which in turn was fed to the upgraded digitizer and field programmable gate array to serve as a sample clock. This clock signal was used within the customized version of ScanImage to synchronize the line trigger to the pulse repetition rate of the laser, thus ensuring a single laser pulse constituted one voxel of the recording. 
     Additionally, the ScanImage customization allowed the user to define channels by integrating temporal windows of the raw PMT signal with respect to a trigger from the laser. The window for each channel was set to integrate the fluorescence signal associated with each beam from the MAxiMuM system such that the channels constitute the de-multiplexed axial planes of the volumetric recording (channel plots  310  in  FIG.  3   ). The microscope recorded frames for each channel separately, in the same fashion as a two-color compatible microscope records separate channels from each PMT. The data streamed to disk consisted of 30 consecutive frames representing each channel, and thus each axial plane, repeated in sequence for each time point in the measurement. 
     Co-Linear Many-Fold Axial Multiplexing (c-MAM) 
     c-MAM builds upon the MAxiMuM spatiotemporal multiplexing module discussed in the preceding examples to provide an improved spatiotemporal multiplexing module that utilizes a co-linear geometry. As will be discussed in the examples which follow, c-MAM may be used for a versatile LBM platform that enables volumetric, cellular resolution, whole-mouse cortical recording of neuroactivity at multi-Hertz rate. For example, the c-MAM platform may be combined with a four-fold spatial multiplexer to provide for single-neuron resolution recordings from volumes as large as 7.5×7.5×0.6 mm 3  at ˜7 Hz or from a volume of ˜3.25×3.25×0.6 mm 3  at ˜28 Hz. The c-MAM platform may also implement synaptic resolution LBM capable of recording the activity of axonal and dendritic processes within a volume of ˜1×1×0.5 mm 3 ˜35 Hz as well as single cell resolution high-speed volumetric recording at ˜400 Hz and beyond, which opens up new possibilities for volumetric optical voltage imaging using GEVIs. While this document references specific recording volumes in various example implementations, these examples are intended to illustrate possible configurations of c-MAM but do not limit c-MAM to these specific example configurations. 
     The c-MAM module provides a fully colinear spatiotemporal multiplexing module geometry that uses an active switching strategy. The modularity of the design of the c-MAM, like that of MAxiMuM, facilitates commercial adoption of the s-LBM and c-MAM module. 
       FIG.  6 A  shows an example implementation of a c-MAM module  601 . The c-MAM module  601  is configured to receive pulsed laser light  602  input from a pulsed laser source. The c-MAM  601  includes a half waveplate (HWP)  605  and a polarizing beam splitter (PBS)  603  are used to couple the incoming pulses into the cavity of the c-MAM  601 . The PBS  605  outputs light pulses with horizontal polarization. 
     The c-MAM uses a fast (˜200 MHz) switching electro optical modulator (EOM)  604  which rotates the plane of the polarization such that the incoming pulse is trapped inside the I-MAM cavity. The EOM  604  is “on” (i.e., V=Vp) when the beam is initially incident, converting the polarization to vertical. The EOM is “off” is then switched off until the next pulse of the beam is incident. In some implementations, the EOM module may be implemented using off-the-shelf EOM modules and customized driver electronics. 
     During each round trip of the pulse, the polarization of the pulse is slightly rotated as the pulse passes through the HWP  605 . A portion of the pulse (a beamlet) is coupled out via the PBS  603 . The overall length of the cavity (˜2 m) in the implementation shown in  FIG.  6 A  is chosen to provide the required (˜6.25 ns) temporal delay between subsequent sub-pulses. Other implementations may have a different configuration to provide a different temporal delay. The temporal delay may be controlled by adjusting the distance between the mirrors and the focal point of the mirrors for curved mirrors or the focal point of the lens where a combination of lenses and flat mirrors are used to implement the c-MAM module  601 . 
     The c-MAM  601  includes focusing mirrors that relay the beam with a slight axial offset Δz. The vertically polarized beam is primarily reflected by the PBS, reentering the EOM  604  at its “off” state (V=0), thereby trapping the pulse and allowing it to “ring down” coupling a small fraction of it (set by the HWP  605 ) to be coupled out. This results in a co-linear column of focused light beads separated by Δz. The c-MAM  601  may be configured to enable the pulse energy of each of the individual beamlets to be arbitrarily controlled by adjusting the angle of the HWP  605 . 
     In the example implementation shown in  FIG.  6 A , the PBS  605  outputs light pulses with horizontal polarization and the EOM  604  converts the polarization of the light to vertical. However, in other implementations the PBS  605  may be configured to output light pulses with vertical polarization and the EOM  604  may be configured to convert the polarization of the light to horizontal. 
     c-MAM provides optimal voxel acquisition conditions that can implement a volumetric mesoscopy platform for Ca2+ imaging capable of recording unprecedentedly large volumes and at high volume speeds. For example, the c-MAM-based Light Beads Mesoscopy (LBM) Ca2+ imaging is capable of record recording ˜34 mm3 at ˜7 Hz when using 4×spatial multiplexing (such as that provided by spatial multiplexing module  640  shown in  FIG.  6 B ). 
     The c-MAM module presented herein may be integrated with a spatial multiplexing module which provides a new mesoscopy platform that may expand the recording volume of MAxiMuM by more than 4-fold, which may allow the recording of the entire dorsal cortex of the mouse brain. For example, c-MAM may be able to record from a target v-FOV that encompasses the entire dorsal volume of the mouse cortex while maintaining single neuron resolution. The recorded volume will allow simultaneous recording from the primary motor cortex, the secondary motor cortex, the primary sensory cortex, the visual areas, the retrosplenial area, the posterior parietal and the association areas. To maintain a sufficiently high-volume acquisition rate over such a large volume, the c-MAM module may be combined with a spatial multiplexing module to generate 4 sets of c-MAM excitations each consisting of 40 temporally multiplexed light beads in the axial direction and use them to simultaneously record from the 4 sub-volumes of the above v-FOV. The number of light beads in this example is 40, but as discussed in the preceding examples, the specific number of beamlets that may be generated by the spatiotemporal multiplexing module  100  or the c-MAM  601  is configurable and neither the spatiotemporal multiplexing module  100  nor the c-MAM  601  are limited to generating a specific number beamlets. 
       FIG.  6 B  shows a spatial multiplexing (s-Mux) module  640  that includes four beam splitters that are arranged to split the incoming pluses into four portions, where each portion is directed to a sub-FOV separated by &gt;1 mm at the sample without incurring differential path length. Spatial multiplexing will be achieved using the unique design of a dedicated beam splitter module, such as spatial multiplexing module  640  shown in  FIG.  6 B , that will allow for equal splitting of power while minimizing dispersion and introducing no pathlengths difference between the spatially multiplexed sub-beams. Using a laser source delivering ˜1-1.5 μJ pulse energies at ˜4 MHz repletion rate and 960 nm provides sufficient pulse energy to effectively excite all 40×4=160 locations in the sample at the same time with sufficient SNR. The signals generated by the four c-MAM excited sample locations may be collected by using a specially designed 4-way optical reflector that divides the entire 7.5 mm×7.5 mm FOV into 2×2 laterally stitched sub-FOVs, the fluorescence signal of each of which will be directed to one of the 4 PMTs and subsequently temporally demultiplexed as shown in  FIG.  6 C .  FIG.  6 C  show an example detection module  670  that includes a “pin-wheel” array of a knife-edge prism mirrors that splits the collected fluorescence into four quadrants, where each quadrant includes a dedicated photomultiplier tube (PMT) detector. While the preceding example shows four-way spatial multiplexing, other implementation mays may provide for other multiples of the beams to be generated and detected. Furthermore, the spatial multiplexing techniques are not limited to detecting fluorescence and may be used to detect light from other imaging techniques which may be implemented using c-MAM. 
       FIG.  7    shows a schematic of an example system  700  that incorporates the c-MAM  601 . The system  700  includes a fiber chirped-pulse amplifier (FCPA)  701  which emits the pulsed laser beam, which passes through an optical parametric chirped-pulse amplifier (OPCPA)  702  as in the preceding examples. To obtain optical access to a 7.5 mm FOV at sufficiently high NA and working distance, the system  700  may include a custom objective and tube lens design which provides a nominal clear FOV of ˜8 mm. Using this combination, the system  700  may obtain sufficiently high NA and low aberrations over the imaging FOVs that allow for lateral PSF sizes as small as ˜0.8-1 μm while maintaining a working distance of ˜5 mm. 
     The output from the OPCA may be provided as an input to the c-MAM  703 , which may operate similarly to the c-MAM  601  discussed in the preceding examples. The output from the c-MAM  704  may be provided as an input to the spatial multiplexing module  704 , and the output from the spatial multiplexing module  704  provided as an input to the optical modulation module  705 . 
     The spatial multiplexing module  704  may be implemented using the spatial multiplexing (s-Mux) module  640  shown in  FIG.  6 B . The detection module  706  may be configured to implement the detection module  670  shown in the preceding examples. Furthermore, the optical modulation module  705  may be used to implement the s-LBM using Closed-Loop Active Switching techniques discussed in the examples which follow. The optical modulation module  705  may dynamically switch on or off individual light beads as will be discussed in the detail below.  FIG.  9   , discussed in detail below, provides an example implementation of an optical modulation module that may used to implement the optical modulation module  905 . Diagram  707  shows four sub-volumes of a sample that may be scanned using the multiplexed output of the spatial multiplexing module  704  and a scanning scheme that may be used for scanning these four sub-volumes. While the spatial multiplexing module  704  is configured to multiplex the four times, other configurations may be configured to multiplex a different number of times. 
       FIG.  8    provides examples of simulated system performance data for the system  700 . Chart  801  shows PSF diameters for nominal and high-resolution mode. Chart  802  shows root-mean-square (RMS) wavefront error across the FOV. Chart  803  shows focal offset across the FOV. 
     Temporal demultiplexing and signal extraction in LBM: The effective voxel acquisition rate of 640 MHz in the 4-fold spatially multiplexed LBS sets high demands on the data acquisition and temporal demultiplexing electronic and software. Since the signals from the 4-fold spatially multiplexed sub-FOV will be collected by 4 separate PMTs, the data acquisition and demultiplexing logic of c-MAM from a single channel will be replicated 4 times. To enable the acquisition and temporal demultiplexing of a single channel of the c-MAM module  601  under the one pulse-per pixel excitation condition, a master clock is generated in the range of ˜2 GHz by multiplying a trigger signal from our pulsed laser system which will be fed to a high bandwidth digitizer and field programmable gate array (FPGA). This clock signal is used within a customized version of the ScanImage acquisition software to synchronize the line trigger to the pulse repetition rate of the laser, thus ensuring a single laser pulse constitutes one voxel of the recording. The fast sample clock allows the system to define and temporally demultiplex the 40 channels by integrating the raw PMT signals over a well-defined number of clock samples (˜12) with respect to a trigger pulse from the laser. 
     Design, development and demonstration of a selective LBM (s-LBM): The design of our proposed c-MAM-based LBM discussed in the preceding examples may be extended by implementing s-LBM in which the individual foci, i.e., the light beads can be switched on and off or have their power level adjusted in a closed-loop, adaptive and iterative fashion during recordings. For example, this approach may reduce the exposure of the sample to ˜⅕ of the average laser power used in the current LBM realization. This will in principle allow for future realization of acquisition schemes from at least 5-times larger volumes, but also enable high-speed volumetric recording from dimmer and low SNR probes such as the GEVIs while maintaining the same overall power-budget. The reduction in the amount of exposure of the sample to laser power may be adjusted by different amounts in other implementations based on the requirements of the acquisition scheme of those implementations. 
     s-LBM Using Closed-Loop Active Switching: 
     The average density of neurons in the rodent brain is ˜92,000 per mm 3  and the average size of neuronal cell bodies is ˜15-20 μm. This means that only ˜20% of the imaged volume is filled by neuronal cell bodies. Thus, when performing large-scale single neuron resolution Ca 2+  imaging, ˜80% of the excitation power is delivered to sample locations that do not overlap with neuronal cell bodies. While both the preliminary implementation of the LBM using MAM as well as its proposed spatially multiplexed version through the collinear c-MAM approach can operate at sample power densities that are within the established limits of tissue heating, it is nevertheless desirable to exploit this potential for reducing power exposure by ˜5-times for several reasons. First, generally a lower level of brain exposure to laser light will result in recoding conditions that are closer to the native physiological conditions. Second, reducing the total power at the sample while maintaining the same voxel SNR will allow in principle for future realization of volumetric Ca 2+  imaging over at least 5-times larger volumes. While such further increase of cortical recoding volume might be less relevant for the mouse brain, it will be highly relevant for applications of our method to non-human primates (NHP). Third, since current GEVIs suffer from a lack of brightness, exhibit very fast response times and are exclusively localized in the cell membrane, they are not compatible with existing volumetric Ca 2+  imaging approaches, at least not over volume sizes that would capture a large population. In this context, the above gain in efficiency can be used to increase voxel SNR by ˜25-times while maintaining the current total power levels. Thus, this enhancement in SNR by the method provided herein allows realization of cellular resolution volumetric voltage imaging at ˜400 Hz or higher, while ideally compensates for the above limitations of the current GEVIs. Lastly, by reducing the number of voxels in the 3D imaging volume that are receiving simultaneous excitations at any given point in time, scatter-induce crosstalk will be reduced between our  4  spatially multiplexed strings of light beads. Thereby compared to the case when all light beads in LBM are receiving excitations, in principle is possible to accommodate for a shorter lateral distance between the spatially multiplexed beams for the same level of tolerable crosstalk. This would allow for implementation of spatial multiplexing within smaller FOV or a higher degree of spatial multiplexing within the mesoscale imaging FOV. 
     To realize s-LBM, after spatial multiplexing, each of the 4 strings of light beads generated by the c-MAM module may be sent through a high-bandwidth (˜160 MHz) acousto-optical modulator (AOM), which will allow for passing or deflecting each individual light bead in each string within the 6.25 ns inter-pulse time of the laser while the temporally multiplexed string of light beads is propagating through each AOM. Thereby, the individual light beads, directed at different axial locations in each of the sub-volumes, can be switched on and off on the time scale of the fluorescence lifetime as they are being laterally scanned. Alternatively, the power level of the individual light beads may be adjusted rather than simply switching on or off a particular light bead. In s-LBM, no a-priory knowledge about the neuron locations is necessary. This information will be generated and iteratively and repeatedly updated during the recording by s-LBM itself. Initially, all light beads will be in their “on” state. After a few recorded volumes, the raw signals from each voxel in the sample will be thresholded depending on a criterion that would indicate the presence of a neuronal cell body at that sample voxel. For all voxels at which the recorded signal is below the threshold criterion, an analog signal will be generated by our data acquisition system and the corresponding sub-pulse will be deflected by the AOM. The “on” and the “off” states of the light beads may be periodically evaluated and updated using a change-point detection-like algorithm that takes the previous history of the state of each light bead into account (see  FIG.  9   ). Thereby, neurons will be identified that have been inactive during the initialization or any subsequent imaging period while voxels that might have been initially incorrectly classified as containing neuronal cell bodies will be turned off in subsequent cycles of the recording. In this fashion, the utilized power level at the sample converges iteratively to a level given by the actual number of neuronal cell bodies in the recoding volume. It is important to note that the s-LBM approach provided herein does not suffer from sample induced motion artifacts for at for two reasons. First, the size of the PSF for recording neuronal cell bodies is ˜2-4-times larger than the diffraction limited PSFs used in random access microscopy. This will result in a higher level of resilience towards sample motion. Second, and more importantly, s-LBM exhibits 3 orders of magnitude higher recorded voxel density and more than 3 orders of magnitude higher voxel acquisition rate compared to random access microscopy. This feature makes within-frame axial motion virtually non-existent for s-LBM while our higher voxel sampling density results in multiple excitations (˜5-25) per neuron, allowing for disambiguation of neuroactivity from sample motion. 
     Preliminary data and biological applications: To demonstrate the feasibility of s-LBM and to compare the obtainable neuronal positions and activity traces with this method to a structural and functional ground truth, a series of experiments and experimentally guided simulations were performed. To obtain spatial and temporal ground truth, high speed Ca 2+  imaging was performed at 28 Hz using a pixel spacing of 0.5 μm for 2 minutes. Neuronal positions were identified, and their time series was extracted using our previously established standard CaiMAN data processing pipeline. The effect of the implementation of the proposed s-LBM was then evaluated within an imaging plane by assuming a larger voxel spacing and implementing the operation modality of s-LBM in the following fashion: the voxel values for 5 subsequent frames were averaged for each voxel and subsequently, based on the cumulative distribution of the voxel values, a threshold was applied. Thereby an initialization condition for all voxel settings (“on” or “off”) were obtained which represented our best estimate as to whether a given voxel contained a neuron or not. This evaluation was repeated after the “polling time” T over the entire imaging time. The results, which were evaluated based on a receiving operator characteristics (ROC) framework. As expected, the sensitivity for identifying a neuron increases with both a lower threshold value and higher polling frequencies. However, even for polling frequencies in the range of 0.5-1 Hz—which are well in-line with the timescales of the closed-loop selective switching hardware and software and for threshold values ˜08.-0.9, the obtainable sensitivities can reach &gt;90%. At these conditions, the need for power at the sample can be reduced to ˜20% of the full power value. A sensitivity of &gt;90% directly implies low values for false negatives while our method also naturally exhibits a low susceptibility to false positives since in our approach, excitation voxels and thereby information is removed from the recording. 
     The s-LBM platform provides a versatile solution that allows volume size to be traded off for spatial resolution while increasing the temporal acquisition rate of the volume. By going from the mesoscopic volume to a volume of ˜1.0×1.0×0.5 mm 3 , the s-LBM platform is able to perform synaptic resolution s-LBM capable of recording the activity of axonal and dendritic processes within this volume at ˜35 Hz as well as single cell resolution high-speed volumetric recording at ˜400 Hz and beyond, opening up new possibilities for volumetric optical voltage imaging using GEVIs. 
       FIG.  9    is a diagram of a selective switching schematic  900  enabling s-LBM.  FIG.  9    illustrates an iterative polling process in which: all beads are turned on for a ˜1 s recording with some total power Po, candidate neurons are identified and localized and the state of each light bead is correspondingly assigned a Boolean “on” or “off” value. This Boolean map modulates an acousto-optic modulator (AOM) which turns off light beads that do not overlap with neurons or other objects of interest in a sample. In other implementations, the Boolean map may be replaced with a map of power values, percentages, or other values that indicate a power level of each individual bead that should be set to rather than turning on or turning off each respective bead. The process may be repeated with the polling frequency F leading to a reduction of the P o  to P P o /5. 
     In operation  905 , the initial scanning is performed in which all beads are turned on until the locations of neurons within the candidate neurons are identified. In operation  910 , a selective-MAM (s-MAM) algorithm may be applied to determine which of the beads should be turned on and which of the beads should be turned off. The beads associated with locations in the sample that do not appear to have a neuron present may be turned off. The s-MAM may determine threshold criterion whether a neuron is present at a particular location by: (1) determining whether a signal associated with light detected at the respective location within the volume of the sample exceeds a threshold; (2) analyzing signals associated with light detected at the respective location in the sample using a factorization algorithm to determine whether the object of interest is present at the respective location, the factorization algorithm may be a Constrained Nonnegative Matrix Factorization (3) determining whether the object of interest is present at the respective location within the volume based on change-point detection of signals associated with light detected at the respective location within the volume; (4) determining whether the object of interest is present at the respective location within the volume based on cumulative history weighting of signals associated with light detected at the respective location in the volume of the sample; or (5) a combination of two or more of these determinations. In operation  915 , an array of binary values representing each of the beads may be generated based on the determination from operation  910 . In operation  920 , the beads may be selectively turned on or off based on the binary values determined in operation  915 . In some implementations, the binary values may be generated as a binary signal that may be provided to the AOM to cause the AOM to selectively deflect the light beads for locations in the sample where no candidate neuron was present. In operation  925 , the sub-pulses corresponding to the beads that are turned off will be deflected by the AOM or other means for deflecting the sub-pulses. The “on” and the “off” states of the light beads will be periodically evaluated and updated. 
       FIG.  10    provides a chart  1005  of simulated sensitivity (1−false negative rate) as a function of the polling frequency F and threshold value. Thresholds are determined by setting a minimum pixel value based on the cumulative distribution of values in the frame.  FIG.  10    also provides a chart  1010  of simulated sensitivity versus reduction in required imaging power for different threshold values. Power is function of threshold. 
       FIG.  11    is a flow diagram of a process  1100  for operating a spatiotemporal multiplexing module to image a sample. The process  1100  may be implemented by the spatiotemporal multiplexing module  100  discussed in the preceding examples. The process  1100  may be used to implement, at least in part, the MAXiMuM techniques or the c-MAM techniques shown in the preceding figures. 
     The process  1100  may include an operation  1110  of receiving a plurality of laser pulses from a pulsed laser source. As shown in  FIGS.  1 A,  2 B, and  6   , the pulsed laser source may provide an input beam to the multiplexing module. The process  1100  may include an operation  1120  splitting each laser pulse into a plurality of beamlets using the spatiotemporal multiplexing module  100 . The spatiotemporal multiplexing module  100  may include a first re-imaging cavity  110  that includes an output coupler, also referred to as a beamsplitter or PRM, for splitting the laser pulse receive from the pulsed laser source into series of beamlets or pulses. The beamsplitter redirects the majority of the light incident on the beamsplitter back through the re-imaging cavity  110  and outputs the remainder of the light from the re-imaging cavity  110  to the secondary re-imaging cavity  115 . 
     The process  1100  may include an operation  1130  introducing a delay between each adjacent beamlet of the plurality of beamlets using the multiplexing module. The plurality of beamlets associated with a respective laser pulse of the plurality of laser pulses is distributed equally across a pulse repetition period associated with the pulsed laser source. As discussed in the preceding examples, the delay is introduced by the beamsplitter redirecting the majority of the beam back through a re-imaging cavity of the multiplexing module, and each round trip through the cavity introduces a temporal delay. 
     The process  1100  may include an operation  1140  of changing a divergence of each subsequent beamlet of the plurality of beamlets associated with each respective laser pulse to introduce a distinguishing feature between each beamlet of the plurality of beamlet to cause each beamlet to focus on a different axial plane or lateral position of the sample. Each round trip through the cavity may also provide a lateral or axial offset in the focal plane in addition to the temporal offset introduced between beamlets. This temporal and/or positional offset information may be used to distinguish between light resulting from fluorescence at different positions within the sample. 
     The process  1100  may include an operation  1150  of outputting the plurality of beamlets associated with each respective laser pulse. The beamlets may be output by the multiplexing module as discussed in the preceding examples. The beamlets output to a microscope and the microscope scans a set of axially separated and temporally distinct foci as shown in  FIG.  2   . The beamlets may be processed by a spatial multiplexing module and/or an optical modulation module as discussed in the preceding examples. 
       FIG.  12    is a flow diagram of a process  1200  for operating a spatial multiplexing module to image a sample. The spatial multiplexing module may be implemented by the spatial multiplexing module  640  shown in  FIG.  6 B . The spatial multiplexing module may be implemented by a dedicated beam splitter that allows for equal splitting of power while minimizing dispersion and introducing no pathlengths difference between the spatially multiplexed sub-beams. 
     The process  1200  may include an operation  1210  of receiving a plurality of laser pulses from a pulsed laser source. As discussed in the preceding examples, the plurality of laser pulses may be multiplexed into a plurality of beamlets. In some implementations, the plurality of spatially separated laser pulses may be focused on a sample to cause an indicator on the sample to fluoresce. However, the spatial multiplexing module may be used with other types of imaging techniques, such as but not limited to voltage imaging. 
     The process  1200  may include an operation  1220  of splitting the plurality of laser pulses into a plurality of laterally separated laser pulses. The spatial multiplexing module  640  may be used to split each of the laser pulses and/or the set of beamlets into multiple laterally separated beamlets that may be used to simultaneously scan more than one portion of a sample simultaneously. The process  1200  may include an operation  1230  of outputting the plurality of laterally separated laser pulses to be focused on a sample to cause an indicator on the sample to fluoresce. The spatial multiplexing module  640  may be used to split each of the laser pulses and/or the set of beamlets into multiple laterally separated beamlets that may be output by the spatial multiplexing module  640 . The laser pulses and/or the set of beamlets into multiple laterally separated beamlets may be directed to the sample to simultaneously scan more than one portion of a sample simultaneously. 
     The spatial multiplexing module  640  may be integrated with an imaging device comprising a two photon or multi-photon excitation scanning microscope. The microscope may be configured to focus each of the plurality of sets of laterally separated beamlets onto a separate region of a sample. The imaging device is further integrated with a spatial resolved detector, such as the detection module  670  shown in  FIG.  6 C . The spatial resolved detector may be configured to split light associated with each respective region of the sample and to direct the light associated each respective region to a separate photodetector associated with that region. In implementations where the beamlets cause an indicator on the sample to fluoresce, the spatial resolved detector may detect light associated with the fluorescence of objects of interest within the sample. 
       FIG.  13    is a flow diagram of a process  1300  for operating an optical modulation module to image a sample. The process  1300  may be implemented by the optical modulation module  705  and the selective switching schematic  900  shown in  FIG.  9   . 
     The process  1300  may include an operation  1310  of receiving a plurality of beamlets associated with each laser pulse of a plurality of laser pulses. Each beamlet is associated with a laser pulse is associated with a different axial or lateral position within a sample. Each laser pulse may be subdivided into a plurality of beamlets using the MaXiMuM module or the c-MAM discussed in the preceding examples. 
     The process  1300  may include an operation  1320  of determining whether an object of interest is present at a respective location within the volume associated with each beamlet of the plurality of beamlets associated with each laser pulse. The s-LBM using Closed-Loop Active Switching techniques discussed above may be used to determine whether an object of interest, such as but not limited to a neuron, is present at a particular location within the volume of the sample. The beamlets or light beads associated with locations where no object of interest are detected in the sample volume may be selectively switched off or the power level of the beamlets or lights beams may be selectively adjusted higher or lower. Furthermore, if location was incorrectly determined to not include an object of interest, the beamlet or light bead associated with that location may be selectively switch on. 
     The process  1300  may include an operation  1330  of selectively adjust the amount of power associated with a set of beamlets from the plurality of beamlets for which no object of interest is located at the respective location in the volume associated with the respective location in the volume of the sample associated with the respective beamlet to reduce an amount of light associated with the set of beamlets from reaching the sample to generate modified beamlets. In the examples discussed in the preceding examples, an acousto-optical modulator (AOM) may be used to deflect the beams to be turned off. In other implementations, other types of optical modulators may be used to selectively turn on or off the beams or light beads to prevent the beamlets or light beads that have been turned off from reaching the sample. 
     The process  1300  may include an operation  1340  of outputting the optically modulated beamlets. As discussed with respect to  FIG.  9   , the process  1300  may be performed iteratively to dynamically determine which beamlets or light beads may be turned on or off. 
     The detailed examples of systems, devices, and techniques described in connection with  FIGS.  1 A- 13    are presented herein for illustration of the disclosure and its benefits. Such examples of use should not be construed to be limitations on the logical process embodiments of the disclosure, nor should variations of user interface methods from those described herein be considered outside the scope of the present disclosure. It is understood that references to displaying or presenting an item (such as, but not limited to, presenting an image on a display device, presenting audio via one or more loudspeakers, and/or vibrating a device) include issuing instructions, commands, and/or signals causing, or reasonably expected to cause, a device or system to display or present the item. In some embodiments, various features described in  FIGS.  1 A- 13    are implemented in respective modules, which may also be referred to as, and/or include, logic, components, units, and/or mechanisms. Modules may constitute either software modules (for example, code embodied on a machine-readable medium) or hardware modules. 
     In some examples, a hardware module may be implemented mechanically, electronically, or with any suitable combination thereof. For example, a hardware module may include dedicated circuitry or logic that is configured to perform certain operations. For example, a hardware module may include a special-purpose processor, such as a field-programmable gate array (FPGA) or an Application Specific Integrated Circuit (ASIC). A hardware module may also include programmable logic or circuitry that is temporarily configured by software to perform certain operations and may include a portion of machine-readable medium data and/or instructions for such configuration. For example, a hardware module may include software encompassed within a programmable processor configured to execute a set of software instructions. It will be appreciated that the decision to implement a hardware module mechanically, in dedicated and permanently configured circuitry, or in temporarily configured circuitry (for example, configured by software) may be driven by cost, time, support, and engineering considerations. 
     Accordingly, the phrase “hardware module” should be understood to encompass a tangible entity capable of performing certain operations and may be configured or arranged in a certain physical manner, be that an entity that is physically constructed, permanently configured (for example, hardwired), and/or temporarily configured (for example, programmed) to operate in a certain manner or to perform certain operations described herein. As used herein, “hardware-implemented module” refers to a hardware module. Considering examples in which hardware modules are temporarily configured (for example, programmed), each of the hardware modules need not be configured or instantiated at any one instance in time. For example, where a hardware module includes a programmable processor configured by software to become a special-purpose processor, the programmable processor may be configured as respectively different special-purpose processors (for example, including different hardware modules) at different times. Software may accordingly configure a processor or processors, for example, to constitute a particular hardware module at one instance of time and to constitute a different hardware module at a different instance of time. A hardware module implemented using one or more processors may be referred to as being “processor implemented” or “computer implemented.” 
     Hardware modules can provide information to, and receive information from, other hardware modules. Accordingly, the described hardware modules may be regarded as being communicatively coupled. Where multiple hardware modules exist contemporaneously, communications may be achieved through signal transmission (for example, over appropriate circuits and buses) between or among two or more of the hardware modules. In embodiments in which multiple hardware modules are configured or instantiated at different times, communications between such hardware modules may be achieved, for example, through the storage and retrieval of information in memory devices to which the multiple hardware modules have access. For example, one hardware module may perform an operation and store the output in a memory device, and another hardware module may then access the memory device to retrieve and process the stored output. 
     In some examples, at least some of the operations of a method may be performed by one or more processors or processor-implemented modules. Moreover, the one or more processors may also operate to support performance of the relevant operations in a “cloud computing” environment or as a “software as a service” (SaaS). For example, at least some of the operations may be performed by, and/or among, multiple computers (as examples of machines including processors), with these operations being accessible via a network (for example, the Internet) and/or via one or more software interfaces (for example, an application program interface (API)). The performance of certain of the operations may be distributed among the processors, not only residing within a single machine, but deployed across several machines. Processors or processor-implemented modules may be in a single geographic location (for example, within a home or office environment, or a server farm), or may be distributed across multiple geographic locations. 
       FIG.  14    is a block diagram  1400  illustrating an example software architecture  1402 , various portions of which may be used in conjunction with various hardware architectures herein described, which may implement any of the above-described features.  FIG.  14    is a non-limiting example of a software architecture and it will be appreciated that many other architectures may be implemented to facilitate the functionality described herein. The software architecture  1402  may execute on hardware such as a machine  1500  of  FIG.  15    that includes, among other things, processors  1510 , memory  1530 , and input/output (I/O) components  1550 . A representative hardware layer  1404  is illustrated and can represent, for example, the machine  1500  of  FIG.  15   . The representative hardware layer  1404  includes a processing unit  1406  and associated executable instructions  1408 . The executable instructions  1408  represent executable instructions of the software architecture  1402 , including implementation of the methods, modules and so forth described herein. The hardware layer  1404  also includes a memory/storage  1410 , which also includes the executable instructions  1408  and accompanying data. The hardware layer  1404  may also include other hardware modules  1412 . Instructions  1408  held by processing unit  1406  may be portions of instructions  1408  held by the memory/storage  1410 . 
     The example software architecture  1402  may be conceptualized as layers, each providing various functionality. For example, the software architecture  1402  may include layers and components such as an operating system (OS)  1414 , libraries  1416 , frameworks  1418 , applications  1420 , and a presentation layer  1444 . Operationally, the applications  1420  and/or other components within the layers may invoke API calls  1424  to other layers and receive corresponding results  1426 . The layers illustrated are representative in nature and other software architectures may include additional or different layers. For example, some mobile or special purpose operating systems may not provide the frameworks/middleware  1418 . 
     The OS  1414  may manage hardware resources and provide common services. The OS  1414  may include, for example, a kernel  1428 , services  1430 , and drivers  1432 . The kernel  1428  may act as an abstraction layer between the hardware layer  1404  and other software layers. For example, the kernel  1428  may be responsible for memory management, processor management (for example, scheduling), component management, networking, security settings, and so on. The services  1430  may provide other common services for the other software layers. The drivers  1432  may be responsible for controlling or interfacing with the underlying hardware layer  1404 . For instance, the drivers  1432  may include display drivers, camera drivers, memory/storage drivers, peripheral device drivers (for example, via Universal Serial Bus (USB)), network and/or wireless communication drivers, audio drivers, and so forth depending on the hardware and/or software configuration. 
     The libraries  1416  may provide a common infrastructure that may be used by the applications  1420  and/or other components and/or layers. The libraries  1416  typically provide functionality for use by other software modules to perform tasks, rather than rather than interacting directly with the OS  1414 . The libraries  1416  may include system libraries  1434  (for example, C standard library) that may provide functions such as memory allocation, string manipulation, file operations. In addition, the libraries  1416  may include API libraries  1436  such as media libraries (for example, supporting presentation and manipulation of image, sound, and/or video data formats), graphics libraries (for example, an OpenGL library for rendering 2D and 3D graphics on a display), database libraries (for example, SQLite or other relational database functions), and web libraries (for example, WebKit that may provide web browsing functionality). The libraries  1416  may also include a wide variety of other libraries  1438  to provide many functions for applications  1420  and other software modules. 
     The frameworks  1418  (also sometimes referred to as middleware) provide a higher-level common infrastructure that may be used by the applications  1420  and/or other software modules. For example, the frameworks  1418  may provide various graphic user interface (GUI) functions, high-level resource management, or high-level location services. The frameworks  1418  may provide a broad spectrum of other APIs for applications  1420  and/or other software modules. 
     The applications  1420  include built-in applications  1440  and/or third-party applications  1442 . Examples of built-in applications  1440  may include, but are not limited to, a contacts application, a browser application, a location application, a media application, a messaging application, and/or a game application. Third-party applications  1442  may include any applications developed by an entity other than the vendor of the particular platform. The applications  1420  may use functions available via OS  1414 , libraries  1416 , frameworks  1418 , and presentation layer  1444  to create user interfaces to interact with users. 
     Some software architectures use virtual machines, as illustrated by a virtual machine  1448 . The virtual machine  1448  provides an execution environment where applications/modules can execute as if they were executing on a hardware machine (such as the machine  1500  of  FIG.  15   , for example). The virtual machine  1448  may be hosted by a host OS (for example, OS  1414 ) or hypervisor, and may have a virtual machine monitor  1446  which manages operation of the virtual machine  1448  and interoperation with the host operating system. A software architecture, which may be different from software architecture  1402  outside of the virtual machine, executes within the virtual machine  1448  such as an OS  1450 , libraries  1452 , frameworks  1454 , applications  1456 , and/or a presentation layer  1458 . 
       FIG.  15    is a block diagram illustrating components of an example machine  1500  configured to read instructions from a machine-readable medium (for example, a machine-readable storage medium) and perform any of the features described herein. The example machine  1500  is in a form of a computer system, within which instructions  1516  (for example, in the form of software components) for causing the machine  1500  to perform any of the features described herein may be executed. As such, the instructions  1516  may be used to implement modules or components described herein. The instructions  1516  cause unprogrammed and/or unconfigured machine  1500  to operate as a particular machine configured to carry out the described features. The machine  1500  may be configured to operate as a standalone device or may be coupled (for example, networked) to other machines. In a networked deployment, the machine  1500  may operate in the capacity of a server machine or a client machine in a server-client network environment, or as a node in a peer-to-peer or distributed network environment. Machine  1500  may be embodied as, for example, a server computer, a client computer, a personal computer (PC), a tablet computer, a laptop computer, a netbook, a set-top box (STB), a gaming and/or entertainment system, a smart phone, a mobile device, a wearable device (for example, a smart watch), and an Internet of Things (IoT) device. Further, although only a single machine  1500  is illustrated, the term “machine” includes a collection of machines that individually or jointly execute the instructions  1516 . 
     The machine  1500  may include processors  1510 , memory  1530 , and I/O components  1550 , which may be communicatively coupled via, for example, a bus  1502 . The bus  1502  may include multiple buses coupling various elements of machine  1500  via various bus technologies and protocols. In an example, the processors  1510  (including, for example, a central processing unit (CPU), a graphics processing unit (GPU), a digital signal processor (DSP), an ASIC, or a suitable combination thereof) may include one or more processors  1512   a  to  1512   n  that may execute the instructions  1516  and process data. In some examples, one or more processors  1510  may execute instructions provided or identified by one or more other processors  1510 . The term “processor” includes a multi-core processor including cores that may execute instructions contemporaneously. Although  FIG.  15    shows multiple processors, the machine  1500  may include a single processor with a single core, a single processor with multiple cores (for example, a multi-core processor), multiple processors each with a single core, multiple processors each with multiple cores, or any combination thereof. In some examples, the machine  1500  may include multiple processors distributed among multiple machines. 
     The memory/storage  1530  may include a main memory  1532 , a static memory  1534 , or other memory, and a storage unit  1536 , both accessible to the processors  1510  such as via the bus  1502 . The storage unit  1536  and memory  1532 ,  1534  store instructions  1516  embodying any one or more of the functions described herein. The memory/storage  1530  may also store temporary, intermediate, and/or long-term data for processors  1510 . The instructions  1516  may also reside, completely or partially, within the memory  1532 ,  1534 , within the storage unit  1536 , within at least one of the processors  1510  (for example, within a command buffer or cache memory), within memory at least one of I/O components  1550 , or any suitable combination thereof, during execution thereof. Accordingly, the memory  1532 ,  1534 , the storage unit  1536 , memory in processors  1510 , and memory in I/O components  1550  are examples of machine-readable media. 
     As used herein, “machine-readable medium” refers to a device able to temporarily or permanently store instructions and data that cause machine  1500  to operate in a specific fashion, and may include, but is not limited to, random-access memory (RAM), read-only memory (ROM), buffer memory, flash memory, optical storage media, magnetic storage media and devices, cache memory, network-accessible or cloud storage, other types of storage and/or any suitable combination thereof. The term “machine-readable medium” applies to a single medium, or combination of multiple media, used to store instructions (for example, instructions  1516 ) for execution by a machine  1500  such that the instructions, when executed by one or more processors  1510  of the machine  1500 , cause the machine  1500  to perform and one or more of the features described herein. Accordingly, a “machine-readable medium” may refer to a single storage device, as well as “cloud-based” storage systems or storage networks that include multiple storage apparatus or devices. The term “machine-readable medium” excludes signals per se. 
     The I/O components  1550  may include a wide variety of hardware components adapted to receive input, provide output, produce output, transmit information, exchange information, capture measurements, and so on. The specific I/O components  1550  included in a particular machine will depend on the type and/or function of the machine. For example, mobile devices such as mobile phones may include a touch input device, whereas a headless server or IoT device may not include such a touch input device. The particular examples of I/O components illustrated in  FIG.  15    are in no way limiting, and other types of components may be included in machine  1500 . The grouping of I/O components  1550  are merely for simplifying this discussion, and the grouping is in no way limiting. In various examples, the I/O components  1550  may include user output components  1552  and user input components  1554 . User output components  1552  may include, for example, display components for displaying information (for example, a liquid crystal display (LCD) or a projector), acoustic components (for example, speakers), haptic components (for example, a vibratory motor or force-feedback device), and/or other signal generators. User input components  1554  may include, for example, alphanumeric input components (for example, a keyboard or a touch screen), pointing components (for example, a mouse device, a touchpad, or another pointing instrument), and/or tactile input components (for example, a physical button or a touch screen that provides location and/or force of touches or touch gestures) configured for receiving various user inputs, such as user commands and/or selections. 
     In some examples, the I/O components  1550  may include biometric components  1556 , motion components  1558 , environmental components  1560 , and/or position components  1562 , among a wide array of other physical sensor components. The biometric components  1556  may include, for example, components to detect body expressions (for example, facial expressions, vocal expressions, hand or body gestures, or eye tracking), measure biosignals (for example, heart rate or brain waves), and identify a person (for example, via voice-, retina-, fingerprint-, and/or facial-based identification). The motion components  1558  may include, for example, acceleration sensors (for example, an accelerometer) and rotation sensors (for example, a gyroscope). The environmental components  1560  may include, for example, illumination sensors, temperature sensors, humidity sensors, pressure sensors (for example, a barometer), acoustic sensors (for example, a microphone used to detect ambient noise), proximity sensors (for example, infrared sensing of nearby objects), and/or other components that may provide indications, measurements, or signals corresponding to a surrounding physical environment. The position components  1562  may include, for example, location sensors (for example, a Global Position System (GPS) receiver), altitude sensors (for example, an air pressure sensor from which altitude may be derived), and/or orientation sensors (for example, magnetometers). 
     The I/O components  1550  may include communication components  1564 , implementing a wide variety of technologies operable to couple the machine  1500  to network(s)  1570  and/or device(s)  1580  via respective communicative couplings  1572  and  1582 . The communication components  1564  may include one or more network interface components or other suitable devices to interface with the network(s)  1570 . The communication components  1564  may include, for example, components adapted to provide wired communication, wireless communication, cellular communication, Near Field Communication (NFC), Bluetooth communication, Wi-Fi, and/or communication via other modalities. The device(s)  1580  may include other machines or various peripheral devices (for example, coupled via USB). 
     In some examples, the communication components  1564  may detect identifiers or include components adapted to detect identifiers. For example, the communication components  1564  may include Radio Frequency Identification (RFID) tag readers, NFC detectors, optical sensors (for example, one- or multi-dimensional bar codes, or other optical codes), and/or acoustic detectors (for example, microphones to identify tagged audio signals). In some examples, location information may be determined based on information from the communication components  1562 , such as, but not limited to, geo-location via Internet Protocol (IP) address, location via Wi-Fi, cellular, NFC, Bluetooth, or other wireless station identification and/or signal triangulation. 
     In the following, further features, characteristics and advantages of the system and method will be described by means of items: 
     Item 1. A multiplexing module configured to perform operations of: receiving a plurality of laser pulses from a pulsed laser source; splitting each laser pulse into a plurality of beamlets; introducing a delay between each adjacent beamlet of the plurality of beamlets, such that the plurality of beamlets associated with a respective laser pulse of the plurality of laser pulses is distributed equally across a pulse repetition period associated with the pulsed laser source; changing a divergence of each subsequent beamlet of the plurality of beamlets associated with each respective laser pulse to introduce a distinguishing feature between each beamlet of the plurality of beamlet to cause each beamlet to focus on a different axial plane or lateral position of the sample; and outputting the plurality of beamlets associated with each respective laser pulse. 
     Item 2. The multiplexing module of item 1, wherein the multiplexing module comprises a re-imaging cavity, and wherein to split each laser pulse into a plurality of beamlets the multiplexing module is configured to perform the operations of: passing each laser pulse through a beam splitter to split the laser pulse into a beamlet of the plurality of beamlets to be output by the multiplexing module and a second portion of the laser pulse to reenter the re-imaging cavity. 
     Item 3. The multiplexing module of item 1, wherein the multiplexing module is further configured to: adjusting a pulse energy by a selected amount for each subsequent beamlet of the plurality of beamlets by selecting a beamsplitter and its alignment having a splitting ratio configured to cause the pulse energy to be adjusted by the selected amount. 
     Item 4. The multiplexing module of item 1, wherein to introduce the delay between each adjacent beamlet of the plurality of beamlets the multiplexing module is configured to perform the operations of: directing the second portion of the laser pulse around a series of mirrors of the re-imaging cavity to introduce the delay between adjacent beamlets. 
     Item 5. The multiplexing module of item 4, wherein the multiplexing module is further configured to perform the operation of: introducing an axial offset between each adjacent beamlet to create an offset in the focal plane for each adjacent beamlet. 
     Item 6. The multiplexing module of item 4, wherein the series of mirrors comprises curved mirrors, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the mirrors. 
     Item 7. The multiplexing module of item 4, wherein the series of mirrors comprise flat mirrors and lenses, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the lenses. 
     Item 8. The multiplexing module of item 4, wherein changing the divergence of each subsequent beamlet causes each beamlet to focus on the different axial plane of the sample, wherein the multiplexing module comprises a second re-imaging cavity, and wherein the second re-imaging cavity is configured to perform operations of: splitting the plurality of beamlets output by the first cavity into a second plurality of beamlets for each respective laser pulse; introducing a delay and a lateral offset between each adjacent beamlet of the plurality of beamlets; increasing the divergence of each subsequent beamlet of the plurality of second beamlets associated with each respective laser pulse; and outputting the plurality of second beamlets. 
     Item 9. The multiplexing module of item 4, wherein changing the divergence of each subsequent beamlet causes each beamlet to focus on the different lateral position of the sample, wherein the multiplexing module comprises a second cavity, and wherein the second cavity is configured to perform operations of: splitting the plurality of beamlets output by the first cavity into a second plurality of beamlets for each respective laser pulse; introducing a delay between each adjacent beamlet of the plurality of beamlets; increasing the divergence of each subsequent beamlet of the plurality of second beamlets associated with each respective laser pulse; and outputting the plurality of second beamlets. 
     Item 10. The multiplexing module of item 1, wherein the multiplexing module comprises a re-imaging cavity, and wherein to split each laser pulse into a plurality of beamlets the multiplexing module is configured to perform operations of: rotating a plane of polarization of each laser pulse by passing the through an electro optical modulator (EOM); directing the laser pulse around the multiplexing module to a half-wave plate (HWP); passing each laser pulse through a half-wave plate (HWP) to change the plane of polarization of the laser pulse by a fixed amount such that a chosen portion of the laser pulse can pass through a polarized beam splitter (PSB); and directing the laser pulse to the PSB to cause a first portion of the laser pulse to pass through the PSB and exit the multiplexing module as a beamlet of the plurality of beamlets and a second portion of the laser pulse which is directed back into the re-imaging cavity. 
     Item 11. The multiplexing module of item 10, wherein the multiplexing module is further configured to: adjusting a pulse energy by a selected amount for each subsequent beamlet of the plurality of beamlets by selecting the HWP having a rotation angle configured to cause the pulse energy to be adjusted by the selected amount for each subsequent beamlet of the plurality of beamlets. 
     Item 12. The multiplexing module of item 10, wherein to introduce the delay between each adjacent beamlets of the plurality of beamlets the multiplexing module is configured to perform operations of: directing the second portion of the laser pulse around a series of mirrors of the re-imaging cavity to introduce the delay between adjacent beamlets. 
     Item 13. The multiplexing module of item 12, wherein the series of mirrors comprises curved mirrors, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the mirrors. 
     Item 14. The multiplexing module of item 12, wherein the series of mirrors comprise flat mirrors and lenses, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the lenses. 
     Item 15. The multiplexing module of item 1, wherein the multiplexing module is integrated with an imaging device comprising a two photon or multi-photon excitation scanning microscope, and wherein the two photon or multi-photon excitation scanning microscope is configured to perform operations of: receiving the plurality of beamlets of the plurality of beamlets associated with each respective laser pulse from the multiplexing module; and converting divergences associated with the plurality of beamlets into axial or lateral foci on the sample. 
     Item 16. The multiplexing module of item 1, wherein to split each laser pulse into a plurality of beamlets, the multiplexing module is configured to perform operations of: splitting each laser pulse into a plurality of beamlets; focusing the plurality of beamlets onto a first knife edge (KE) mirror that is vertically oriented which directs the plurality of beamlets into a re-imaging cavity of the multiplexing module to cause the beamlets to make a round trip of the multiplexing module. 
     17. The multiplexing module of item 16, wherein the multiplexing module is configured to perform operations of: splitting off a respective beamlet of the plurality of beamlets using a second KE mirror that is vertically oriented for each roundtrip around the multiplexing module; and outputting the respective beamlet after corresponding number of roundtrips associated with its intended delay from the multiplexing module. 
     Item 18. A method of operating a multiplexing module, the method comprising: receiving a plurality of laser pulses from a pulsed laser source; splitting each laser pulse into a plurality of beamlets; introducing a delay between each adjacent beamlet of the plurality of beamlets, such that the plurality of beamlets associated with a respective laser pulse of the plurality of laser pulses is distributed equally across a pulse repetition period associated with the pulsed laser source; changing a divergence of each subsequent beamlet of the plurality of beamlets associated with each respective laser pulse to introduce a distinguishing feature between each beamlet of the plurality of beamlet to cause each beamlet to focus on a different axial plane or lateral position of the sample; and outputting the plurality of beamlets associated with each respective laser pulse. 
     Item 19. The method of item 18, wherein the multiplexing module comprises a re-imaging cavity, and wherein splitting each laser pulse into a plurality of beamlets further comprises: passing each laser pulse through a beam splitter to split the laser pulse into a beamlet of the plurality of beamlets to be output by the multiplexing module and a second portion of the laser pulse to reenter the re-imaging cavity. 
     Item 20. The method of item 18, further comprising: adjusting a pulse energy by a selected amount for each subsequent beamlet of the plurality of beamlets by selecting a beamsplitter and its alignment having a splitting ratio configured to cause the pulse energy to be adjusted by the selected amount. 
     Item 21. The method of item 18, wherein introducing the delay between each adjacent beamlet of the plurality of beamlets the multiplexing module further comprises: directing the second portion of the laser pulse around a series of mirrors of the re-imaging cavity to introduce the delay between adjacent beamlets. 
     Item 22. The method of item 21, further comprising: introducing an axial offset between each adjacent beamlet to create an offset in the focal plane for each adjacent beamlet. 
     Item 23. The method of item 21, wherein the series of mirrors comprises curved mirrors, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the mirrors. 
     Item 24. The method of item 21, wherein the series of mirrors comprise flat mirrors and lenses, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the lenses. 
     Item 25. The method of item 21, wherein changing the divergence of each subsequent beamlet causes each beamlet to focus on the different axial plane of the sample, wherein the multiplexing module comprises a second re-imaging cavity, and the method further comprising: splitting the plurality of beamlets output by the first cavity into a second plurality of beamlets for each respective laser pulse; introducing a delay and a lateral offset between each adjacent beamlet of the plurality of beamlets; increasing the divergence of each subsequent beamlet of the plurality of second beamlets associated with each respective laser pulse; and outputting the plurality of second beamlets. 
     Item 26. The method of item 21, wherein changing the divergence of each subsequent beamlet causes each beamlet to focus on the different lateral position of the sample, wherein the multiplexing module comprises a second cavity, and the method further comprising: splitting the plurality of beamlets output by the first cavity into a second plurality of beamlets for each respective laser pulse; introducing a delay between each adjacent beamlet of the plurality of beamlets; increasing the divergence of each subsequent beamlet of the plurality of second beamlets associated with each respective laser pulse; and outputting the plurality of second beamlets. 
     Item 27. The method of item 18, wherein the multiplexing module comprises a re-imaging cavity, and wherein splitting each laser pulse into a plurality of beamlets further comprises: rotating a plane of polarization of each laser pulse by passing the through an electro optical modulator (EOM); directing the laser pulse around the multiplexing module to a half-wave plate (HWP); passing each laser pulse through a half-wave plate (HWP) to change the plane of polarization of the laser pulse by a fixed amount such that a chosen portion of the laser pulse can pass through a polarized beam splitter (PSB); and directing the laser pulse to the PSB to cause a first portion of the laser pulse to pass through the PSB and exit the multiplexing module as a beamlet of the plurality of beamlets and a second portion of the laser pulse which is directed back into the re-imaging cavity. 
     Item 28. The method of item 27, further comprising: adjusting a pulse energy by a selected amount for each subsequent beamlet of the plurality of beamlets by selecting the HWP having a rotation angle configured to cause the pulse energy to be adjusted by the selected amount for each subsequent beamlet of the plurality of beamlets. 
     Item 29. The method of item 27, wherein introducing the delay between each adjacent beamlets of the plurality of beamlets further comprises: directing the second portion of the laser pulse around a series of mirrors of the re-imaging cavity to introduce the delay between adjacent beamlets. 
     Item 30. The method of item 29, wherein the series of mirrors comprises curved mirrors, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the mirrors. 
     Item 31. The method of item 29, wherein the series of mirrors comprise flat mirrors and lenses, and wherein the delay is configurable by changing a distance between the mirrors and focal lengths of the lenses. 
     Item 32. The method of item 18, wherein the multiplexing module is integrated with an imaging device comprising a two photon or multi-photon excitation scanning microscope, the method further comprising: receiving, at the two photon or multi-photon excitation scanning microscope, the plurality of beamlets of the plurality of beamlets associated with each respective laser pulse from the multiplexing module; and converting axial divergences associated with the plurality of beamlets into axial foci on the sample using the two photon or multi-photon excitation scanning microscope. 
     Item 33. The method of item 18, wherein splitting each laser pulse into a plurality of beamlets further comprises: splitting each laser pulse into a plurality of beamlets; and focusing the plurality of beamlets onto a first knife edge (KE) mirror that is vertically oriented which directs the plurality of beamlets into a re-imaging cavity of the multiplexing module to cause the beamlets to make a round trip of the multiplexing module. 
     Item 34. The method of item 33, further comprising: splitting off a respective beamlet of the plurality of beamlets using a second KE mirror that is vertically oriented for each roundtrip around the multiplexing module; and outputting the respective beamlet after corresponding number of roundtrips associated with its intended delay from the multiplexing module. 
     Item 35. A spatial multiplexing module configured to perform the operations of: receiving a plurality of laser pulses from a pulsed laser source; splitting the plurality of laser pulses into a plurality of spatially separated laser pulses; and outputting the plurality of spatially separated laser pulses, wherein the spatial multiplexing module provides for equal splitting of power while minimizing dispersion and introduces no pathlength difference between the plurality of laser pulses. 
     Item 36. The spatial multiplexing module of item 35, wherein the plurality of spatially separated laser pulses is to be focused on a sample to cause an indicator on the sample to fluoresce. 
     Item 37. The spatial multiplexing module of item 35, wherein receiving the plurality of laser pulses from the pulsed laser source further comprises receiving a plurality of beamlets associated with each laser pulse of the plurality of laser pulses, and wherein splitting the plurality of laser pulses into the plurality of spatially separated laser pulses further comprises splitting the plurality of beamlets into a plurality of sets of spatially separated beamlets, wherein the plurality of sets of spatially separated beamlets include a same number of beamlets as the plurality of beamlets. 
     Item 38. The spatial multiplexing module of item 37, wherein outputting the plurality of spatially separated laser pulses further comprises outputting the plurality of laterally separated sets of beamlets. 
     Item 39. The spatial multiplexing module of item 37, wherein the spatial multiplexing module is integrated with an imaging device comprising a two photon or multi-photon excitation scanning microscope, and wherein the two photon or multi-photon excitation scanning microscope is configured to perform operations of: focusing each of the plurality of sets of laterally separated beamlets onto a separate region of a sample. 
     Item 40. The spatial multiplexing module of item 39, wherein the imaging device is further integrated with a spatial resolved detector configured to perform operations of: splitting light associated with each respective region; and directing the light associated each respective region to a separate photodetector associated with that region. 
     Item 41. A method of operating a spatial multiplexing module, the method comprising: receiving a plurality of laser pulses from a pulsed laser source; splitting the plurality of laser pulses into a plurality of spatially separated laser pulses; and outputting the plurality of spatially separated laser pulses, wherein the spatial multiplexing module provides for equal splitting of power while minimizing dispersion and introduces no pathlength difference between the plurality of laser pulses. 
     Item 42. The method of item 41, wherein the plurality of spatially separated laser pulses is to be focused on a sample to cause an indicator on the sample to fluoresce. 
     Item 43. The method of item 41, wherein receiving the plurality of laser pulses from the pulsed laser source further comprises receiving a plurality of beamlets associated with each laser pulse of the plurality of laser pulses, and wherein splitting the plurality of laser pulses into the plurality of spatially separated laser pulses further comprises splitting the plurality of beamlets into a plurality of sets of spatially separated beamlets, wherein the plurality of sets of spatially separated beamlets include a same number of beamlets as the plurality of beamlets. 
     Item 44. The method of item 43, wherein outputting the plurality of spatially separated laser pulses further comprises outputting the plurality of laterally separated sets of beamlets. 
     Item 45. The method of item 44, wherein the spatial multiplexing module is integrated with an imaging device comprising a two photon or multi-photon excitation scanning microscope, and wherein the two photon or multi-photon excitation scanning microscope is configured to perform operations of: focusing each of the plurality of sets of laterally separated beamlets onto a separate region of a sample. 
     Item 46. The method of item 44, wherein the imaging device is further integrated with a spatial detector configured to perform operations of: splitting light associated with each respective region; and directing the light associated each respective region to a separate photodetector associated with that region. 
     Item 47. An optical modulation module configured to perform the operations of: receiving a plurality of beamlets associated with each laser pulse of a plurality of laser pulses, wherein each beamlet associated with a laser pulse is associated with a different axial or lateral position within a volume of a sample; determining whether an object of interest is present at a respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse; selectively adjust the amount of power associated with a set of beamlets from the plurality of beamlets for which no object of interest is located at the respective location in the volume associated with the respective location in the volume of the sample associated with the respective beamlet to reduce an amount of light associated with the set of beamlets from reaching the sample to generate modified beamlets; and outputting the modified beamlets. 
     Item 48. The optical modulation module of item 47, wherein to reduce an amount of power associated with a set of beamlets the optical modulation module is configured to perform an operation of: deflecting the set of beamlets from the plurality of beamlets to prevent the set of beamlets from reaching the sample. 
     Item 49. The optical modulation module of item 47, wherein to determine whether an object of interest is present at a respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse the optical modulation module is configured to perform an operation of: determining whether a signal associated with light detected at the respective location within the volume of the sample exceeds a threshold. 
     Item 50. The optical modulation module of item 47, wherein the object of interest in the sample is a neuron. 
     Item 51. The optical modulation module of item 47, wherein to determine whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse the optical modulation module is configured to perform an operation of: analyzing signals associated with light detected at the respective location in the sample using a factorization algorithm to determine whether the object of interest is present at the respective location. 
     Item 52. The optical modulation module of item 51, wherein the factorization algorithm is a Constrained Nonnegative Matrix Factorization. 
     Item 53. The optical modulation module of item 47, wherein to determine whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse the optical modulation module is configured to perform an operation of: determining whether the object of interest is present at the respective location within the volume based on change-point detection of signals associated with light detected at the respective location within the volume. 
     Item 54. The optical modulation module of item 47, wherein to determine whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse the optical modulation module is configured to perform an operation of: determining whether the object of interest is present at the respective location within the volume based on cumulative history weighting of signals associated with light detected at the respective location in the volume of the sample. 
     Item 55. The optical modulation module of item 47, wherein to determine whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse the optical modulation module is configured to perform an operation of: focusing the optically modulated beamlets on the sample to cause an indicator on the sample to fluoresce. 
     Item 56. A method for operating an optical modulation module, the method comprising: receiving a plurality of beamlets associated with each laser pulse of a plurality of laser pulses, wherein each beamlet associated with a laser pulse is associated with a different axial or lateral position within a volume of a sample; determining whether an object of interest is present at a respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse; selectively adjust the amount of power associated with a set of beamlets from the plurality of beamlets for which no object of interest is located at the respective location in the volume associated with the respective location in the volume of the sample associated with the respective beamlet to reduce an amount of light associated with the set of beamlets from reaching the sample to generate modified beamlets; and outputting the modified beamlets. 
     Item 57. The method of item 56, wherein reducing an amount of power associated with a set of beamlets further comprises: deflecting the set of beamlets from the plurality of beamlets to prevent the set of beamlets from reaching the sample. 
     Item 58. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: determining whether a signal associated with fluorescence associated with a respective location within the sample exceeds a threshold. 
     Item 59. The method of item 56, wherein the object of interest in the samples is a neuron. 
     Item 60. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: determining whether a signal associated with light detected at the respective location within the volume of the sample exceeds a threshold. 
     Item 61. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: analyzing signals associated with light detected at the respective location in the sample using a factorization algorithm to determine whether the object of interest is present at the respective location. 
     Item 62. The method of item 61, wherein the factorization algorithm is a Constrained Nonnegative Matrix Factorization. 
     Item 63. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: determining whether the object of interest is present at the respective location within the volume based on change-point detection of signals associated with light detected at the respective location within the volume. 
     Item 64. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: determining whether the object of interest is present at the respective location within the volume based on cumulative history weighting of signals associated with light detected at the respective location in the volume of the sample. 
     Item 65. The method of item 56, wherein determining whether the object of interest is present at the respective location within the volume of the sample associated with each beamlet of the plurality of beamlets associated with each laser pulse further comprises: focusing the optically modulated beamlets on the sample to cause an indicator on the sample to fluoresce using the two photon or multi-photon excitation scanning microscope. 
     While various embodiments have been described, the description is intended to be exemplary, rather than limiting, and it is understood that many more embodiments and implementations are possible that are within the scope of the embodiments. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any embodiment may be used in combination with or substituted for any other feature or element in any other embodiment unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the embodiments are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims. 
     While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings. 
     Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain. 
     The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections  101 ,  102 , or  103  of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed. 
     Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims. 
     It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. 
     The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various examples for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claims require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed example. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.