WO2013184918A1 - Microscope rapide pour la mesure de signaux optiques dynamiques - Google Patents

Microscope rapide pour la mesure de signaux optiques dynamiques Download PDF

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Publication number
WO2013184918A1
WO2013184918A1 PCT/US2013/044515 US2013044515W WO2013184918A1 WO 2013184918 A1 WO2013184918 A1 WO 2013184918A1 US 2013044515 W US2013044515 W US 2013044515W WO 2013184918 A1 WO2013184918 A1 WO 2013184918A1
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Prior art keywords
pattern
emission
imaging device
emission pattern
microscopy
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PCT/US2013/044515
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English (en)
Inventor
Matthew Shtrahman
Daniel AHARONI
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The Regents Of The University Of California
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Publication of WO2013184918A1 publication Critical patent/WO2013184918A1/fr

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/067—Electro-optic, magneto-optic, acousto-optic elements
    • G01N2201/0675—SLM

Definitions

  • a method includes instructing a pattern generator to provide an excitation pattern to a sample under test, and controlling a movable mechanism that receives an emission pattern from the sample under test responsive to the excitation pattern.
  • the controlling includes causing the movable mechanism to sweep a representation of the emission pattern along a path, thereby creating at least one streak, and synchronizing the motion of the movable mechanism with the frame rate of an imaging device, such that a full sweep of the representation of the emission pattern along the path corresponds to approximately one frame of the imaging device.
  • the variable intensity of each streak represents a corresponding time varying intensity of a portion of the emission pattern.
  • the varying intensity of a portion of the emission pattern may be captured with sub-millisecond resolution.
  • the excitation pattern may be a plurality of excitation points or other geometries of interest, and the resulting emission pattern may be a plurality of corresponding point source emissions.
  • the pattern generation device is a spatial light modulator (SLM) that focuses energy received from a light source into the excitation pattern by adjusting phase of the wavefront of coherent light.
  • the movable mechanism is a galvanometer that rotates about an axis.
  • a microscopy device includes a reflective surface configured to receive an emission from a microscope and reflect the emission to an imaging device, and a focusing device in the path between the reflective surface and the imaging device, wherein the emission from the microscope is adjusted by at least one of the reflective surface or the focusing device to increase the temporal resolution of a recorded time varying signal.
  • the reflective surface of the microscopy device may be movable, and the emission from the microscope is adjusted by movement of the reflective surface during a period of time, such that the emission is caused to be reflected along a path on the imaging device, thereby recording on the imaging device a streak representing variable intensity of the emission over the period of time.
  • the reflective surface may be movable about or along an axis or arbitrary trajectory. The streak may be in a line or arbitrary path.
  • the focusing device may alter the magnification of one or more dimensions of an incidence area of the emission from the microscope as reflected by the reflective surface.
  • a microscopy system in another embodiment, includes a control mechanism, and an excitation mechanism configured to apply a pattern of energy to a sample under analysis, wherein the control mechanism controls the excitation mechanism to provide the pattern.
  • the microscopy system also includes a movable mechanism configured to receive an emission pattern from the sample under analysis, the emission pattern being responsive to the applied pattern of energy, wherein the control mechanism controls a movement of the movable mechanism such that the movable mechanism provides a representation of the emission pattern swept along a defined path.
  • the microscopy system may further include an imaging device, wherein the emission pattern is swept along the defined path across the imaging device, and the control mechanism controls a frame rate of the imaging device such that a full sweep across the imaging device occurs substantially within one frame.
  • the microscopy system may further include a memory, wherein multiple sequential frames taken by the imaging device are stored in the memory and represent corresponding multiple sequential sweeps of the image across the imaging device.
  • the pattern of energy may be controlled by controlling the phase or amplitude of the energy.
  • the movable mechanism is a galvanometer
  • the representation of the emission pattern is a reflection of the emission pattern.
  • the microscopy system may further include a lens or mirror structure that receives the representation of the emission pattern from the movable mechanism and alters at least one dimension of the representation.
  • FIG. 1A illustrates an example system for analyzing a sample.
  • FIG. IB illustrates an example system for analyzing a sample.
  • FIG. 2 illustrates an example of a microscopy system.
  • FIG. 3 illustrates an example of a sweep mode in a microscopy system.
  • FIG. 4 illustrates an example of an emission path unit in a microscopy system.
  • This disclosure describes a system for recording multiple sources of emission in parallel at high resolution. Prolonged measurements of spatially distributed signals are recorded with sub-millisecond resolution. A continuous, patterned excitation is generated and directed to user- selected regions of interest in a sample under analysis. The responsive emission pattern from the sample under analysis is recorded by sweeping the emission pattern across a sensor in a recorder, synchronizing the sweeps with the frame rate of the recorder, thereby allowing for increased temporal resolution. For example, synchronization may allow for a full sweep to occur substantially within one frame of the recorder, where substantially may indicate that a full sweep occurs within +/-20%, +/- 10%, +1-5%, or +/- 1 % of a full frame exposure time.
  • the system is a microscopy system.
  • the microscopy system may be used, for example, for studying biological phenomena with collective properties, such as for measuring neuronal network activity.
  • the microscopy system may be optimized to record ensemble neuronal activity with temporal precision appropriate to resolve action potential activity in single neurons.
  • a flexible design is adaptable for use in a variety of experiments, including in vivo brain imaging and other biomedical applications.
  • the system described in this disclosure overcomes the failures of existing devices to provide high temporal resolution for multiple points of interest simultaneously.
  • the system described in this disclosure may be used to measure action potentials simultaneously from large numbers of individual neurons, networks of primary cultured neurons, and other groups of subjects of interest.
  • neuronal networks may be stimulated to fire collectively via field stimulation and action potentials recorded.
  • spontaneous and subtle induced recurrent network activity that more closely approximates physiological activity may be measured.
  • the powerful excitatory inputs from climbing fibers onto cerebellar Purkinje cells offers an opportunity to study dendritic integration in sub-cellular structures.
  • the dendritic trees of Purkinje cells are perhaps the most elaborate in the brain, and each Purkinje cell receives inputs from a single climbing fiber, which ramifies extensively on the proximal dendrite.
  • the manner in which an action potential invades the climbing fiber ramification impacts the synchrony of transmitter release and thereby the degree of saturation of postsynaptic receptors.
  • the invasion of the electrical signal into this giant presynaptic terminal has not been visualized.
  • the system described in this disclosure provides the capability to visualize this process and provides insight into the mechanism of transmitter saturation at the failsafe synapse.
  • FTM fluorescent trails microscope
  • FIG. 1A represents in block diagram form an example of a system 100 for analyzing a sample under analysis 1 10.
  • An excitation mechanism 105 provides an excitation to sample under analysis 1 10, which, in response to the excitation, makes an emission.
  • a detection and recordation mechanism 1 15 detects and records the emission.
  • a control mechanism 120 controls components of system 100.
  • FIG. IB illustrates an example of a system 100 capable of performing high resolution microscopy in accordance with this disclosure.
  • FIG. IB illustrates examples of excitation mechanism 105, detection and recordation mechanism 1 15, and control mechanism 120.
  • System 100 is illustrated for convenience and understanding as blocks connected together in a particular configuration. Each block is representative of one or more components. It will be apparent from this disclosure that other configurations are also within the scope of the disclosure. For example, additional or fewer blocks may be included, one or more blocks may be omitted, multiple blocks may be combined, there may be multiple instantiations of one block, and blocks may be connected differently than illustrated.
  • the connections as illustrated between blocks may be, for example, mechanical, electrical, optical, or wireless communication connections, or connections through software.
  • excitation mechanism 105 may represent three physical devices.
  • control mechanism 120 may be part of excitation mechanism 105 or part of detection and recordation mechanism 1 15.
  • one or more blocks of excitation mechanism 105 and detection and recordation mechanism 1 15 may be combined into a separate physical device, as described below.
  • excitation mechanism 105 includes an excitation source 125, a collimator 130, a filter 135, a pattern generator 140, and a position and focus mechanism 145.
  • FTM position and focus mechanism
  • Excitation source 125 provides a stimulus for excitation of sample under analysis 1 10.
  • source 125 may be an energy source that emits a stimulus in the form of a substantially periodic waveform, where the frequency band of the stimulus is within the electromagnetic spectrum, such as within or including portions of the visible spectrum, infrared spectrum, ultraviolet spectrum, radio spectrum, gamma ray spectrum, or other portion of the electromagnetic spectrum.
  • system 100 includes an FTM, described in detail below, in which source 125 is a laser, emitting a stimulus that is a focused beam of light.
  • Collimator 130 collimates the waveform from source 125.
  • lenses and a pinhole may be used to collimate the light from the laser.
  • Filter 135 may amplify, attenuate, spatially filter, or otherwise adjust the stimulus.
  • lenses may be used to expand the laser beam to a desired size and geometry.
  • Pattern generator 140 transforms the stimulus into a patterned stimulus.
  • a wavefront modulator such as an SLM or the like may be used to impose a desired stimulus pattern by modulating the frequency, phase, or amplitude of the laser beam such that portions of sample under analysis 1 10 are stimulated more or less depending on the applied pattern.
  • Position and focus mechanism 145 allows the patterned stimulus to be directed and/or focused at a desired portion of sample under analysis 1 10, and may include a rotational mechanism of 'n' degrees of freedom to allow the stimulus to be directed at sample under analysis 1 10 from an angle.
  • one or more components may be rotatable for directing the laser beam to different portions of sample under analysis 1 10 or directing the laser beam in different angles of incidence. Further in the FTM example, the laser beam may be directed at different focal depths within sample under analysis 1 10.
  • detection and recordation mechanism 1 15 includes a response detector 150, a response sweeper 155, a response shaper 160, a response recorder 165, and a memory 170.
  • a non-limiting example of an FTM is discussed by way of illustration for each block.
  • Response detector 150 detects the response of sample under analysis 1 10 to the stimulation.
  • the response may be in a pattern as a result of a pattern being applied to sample under analysis 1 10.
  • an emission pattern of one or more points of light appears in sample under analysis 1 10 in response to the stimulus, and the one or more points of light are detected via reflective surfaces in an emission path.
  • Response sweeper 155 receives the response pattern and sweeps the response pattern back and forth along or about an axis.
  • the received emission pattern is reflected off a surface that rotates about an axis through an arc of travel, such that the received pattern is swept back and forth across a defined path.
  • Response shaper 160 shapes the response pattern.
  • the emission pattern is received as a light pattern in a two-dimensional plane with a defined area, and lenses or mirrors shape the defined area by reducing one dimension of the area, such as by "squashing" a square-shaped area into a thin rectangular-shaped area, squashing a circular or elliptical-shaped area into a strip, or the like.
  • Response recorder 165 receives the swept pattern and records the pattern.
  • Response recorder 165 may be rotated with one or more degrees of freedom to orient a recorded response according to the position of other components of system 100.
  • each sweep of the response pattern across the defined path corresponds to one recorded image.
  • a light detector records the intensity of the point of light as a streak across a detection surface, such that the streak indicates a change in the intensity of the light over the time period of one sweep.
  • clustering techniques may be used to enhance the accuracy of assignment of point sources to the appropriate location and time.
  • Memory 170 may store information from response recorder 165. In the FTM example, recorded images may be stored. [0033] Detection and recordation of a response of sample under analysis 1 10 to a stimulus has thus been described with respect to blocks 150-170 of block 1 15.
  • Control mechanism 120 represents a control function for system 100, and may be implemented as mechanical control, electrical control, or a combination of mechanical and electrical control, and further may include manual controls. Control mechanism 120 that is implemented at least in part with electrical control may include analog components, digital components, or a combination of analog and digital components.
  • control mechanism 120 may be, or may include, a processor such as a microprocessor, field programmable gate array (FPGA), application-specific integrated circuit (ASIC), or other processor.
  • a processor executes instructions, which may be, for example, stored in a memory such as memory 170, or implemented in the construction of the processor.
  • Control mechanism 120 may be in communication with an external device and further may be controlled by the external device.
  • An example of an external device is a computer.
  • Two or more blocks of system 100 may be in communication with each other. Communication may be, for example, via wires, traces on a circuit board, or a wireless communication link. Communication may follow a standard or proprietary protocol. In some implementations, communication may be made via one or more buses. For example, there may be a dedicated bus between control mechanism 120 and memory 170, and a shared bus between control mechanism 120 and other blocks of system 100, such that memory 170 may be accessed by control mechanism 120. Components of the blocks of system 100 may be directly wired together. For example, a pin of a processor in control mechanism 120 may be connected to a pin of an integrated circuit for controlling a component of position and focus mechanism 145. For another example, position and focus mechanism 145 may be connected to response detector 150 such that positioning of response detector 150 may adjust to an adjustment of the focal point of the stimulus.
  • collimator 130, filter 135, pattern generator 140, position and focus mechanism 145, and response detector 150 are components of a microscope, and response recorder 165 and memory 170 are components of a camera.
  • response sweeper 155 and response shaper 160 are included in a microscope or a camera or may be, alone or together, embodied in a physical device that may be attached to a microscope and/or a camera. Many other combinations of the blocks illustrated in FIG. IB are also possible.
  • An FTM is one example of system 100.
  • FIG. 2 illustrates the components of the FTM in a representative configuration.
  • the excitation path includes a laser 205 sourcing a beam of light.
  • Laser 205 may be, for example, a solid state 473 nanometer (nm) laser in an open loop or closed loop configuration.
  • the beam of light from laser 205 is modulated by a modulator 210, which may be, for example, a custom modified Pockels cell or an acousto-optical tunable filter (AOTF).
  • Reflectors 215 represent fully or partially reflective surfaces, such as mirrors and beam splitters, for reflecting light along a desired path.
  • Spatial filter / beam expander 225 includes pinhole 240, lens 230 and lens 231 , which together collimate, spatially filter, and expand the light beam.
  • a half-wavelength plate 240 allows a wavefront modulator 245 to be switched from a diffractive element ("on") to a purely reflective element ("off).
  • the light beam as filtered and expanded fills wavefront modulator 245, which modulates the wavefront to create excitation patterns within a sample 255.
  • Wavefront modulator 245 may be, for example, an SLM. Pattern generation may be alternatively provided by a micro mirror array, a light emitting diode (LED) array, or a diffraction grating.
  • Wavefront modulator 245 may be computer addressable to control the generation of the excitation pattern.
  • a beam splitter 216 deflects a portion of the laser power to a photodetector 250, which provides intensity feedback in a closed loop control system to correct for fluctuations introduced by wavefront modulator 245 or by laser 205.
  • Lenses 232 and 233 act as a telescope to image wavefront modulator 245 onto a pair of galvanometers 217 and 218.
  • Galvanometers 217 and 218 may be used for raster scanning via scan lenses 234 and 235, For example, galvanometers 217 and 218 may be used to create a traditional laser scanning image, to decide where to place the points of interest by pattern generator 140, as discussed below. Alternatively to raster scanning, a wide field excitation source such as a light emitting diode (LED) or arc lamp may be used to create the image. A region of interest on sample 255 may be excited continuously without scanning using pattern generator 140, thereby minimizing shot noise by maximizing the dwell time for a given frame rate. One or both of galvanometer 217 and 218 may be replaced with an acoustic optical modulator or other beam steering device.
  • LED light emitting diode
  • arc lamp may be used to create the image.
  • a region of interest on sample 255 may be excited continuously without scanning using pattern generator 140, thereby minimizing shot noise by maximizing the dwell time for a given frame rate.
  • One or both of galvanometer 217 and 218 may
  • Dichroic mirror 219 reflects the excitation light beam to the back aperture of an objective 236 such that optical probes in sample 255 are excited and produce an emission pattern in response.
  • Dichroic mirror 219 allows the emission light to pass, and a beam splitter 220 routes the emission pattern to a galvanometer 221 or to a photomultiplier tube (PMT) 260, or splits the emission between galvanometer 221 and PMT 260 in a test mode, as discussed below.
  • PMT photomultiplier tube
  • Orthogonal cylindrical lenses 238 and 239 with different focal lengths compress or squash the emission pattern in one dimension. For example, if the emission pattern reflected by galvanometer 221 has an area in the shape of a square, lenses 238 and 239 may squash the square into a thin rectangle. Cylindrical lenses 238 and 239 may be replaced with cylindrical mirrors.
  • Galvanometer 221 is a single axis galvanometer that rotates about the axis to direct the reflected image along a straight line path across an image detector 265, such as a CCD or CMOS sensor.
  • the motion of galvanometer 221 in one sweep causes the emissions from the optical probes to create streaks, where each streak indicates intensity of the emission captured continuously during a time equal to the duration of the sweep.
  • the streak provides information on how the intensity of the emission changes during the time of the sweep.
  • Galvanometer 221 may decelerate and accelerate rapidly near the end of travel, potentially introducing nonlinearities or discontinuities into the streaks. Such nonlinearities or discontinuities may be compensated for in post-processing.
  • Image detector 265 may capture light information using pixels. For example, pixels may be oriented in rows and columns. Wavefront modulator 245 may be used to create a pattern of point excitations such that the corresponding responses from the optical probes are point sources, each point source in a separate row. For this configuration, an image captured by image detector 265 includes one streak for each point source. Point excitations and point sources are discussed by way of example but are not limiting. Excitations may be other shapes, and of any size within the dimensional limits of sample 255.
  • excitation light may be restricted to regions containing electrically active membrane while avoiding fluorescently labeled background structures that contribute noise to the image.
  • background structures often make up a large portion of the field of view.
  • bandwidth in the form of unused image pixels can be utilized to record dynamic information from regions of interest.
  • raster scanning devotes significant resources to recording fluorescence signals for biologically inconsequential tissue.
  • Laser 205 and light modulator 210 may be combined.
  • Laser 205 may be replaced by another source of light.
  • Light modulator 210 may be omitted.
  • Spatial filter / beam expander 225 may be replaced with a suitable alternative.
  • Half- wavelength plate 240 may be omitted.
  • Galvanometers 217 and 218 may be replaced by a reflector 215.
  • Lenses 234 and 235 may be omitted. Components between lens 233 and dichroic mirror 219 may be omitted.
  • Lens 237 and PMT 260 may be omitted.
  • Lenses 238 and 239 may be replaced by a tube lens.
  • microscopy capabilities provided by the microscopy system described in this disclosure allow for single or multiple photon excitation.
  • the system may be adapted for use in a variety of experimental preparations, including substrate adherent single molecule experiments, cell culture, tissue slice, and potentially in vivo brain imaging.
  • the microscopy system further allows for illumination of near diffraction-limited regions of interest within a sample, allowing for measurements in subcellular structures and volumes for single molecule experiments and fluorescence correlation spectroscopy (FCS).
  • the microscopy system may be optimized to record ensemble neuronal activity with temporal precision to resolve action potentials in collections of individual neurons.
  • the microscopy system may be optimized to measure photon flux (photometry) from multiple arbitrary fixed locations simultaneously within a system. For example, in a biological system large numbers of fluorescently tagged cells or biological molecules of interest may be probed. Such an implementation provides a solution to record continuous photon flux from large numbers of arbitrary locations, allowing for high throughput experiments. This advance will promote an understanding of biological phenomena ranging from systems neuroscience to collective gene expression, as well as high throughput biological assays involving dynamic optical signals from multiple targets.
  • An example of using a microscopy system such as the FTM of FIG. 2 is illustrative of how the system provides improvement over other techniques.
  • an image is taken of sample under analysis 255 using high resolution imaging. For example, a scanning high resolution raster image or an epifluorescent image of sample under analysis 255 labeled with a fluorescent probe may be taken. Fluorescently labeled structures of interest are isolated and used to design a custom pattern of near diffraction-limited excitation points. Using holographic techniques, the custom pattern of excitation light is created in the sample plane by wavefront modulator 245.
  • the resulting pattern of point source emissions from the optical probes is reduced (“squashed") in one dimension with the use of cylindrical lenses or mirrors and imaged onto a portion of image detector 265.
  • Mirror-mounted, high precision, linear galvanometer 221 creates streaks by sweeping the point source emissions in a line across image detector 265.
  • Use of the microscopy system in this way, with both sweep and squash, is termed "combined mode.”
  • the frequency of the galvanometer oscillation may be half of the camera frame rate, and is synchronized such that each scan transit is captured by a single exposure.
  • the resulting movie of fluorescent streaks can capture continuous temporal information with sub-millisecond resolution in sub-micron sized cellular compartments.
  • the image of point source emissions from the optical probes is not “squashed” in one dimension.
  • one or more dimensions of the excitation pattern from wavefront modulator 245 and the resulting emission pattern are sized such as to be less than corresponding dimensions of image detector 265.
  • the emission pattern may be selected to be 50 rows by 100 columns.
  • the length of each streak will be approximately 50 rows of pixels.
  • the emission pattern may reduced in two directions.
  • the ratio of a given emission pattern dimension to a given image detector 265 dimension will depend, among other things, on the size and resolution of image detector 265, the resolution of the optical probes, and the desired temporal resolution of the measurement. Use of the microscopy system in this way is termed "sweep mode.”
  • the image of point source emissions is not swept by galvanometer 221.
  • Lenses 238 and 239 reduce one or more dimensions of the emission pattern such that the emission pattern is selectively applied to a portion of image detector 265.
  • processing of the image received at image detector 265 may be performed much faster by selectively processing the area where the image is received. For example, if the emission pattern is reduced by lenses 238 and 239 to fill an image space of 25 out of 100 rows on image detector 265, then the 75 empty rows need not be processed, and the effective temporal resolution of image detector 265 is increased.
  • Use of the microscopy system in this way is termed "squash mode.”
  • One camera analyzed for potential improved results using the techniques in this disclosure was a Hamamatsu C9100-14 ImageEM camera. The calculations were based on using combined mode in which the image would be squashed to 20% of a dimension of the camera sensor, and swept across the remaining 80% of the sensor. Utilizing 80% of the 1024 x 1024 pixel sensor with a frame rate of 9.5 frames per second (fps) would theoretically yield a temporal resolution of 0.13 milliseconds (ms) per pixel or approximately 8 kHz.
  • a Hamamatsu C9100-13 ImageEM camera utilizing 80% of the 512 x 512 pixel sensor with a frame rate of 32 fps, would theoretically yield a temporal resolution of 0.08 ms per pixel or approximately 13 kHz. Thus, even the fastest neuronal signals may be captured using the described technique.
  • the microscopy system described in this disclosure increases temporal resolution for a variety of imaging devices.
  • Table 1 provides a summary of some theoretical results for some imaging devices. The results are from creating streaks by sweeping across 80% of an imaging chip. Faster rates can be achieved by lengthening the streaks.
  • the temporal resolutions achieved are many orders of magnitude higher than multifocal muliphoton microscopy (MMM) or light sheet microscopy (LSM). Further, pixel dwell time is approximately 20 times longer than typical MMM experiments running at speeds slower than video rate. The time resolution achieved is comparable to neuronal electrophysiological recordings that are typically filtered at 5 kHz, but with orders of magnitude additional effective bandwidth, allowing one to concurrently capture activity from hundreds of cells.
  • MMM multifocal muliphoton microscopy
  • LSM light sheet microscopy
  • FIG. 3 illustrates an example of how streaks might appear on image detector 265 using the microscopy system described in this disclosure in sweep mode.
  • a fluorescent response pattern of point source emissions is reflected by a galvanometer onto an image detector.
  • the galvanometer is rotated in one direction through a defined arc, the point source emissions create a set of parallel streaks on the image detector.
  • a video sequence may be created of successive sweeps.
  • an approximately 250 ⁇ x 250 ⁇ field of view containing near diffraction limited spots (FWHM of approximately 0.5 ⁇ in size) is imaged and swept across a 13.3 millimeter (mm) x 13.3 mm 1024 x 1024 pixel electron multiplying CCD (EMCCD) chip with a magnification of approximately 50x in the vertical direction and approximately l Ox in the horizontal direction.
  • the spots have a FWHM size in the image plane of approximately 0.4 horizontal by 2 vertical pixels. Maintaining a vertical space between adjacent fluorescent streaks of 2.5 times the spot size, approximately 150 points may be recorded while still avoiding overlap between streaks. Deconvolution techniques may increase the number of points.
  • the position of excitation points may be optimized to allow measurements from cell bodies that are immediately adjacent. This process can be globally optimized to record from the maximum number of cells of interest within a network.
  • FIG. 4 illustrates a prototype of a unit for use in the emission path of a microscopy system such as system 100. Numbering in FIG. 4 relates prototype components to the illustration of FIG. 2.
  • the system shown in FIG. 4 is reconfigurable in that all of the components may be moved into and out of the light path.
  • an optional tube lens 242 may be inserted into the path to replace cylindrical lenses 238 and 239.
  • the unit is used in conjunction with a microscope having the capability of pattern generation, such as a microscope including a wavefront modulator 245 or the like.
  • the unit is attached to the microscope, optionally after removing the microscope tube lens.
  • a camera is attached to the unit and positioned to receive images reflected from a galvanometer in the unit.
  • a timing circuit synchronizes the camera frames with the motion of the galvanometer.
  • Other configurations are encompassed within the scope of this disclosure.
  • the microscopy system described may be used in, for example, cellular neuroscience, biochemistry, cellular and developmental biology, physiology, analytical chemistry, soft condensed matter fields, and many other areas.
  • the concepts described in this disclosure may be adapted for a two-photon platform.
  • the microscopy system may be placed into a test mode. Referring back to FIG. 2, in a testing configuration, a continuous wave single-photon laser 205 is modulated by light modulator 210 to create sinusoidal or pulsed patterns, or patterns that mimic physiological signals.
  • the modulated laser beam is used to excite single point sources such as 100 nm fluorescent beads on sample under analysis 255.
  • the resulting emission light is split by beam splitter 220 such that half of the light is imaged through lens 237 onto a single point detector such as PMT 260 capable of resolving megahertz (MHz) signals.
  • the remaining light is swept across image detector 265 by galvanometer 221 as described above, allowing for simultaneous calibration of the dynamic signals.
  • the 'temporal PSF' of the system is measured by imaging the fluorescent streak resulting from a laser pulse such as a delta function pulse.
  • the fluorescent streak recorded by image detector 265 is a convolution of the actual dynamic fluorescent signal with the PSF of the microscope.
  • the fluorescent streak and the PSF may be deconvolved to recover the original dynamic signal and the single pixel temporal resolution.
  • single 10 nm or smaller fluorescent beads may be embedded into sample under analysis 255 and the above measurements repeated at various depths.
  • deconvolution may be used in the orthogonal axis to limit "cross talk" between streaks, which is particularly important in scattering media.
  • the microscopy system described in this disclosure allows for the first simultaneous recordings of spiking from large numbers of visually identified neurons within an interconnected network, a technical capability with transformative potential for neuroscience.
  • the microscopy system has the potential to accelerate by orders of magnitude many camera based techniques.
  • the technology will allow time resolved point measurement techniques such as FCS to be performed simultaneously at hundreds of multiple sites.
  • the microscopy system facilitates the study of spatial correlations inaccessible to single point measurements. Spatial correlation is important for understanding many diverse biological phenomena with collective properties.
  • An embodiment of the invention relates to a non-transitory computer-readable storage medium having computer code thereon for performing various computer-implemented operations.
  • the term "computer-readable storage medium” is used herein to include any medium that is capable of storing or encoding a sequence of instructions or computer codes for performing the operations, methodologies, and techniques described herein.
  • the media and computer code may be those specially designed and constructed for the purposes of the invention, or they may be of the kind well known and available to those having skill in the computer software arts.
  • Examples of computer-readable storage media include, but are not limited to: magnetic media such as hard disks, floppy disks, and magnetic tape; optical media such as CD-ROMs and holographic devices; magneto-optical media such as optical disks; and hardware devices that are specially configured to store and execute program code, such as ASICs, programmable logic devices (PLDs), and ROM and RAM devices.
  • magnetic media such as hard disks, floppy disks, and magnetic tape
  • optical media such as CD-ROMs and holographic devices
  • magneto-optical media such as optical disks
  • hardware devices that are specially configured to store and execute program code such as ASICs, programmable logic devices (PLDs), and ROM and RAM devices.
  • Examples of computer code include machine code, such as produced by a compiler, and files containing higher-level code that are executed by a computer using an interpreter or a compiler.
  • an embodiment of the invention may be implemented using Java, C++, or other object-oriented programming language and development tools. Additional examples of computer code include encrypted code and compressed code.
  • an embodiment of the invention may be downloaded as a computer program product, which may be transferred from a remote computer (e.g., a server computer) to a requesting computer (e.g., a client computer or a different server computer) via a transmission channel.
  • a remote computer e.g., a server computer
  • a requesting computer e.g., a client computer or a different server computer
  • Another embodiment of the invention may be implemented in hardwired circuitry in place of, or in combination with, machine-executable software instructions.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

Un mode de réalisation de l'invention concerne un procédé consistant à donner l'instruction à un générateur de motifs de fournir un motif d'excitation à un échantillon testé, et à commander un mécanisme mobile qui reçoit un motif d'émission de l'échantillon testé en réponse au motif d'excitation. L'opération de commande consiste à amener le mécanisme mobile à balayer une représentation du motif d'émission le long d'un trajet, de manière à créer au moins une strie, et à synchroniser le mouvement du mécanisme mobile avec la fréquence d'image d'un dispositif d'imagerie, de telle sorte qu'un balayage complet de la représentation du motif d'émission le long du trajet corresponde approximativement à une image du dispositif d'imagerie. L'intensité variable de chaque strie représente une intensité variant dans le temps correspondante d'une partie du motif d'émission. L'intensité variable d'une partie du motif d'émission peut être capturée avec une résolution de l'ordre de la sous-milliseconde.
PCT/US2013/044515 2012-06-08 2013-06-06 Microscope rapide pour la mesure de signaux optiques dynamiques WO2013184918A1 (fr)

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Cited By (1)

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JP2016197045A (ja) * 2015-04-03 2016-11-24 国立大学法人 東京大学 フォトルミネセンス寿命測定装置及び測定方法

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US20050157292A1 (en) * 2002-05-29 2005-07-21 Hamamatsu Photonics K.K. Fluorescence lifetime distribution image measuring system and its measuring method
US20100090127A1 (en) * 2007-01-30 2010-04-15 Ge Healthcare Bio-Sciences Corp. Time resolved fluorescent imaging system
US20100314533A1 (en) * 2007-12-21 2010-12-16 Koninklijke Philips Electronics N.V. Scanning microscope and method of imaging a sample

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US20050157292A1 (en) * 2002-05-29 2005-07-21 Hamamatsu Photonics K.K. Fluorescence lifetime distribution image measuring system and its measuring method
US20100090127A1 (en) * 2007-01-30 2010-04-15 Ge Healthcare Bio-Sciences Corp. Time resolved fluorescent imaging system
US20100314533A1 (en) * 2007-12-21 2010-12-16 Koninklijke Philips Electronics N.V. Scanning microscope and method of imaging a sample

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016197045A (ja) * 2015-04-03 2016-11-24 国立大学法人 東京大学 フォトルミネセンス寿命測定装置及び測定方法

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