WO2016007954A1 - Dispositif de direction pour systèmes d'imagerie à excitation à deux photons - Google Patents

Dispositif de direction pour systèmes d'imagerie à excitation à deux photons Download PDF

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Publication number
WO2016007954A1
WO2016007954A1 PCT/US2015/040173 US2015040173W WO2016007954A1 WO 2016007954 A1 WO2016007954 A1 WO 2016007954A1 US 2015040173 W US2015040173 W US 2015040173W WO 2016007954 A1 WO2016007954 A1 WO 2016007954A1
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Prior art keywords
steering mirror
light
movable steering
pulse
movable
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Spencer L. SMITH
Jeffrey STIRMAN
Michael KUDENOV
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University of North Carolina at Chapel Hill
North Carolina State University
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University of North Carolina at Chapel Hill
North Carolina State University
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • the subject matter disclosed herein relates to multiphoton excitation laser scanning microscopy. More particularly, the subject matter disclosed herein relates to two-photon excited imaging systems, steering devices for two- photon excitation imaging systems, and related methods having an increased field of view (FOV) capable of high speed imaging in at least two regions of interest (ROIs) within the FOV.
  • FOV field of view
  • two-photon population calcium imaging in vivo offers many distinct advantages over alternative approaches, such as metal electrodes.
  • Two-photon calcium imaging provides unambiguous identification of recorded neurons, particularly when genetically encoded indicators are employed.
  • Two-photon calcium imaging can be used to record activity in tens to hundreds of neurons by imaging somata, dendrites, and/or axons in local populations.
  • two-photon imaging can penetrate hundreds of microns into the brain through an intact dura mater. Not only has this approach revealed cellular- level stimulus selectivity, but it can be used to measure neural activity in awake rodents (e.g., mice) performing a psychophysics task or navigating in a virtual reality environment.
  • awake rodents e.g., mice
  • two-photon excitation imaging systems steering devices for two-photon excitation imaging systems, and related methods are provided.
  • the two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can comprise an increased field of view (FOV) capable of high speed imaging in two discrete regions of interest (ROI).
  • FOV field of view
  • a steering device for a two-photo excitation imaging system can comprise a first movable steering mirror, a second movable steering mirror, and at least one component in communication with the first movable steering mirror and the second movable steering mirror for independently repositioning the first and/or second movable steering mirror in XY axes.
  • a two-photon excitation imaging system can comprise a source for producing light pulses, a steering device comprising: a first movable steering mirror disposed in a first pathway along which a first pulse of light is directed, without delay, to the first movable steering mirror; a second movable steering mirror disposed in a second pathway along which a second pulse of light is directed to the second movable steering mirror; and at least one component in communication with the first movable steering mirror and the second movable steering mirror for independently repositioning the first and/or second movable steering mirror in XY axes.
  • the system can also comprise a recombination relay configured to recombine the first and second pulses of light, wherein the first movable steering mirror and the second movable steering mirror are configured to direct the first and second pulses of light, respectively, to the recombination relay, and an objective through which the first and second pulses of light are directed through to a specimen, after the first and second pulses of light are recombined.
  • a two-photon excitation imaging method is provided.
  • the method can comprise at a two-photon excitation imaging system including a source for producing light pulses, a steering device comprising first movable steering mirror, a second movable steering mirror, and at least one component in communication with the first movable steering mirror and the second movable steering mirror for independently repositioning the first and/or second movable steering mirror in XY axes, providing a first pulse of light and a second pulse of light, directing, without delay, the first pulse of light along a first pathway to the first movable steering mirror and the second pulse of light along a second pathway to a second movable steering mirror, recombining the first and second pulses of light, and directing the recombined first and second pulses of light through an objective to a specimen.
  • a steering device comprising first movable steering mirror, a second movable steering mirror, and at least one component in communication with the first movable steering mirror and the second movable steering mirror for independently repositioning the first and/or second movable steering
  • Figure 1 A illustrates a schematic of an exemplary two-photon excitation imaging system according to some embodiments of the subject matter described herein;
  • Figure 1 B illustrates a schematic of an exemplary afocal relay of the two- photon excitation imaging system according to Figure 1 A
  • Figure 1 C illustrates a schematic of two exemplary and separate regions of interest (ROIs) within a field of view (FOV) generated by the two-photon excitation imaging system according to Figure 1 A;
  • ROIs regions of interest
  • Figure 1 D illustrates a schematic of two exemplary ROIs generated within a neural area FOV by an exemplary two-photon excitation imaging system according to some embodiments of the subject matter described herein;
  • Figure 2A illustrates a schematic of an exemplary objective, scanning lens, and tube lens of a two-photon excitation imaging system according to some embodiments of the subject matter described herein;
  • Figure 2B illustrates a schematic of the exemplary objective of the two- photon excitation imaging system according to Figure 2A;
  • Figure 3 illustrates a schematic of an exemplary steering device for use in a two-photon excitation imaging system according to some embodiments of the subject matter described herein;
  • Figure 4 illustrates a flow diagram of an exemplary method for two- photon excitation imaging according to some embodiments of the subject matter described herein.
  • the present subject matter provides two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods.
  • the present subject matter provides two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods that can comprise an increased field of view (FOV) capable of high speed imaging in two discrete regions of interest (ROIs).
  • FOV field of view
  • the present subject matter provides a novel two-photon excitation imaging system with a field of view greater than 3000 ⁇ , resulting in an imaging area over 18-fold greater than that of conventional multiphoton imaging (> 7 square mm), while still achieving cellular resolution.
  • multiplexed excitation and high-speed photon counting can be used.
  • multiplex can refer to splitting a single pulse, delaying one pathway of the split pulse, and then recombining the two pulses.
  • Two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can be advantageous for several reasons.
  • the wide FOV can offer extended overview imaging (e.g. of extended cortical networks). Since an excitation volume can be preserved across the FOV, the two-photon excitation imaging systems, steering devices for two-photon excited imaging systems, and related methods can provide access to over seven-fold more spatial information than conventional multiphoton imaging systems.
  • the two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can provide for two imaging pathways which can allow for simultaneous imaging of spatially distinct ROIs within a FOV.
  • the two- photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can be flexible since imaging pulses can be freely repositioned during a single imaging session, e.g., two ROIs can be positioned in XYZ directions within the increased FOV simply by manipulating motorized steering mirrors disposed within the system, which can allow a user, within a single specimen and imaging session, to image multiple combinations of extended cortical networks without having to move the specimen.
  • the two ROIs can have independent control over a plane of focus. Each ROI can be as large or small as needed for the imaging session depending on the scan speed required.
  • both imaging pathways can be set to a maximum FOV, and set to different image depths (e.g., range is approximately 100 ⁇ ).
  • the two ROIs can be smaller and spatially separated.
  • two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can be used in several ways. Firstly, a number of light pulses can be extended, as long as an interval between any light pulse pulses (across all multiplexed pulses) is longer than a fluorescence lifetime of a fluorophore imaged.
  • spatially patterned optogenetic stimulation can be applied through a wide FOV objective, using a dichroic mirror disposed before any filters and a photomultiplier tube (PMT) on a detection arm of a scope.
  • Optogenetic stimulation can be applied in any pattern across an entire FOV, even if calcium imaging is only targeted to two specific ROIs.
  • the two- photon excitation imaging system can be used in conjunction with microendoscopes. Lateral views of a full cortical depth can be obtained using implanted microprisms with the system.
  • two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods as discussed herein can provide neuroscience with a flexible way to image cellular-level neuronal activity not only across multiple cortical areas, but also any model system that extends beyond spatial limits of conventional multiphoton or two- photon microscopy.
  • Two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods can enable measurements of inter-area correlations at a single neuron level and monitoring of activity across extended neuronal circuits.
  • two-photon excitation imaging systems, steering devices, and related methods of the present subject matter are not limited to the uses, configurations, advantages, etc., discussed herein.
  • two-photon excitation imaging system 100 can comprise a two-photon microscope, such as, for example, a Twin Region, Panoramic two-photon (Trepan2p) microscope, configured to provide a wide FOV and two imaging pulses that can be synchronously scanned, enabling simultaneous two-photon calcium imaging in spatially separate cortical areas of a specimen; thereby doubling temporal resolution.
  • two-photon excitation imaging system 100 comprises a Trepan2p microscope, only a single, conventional microscope objective is needed, which can allow for flexible placement of sensory stimulation and behavior apparatus around a subject or specimen 104.
  • custom objectives such as a 0.8 numerical aperture (NA) objective 102 (see Figs. 2A-2B) can be utilized in system 100.
  • a working distance e.g., approximately eight mm
  • additional devices and/or systems such as, for example, a simultaneous electrophysiology device and head-fixed behavior experimental systems.
  • Two-photon excitation imaging system 100 can comprise, in some aspects, a light source, generally deisgnated110.
  • Light source 110 can produce a pulse incident on a desired specimen 104.
  • Light source 110 can be a laser, such as, for example, a Ti: Sapphire laser, which can produce laser pulses following a directed pathway 112.
  • the laser pulses can be delivered at approximately 12.5 ns intervals (e.g., 80 MHz) before being multiplexed.
  • the laser pulses can be attenuated and split into two pulses, each following its own directed pathway 114, 116, using two sets of polarization optics.
  • the laser pulses produced by light source 110 can be directly detected by a photodiode (not shown) using a pick-off mirror (not shown).
  • the photodiode can also be used to synchronize photon-counting electronics, such as, for example a photon counting unit (e.g., 198).
  • synchronization output from light source 110 can also be used to synchronize photon-counting electronics.
  • Power attenuation mechanism 120 can comprise at least one pulse block 130, a first half wave plate 132A, a second half wave plate 132B, and a first polarizing pulse splitting (PBS) cube 134A.
  • Pulse block 130 can absorb excess laser power.
  • First PBS cube 134A can be disposed in between first half wave plate 132A and second half wave plate 132B relative to the pathway 112 of laser pulses, where first half wave plate 132A can be positioned before first PBS cube 134A, while second half wave plate 132B can be positioned after first PBS cube 134A.
  • second half-wave plate 132B can be used to determine a power ratio sent to each pathway 114, 116 once light pulses have been split into two separate imaging pulses or pulses.
  • Two imaging pulses can be, for example, directed to two different ROIs (e.g., two different cortical networks of a brain). In this example, one brain area might be deeper than the other, and thus require more power for imaging than the other brain area.
  • the power ratio between two pathways 114, 116 can be adjustable.
  • a mirror 136 can be provided after power attenuation mechanism 120 in order to direct pulses to a pulse expander, generally designated 140.
  • pulse expander 140 can be disposed after second half-wave plate 132B in order to expand a size of the light pulses.
  • a second pulse expander (not shown) can be utilized in system 100 after pulse recombination to overfill a back aperture of objective 102.
  • a PBS cube can be implemented in order to split the light pulses traveling along pathway 112 into two separate pulses or pulses following two separate pathways 114, 116.
  • second PBS cube 134B is positioned after pulse expander 140 relative to pathway 112 of pulses and is configured to split pulses into a first pulse and a second pulse, each pulse following its own individual pathway 114, 116, respectively.
  • the first and second pulses can be configured with similar or different polarization.
  • the first pulse can be configured with p- polarization
  • the second pulse can be configured with s-polarization.
  • the first pulse can follow a first pathway 114 and can be configured to pass to a first steering mirror 160 without delay, while the second pulse can follow a second pathway 116 and can be configured to pass to a second steering mirror 162 with a predetermined delay.
  • First pathway 114 can be a similar or different length than second pathway 116.
  • second pathway 116 can be longer in distance than first pathway 114, such that it takes a light pulse of similar properties a longer period of time to travel second pathway 116 compared to first pathway 114.
  • second pathway 116 can be shorter in distance than first pathway 114.
  • first pathway 114 can be configured without a predetermined delay portion, such that the first pulse travels without delay through first pathway 114 before reaching first steering mirror 160.
  • second pathway 116 can be configured as a delay path, such that the second pulse travels through the delay path of second pathway 116 prior to reaching second steering mirror 162.
  • the second pulse can pass between a plurality of mirrors 150.
  • Plurality of mirrors 150 can for example include five (5) mirrors 152A-152E spaced apart in such a manner that the second pulse travels from first mirror 152A to second mirror 152B to third mirror 152C to fourth mirror 152D to fifth mirror 152E.
  • Steering mirrors 160, 162 can each be separately and independently repositionable and/or movable relative to one another in an XY axis.
  • 'repositioning' of steering mirrors 160, 162 can comprise independently pivoting each steering mirror 160, 162 such that a center of a mirror surface of each of mirrors 160, 162 does not translate.
  • steering mirrors 160, 162 can be configured to direct respective light pulses to different regions of a FOV (e.g., FOV 106, Figs. 1 C) without 'clipping' of these light pulses.
  • FOV e.g., FOV 106, Figs. 1 C
  • steering mirrors 160, 162 can define smaller ROIs within a larger FOV.
  • steering mirrors 160, 162 can comprise a large semi-diameter steering mirror, including a semi-diameter of approximately 3.0 mm, although larger and smaller diameters are also contemplated.
  • steering mirrors 160, 162 can be motorized and can be remotely movable by a user for independently repositioning first and/or second movable steering mirrors 160, 162.
  • steering mirrors 160, 162 can comprise a servomotor configured to communicate with remote controls (see, Fig. 3).
  • steering mirrors 160, 162 can be manually movable via at least one component in communication with first and second steering mirrors 160, 162, such as, for example, a joystick and/or other manual control mechanism.
  • first and second steering mirrors 160, 162 are independently controllable by a motor, the mirrors can be moved along in XY axes at least, approximately 20 degrees or more, to reposition a deflection angle of the first and second pulses.
  • first steering mirror 160 can result in changing a deflection angle ⁇ - ⁇ of the first pulse along the XY axes
  • second steering mirror 162 can result in independently changing a deflection angle ⁇ 2 of the second pulse along the XY axes prior to recombination.
  • Deflection angles ⁇ 1 ; ⁇ 2 can determine the central locations (e.g., ⁇ , ⁇ ; X2, Y2) of individual ROIs within the larger FOV (see, Figs. 1 C-1 D). Consequently, it will be apparent to one of skill in the art that moving steering mirrors 160, 162 can cause each corresponding ROI to similarly move along the XY axes within the FOV.
  • adjusting a position (e.g., focal plane) of pathways 114, 116 along the Z-axis may be accomplished without repositioning either first or second steering mirrors 160, 162.
  • electro-optical lenses such as, for example, electrically tunable lenses (ETL) can be used to adjust a position (e.g., focal plane) of pathways 114, 116 along the Z-axis relative to an imaging plane.
  • ETL electrically tunable lenses
  • a first ETL 164A can be disposed in first pathway 114 prior to first steering mirror 160 for adjusting a focal plane of first pathway 114
  • a second ETL 164B can be disposed in second pathway 116 prior to second steering mirror 162 for adjusting a focal plane of second pathway 116.
  • First ETL 164A and second ETL 164B can each be independently controllable such that a focal plane of each pathway in the Z-axis is independently adjustable.
  • First and/or second ETLs 164A, 164B can be disposed after first and/or second steering mirror 160, 162, respectively, to adjust convergence and/or divergence of the first and/or second pulses along the Z-axis.
  • a single angle translator may be used to adjust a focal plane of first and/or second pathways 114, 116 of the first and/or second pulses along the Z-axis.
  • a pulse or pulse recombination relay 170 can be implemented to recombine the first and second pulses after a delay has been imposed upon one or more of the pulses.
  • Recombination relay 170 can comprise relay units 172A-C and a third PBS cube 134C.
  • Third PBS cube 134C can be positioned centrally in relation to relay units 172A-C of recombination relay 170.
  • Each of the three relay units 172A-C can be configured with a plurality of lenses to greatly reduce aberrations (e.g., astigmatism), which can allow for large angles (e.g., ⁇ 1; ⁇ 2 ⁇ 0) to be imparted on the individual imaging pathways 114, 116.
  • the plurality of lenses can comprise an achromatic lens and a positive meniscus lens configured such that a convex surface of the meniscus lens faces crown glass of the achromatic lens.
  • each relay unit 172A-C can comprise a 50 mm achromatic lens and a 100 mm positive meniscus lens.
  • First steering mirror 160 can be configured to direct the first pulse from first pathway 114 to first relay unit 172A, which can then direct the first pulse to third PBS cube 134C.
  • Second steering mirror 162 can be configured to direct the second pulse from second pathway 116 to second relay unit 172B, which can then also direct the second pulse to third PBS cube 134C for recombination.
  • the light pulses are delivered from light source 110 at an 80 MHz pulse rate (12.5 ns intervals).
  • Splitting the light pulses at second PBS cube 134B results in a first pulse traveling along first pathway 114 without delay to first steering mirror 160, while a second pulse experiences a 6.25 ns delay along second pathway 114 before reaching second steering mirror 162.
  • the 6.25 ns delay applied to the second pulse results in perfectly interleaved pulses upon recombination, i.e., first and second pulses can be evenly spaced in time at 160 MHz after recombination.
  • the first and second pulses can be directed to a third relay unit 172C of recombination relay 170.
  • the first and the second pulses can then be relayed to X and Y galvanometer scanner, generally designated 180.
  • X and Y galvanometer scanner 180 can comprise X and Y scan mirrors 182A and 182B, respectively, that are connected via a first relay unit 184A and a second relay unit 184B.
  • Scan mirrors 182A and 182B can be separated by a displacement distance, such as, for example, approximately 6 mm.
  • the first and second pulses can be simultaneously raster scanned, where a field size is determined by scan amplitude, and with independently controlled spatial positions in the X and Y axes, as determined by ⁇ 1 ; ⁇ 2 .
  • first relay unit 184A and second relay unit 184B can be configured with a plurality of lenses.
  • the plurality of lenses can comprise an achromatic lens and a positive meniscus lens configured such that a convex surface of the meniscus lens faces crown glass of the achromatic lens.
  • each relay unit 184A-B can comprise a 50 mm achromatic lens and a 100 mm positive meniscus lens.
  • X and Y galvanometer scanner 180 can comprise alternative embodiments having different types, sizes, etc., of lens in first relay unit 184A and second relay unit 184B.
  • axial movement of the meniscus lens of first relay unit 184A and/or second relay unit 184B can cause a small change in pulse convergence that can alter a focal plane for a particular path up to approximately 100 ⁇ . Therefore, each path can be fully independently positionable in the XY axes (e.g., range of approximately 3500 ⁇ ) and Z-axis (e.g., range of approximately 500 ⁇ ). This allows simultaneously scanning two different ROIs within a full FOV.
  • a scan lens 190 in combination with a tube lens 192 can direct the temporally recombined pulses towards a back aperture of objective 102 in order to expand the beam and provide low aberration beam scanning at the back aperture of objective 102.
  • scan lens 190 and tube lens 192 can form a 4x telescope for this purpose (See, Fig. 2A), which in turn can determine a size of the FOV.
  • a scan angle realized at the back aperture of objective 102 can be made smaller by an inverse factor of beam expansion.
  • a 4x telescope composed of, for example, scan lens 190 and tube lens 192, can result in a scan angle being reduced to one-quarter of the scan angle of scan mirrors 182A, 182B of galvanometer 180.
  • Beam magnification, scan angles, and diameters of clear apertures of scan lens 190 and tube lens 192 can be optimized, along with aberration correction of scan lens 190 and tube lens 192, in order to ensure a wide FOV.
  • a FOV generally designated 106, can be formed having a diameter based on a magnification of objective 102.
  • the magnification is a function of the focal length of objective 102, smaller focal length corresponding to a greater magnification (see, Fig. 2).
  • a diameter of FOV 106 By decreasing a magnification of objective 102, a diameter of FOV 106 can be increased. Conversely, by increasing a magnification of objective 102, a diameter of FOV 106 can be decreased.
  • FOV 106 two separate ROIs, first ROI 108A formed by first pathway 114 and second ROI 108B formed by second pathway 116 can be scanned by system 100.
  • ROIs 108A and 108B can be repositioned within FOV 106 during a scanning session by repositioning steering mirrors 160, 162.
  • repositioning at least one of steering mirrors 160, 162 can result in changing a deflection angle ⁇ - ⁇ of the first pulse along the XY axis, while independent movement (e.g., tilt-tip) of second steering mirror 162 can result in independently changing a deflection angle ⁇ 2 of the second pulse along the XY axis prior to recombination.
  • Angles ⁇ - ⁇ , ⁇ 2 can determine the central locations (e.g., Xi, Yi ; X2, Y2) of individual ROIs 108A, 108B within FOV 106.
  • a focal plane parallel to an imaging plane is represented.
  • ETLs 164A, 164B can be used to individually adjust or reposition the focal plane of pathways 114, 116 along the Z-axis.
  • FOV 106 encompasses a neural area of specimen 104.
  • a primary visual cortex V1 surrounded by higher visual areas e.g., posteromedial, anteromedial, anterior, rostrolateral, anterolateral, lateromedial, laterointermediate
  • FOV 106 Portions of primary and secondary somatosensory cortex S1 , S2 and auditory cortex AU are also encompassed by FOV 106.
  • Changing a magnification and/or other components of system 100 may result in a diameter of FOV 106 increasing and/or decreasing.
  • first ROI 108A can be positioned in one portion of primary visual cortex V1
  • second ROI 108B can be positioned in one portion of the anteromedial area.
  • repositioning of steering mirrors 160 and/or 162 can result in a central location of ROIs 108A and/or 108B changing.
  • multiple ROIs within one or more FOVs can be scanned, without moving system 100 or specimen 104.
  • system 100 can be configured to transmit approximately 13% of entering power.
  • power does not have to be limited in this configuration, such that decreasing overfilling of objective 102 and scan mirrors 182A-B of galvanometer 180 can increase power transmission, though with a possible decrease in excitation efficiency.
  • fluorescence can be collected using, for example, a dichroic mirror 194, a plurality of lenses 196A and 196B, and a PMT 198 of system 100.
  • PMT 198 can comprise, for example, a GaAsP PMT having a high bandwidth amplifier (not shown) for measuring fluorescence signals from both pathways 114, 116.
  • a photodiode (not shown) can be used to directly detect light pulses and synchronize photon counting electronics, e.g., PMT 198.
  • fluorescence events can be reliably assigned to an appropriate pathway (e.g., first pathway 114 or second pathway 116, with minimal channel crosstalk (e.g., 1 .5 - 6.6%).
  • Photon counting can provide an increase in signal to noise over analog integration for dim signals, which can be common with in vivo two-photon calcium imaging data.
  • a digital acquisition board (not shown) can generate mirror command signals, and frame synchronization signals for PMT 198.
  • custom software can control steering mirrors 160, 162, communicate with PMT 198, and can organize photon counting data into images.
  • the pulsing of light source 1 10 can be measured using a photodiode (not shown) to determine which fluorescence signals came from which pathway.
  • FIG. 2A a schematic, generally designated 200, a scan engine composed of a scan lens 202, a tube lens 204, and an objective 206 is illustrated.
  • a central optical axis is illustrated, in Figure 2A, by the broken line, while a maximum scan angle is illustrated by the solid line.
  • Scan lens 202, tube lens 204, and objective 206 may each be utilized in a two-photon excitation imaging system, such as system 100.
  • a ratio TL F L/SL F L results in a factor by which an excitation beam is expanded.
  • An inverse of this factor is the factor by which a scan angle ⁇ is decreased to result in ⁇ 2 .
  • an algorithm such as, 2 * OBJFL * tan(0 2 ) can be used.
  • a magnification of objective 206 can be a function of its focal length. Thus, both the focal length and/or magnification of objective 206, as well as the scan angle can influence the FOV.
  • FIG. 2B A schematic of one embodiment of objective 206 is illustrated in Figure 2B.
  • a standard commercial objective can be utilized in system 200, while in other aspects a custom objective can be used.
  • a custom objective e.g., objective 206 can comprise an effective focal length (EFL) of 27.55 mm and an NA of 0.4.
  • Objective 206 can be used in conjunction with scan lens 202, tube lens 204, and any associated relays (e.g., recombination relay 170, afocal relay in galvanometer 180, Figure 1 ) to minimize aberrations to result in single neuron resolution with two-photon excitation across a wider FOV.
  • relays e.g., recombination relay 170, afocal relay in galvanometer 180, Figure 1
  • a quality of a point spread function (PSF) of two-photon excitation imaging system is crucial to its efficiency, rejection of out of plane fluorescence, and image quality.
  • a 0.80 NA objective e.g. 102
  • a small PSF can result. This can provide a small excitation volume, good optical sectioning, and reduced contamination from out-of-plane fluorescent neuropil.
  • Such a performance can be excellent on axis, but can degrade at large scan angles.
  • performance of two-photon excitation imaging system 100 can be evaluated by measuring the off-axis excitation volume as a full-width at half- maximum (FWHM) of an intensity profile.
  • FWHM full-width at half- maximum
  • a high performance objective and optimized relay lens systems can also lead to a flat FOV. Accordingly, configuring scan lens 202, tube lens 204, and objective 206 in a manner similar to that of Figure 2A enables a wide FOV, with only marginally decreased magnification and/or NA.
  • Steering device 300 can be used in a two-photon excitation imaging system, generally designated 316.
  • System 316 may comprise, in some aspects, components similar to that of system 100 (see, e.g., Fig. 1 A).
  • Steering device 300 can comprise a plurality of steering mirrors 312-314 controllable by at least one motorized component 310 interfacing with a computing platform 302 in order to impart a solid angle deflection (e.g., ⁇ - ⁇ , Q 2 ) to separate light pulses prior to recombination.
  • Other entities that may be associated with the above-described steering device 300 may include a separate motorized component (not shown) for controlling an electro-optical lens (e.g., ETLs 164A-B), which may also interface with computing platform 302.
  • ETLs 164A-B electro-optical lens
  • a computing platform 302 can be configured to control at least one motorized component 310 of steering device 300 and/or system 316 through an interface 308.
  • Computing platform 302 may represent any suitable entity or entities (e.g., a control platform or a server farm) for controlling at least one motorized component 310.
  • computing platform 302 may control at least one motorized component 310, which in turn, may adjust steering mirrors 312-314 of system 316.
  • computing platform 302 may be a stand-alone tool, device, or software executing on a processor, and may not be integrated into steering device 300 and/or system 316.
  • computing platform 302 may be a single node or may be distributed across multiple computing platforms or nodes.
  • computing platform 302 becomes a special purpose computing platform that can improve the technological field of two-photon excitation imaging by independently repositioning steering mirrors to thereby independently reposition temporally multiplexed excitation pathways to simultaneously image separate ROIs within an expanded FOV.
  • Computing platform 302 may include control module (CM) 304.
  • CM 304 may be any suitable entity (e.g., software executing on a processor) for performing one or more aspects associated with two-photon excitation imaging.
  • CM 304 may include functionality for controlling at least one motorized component 310, controlling at least one electro-optical lens (e.g., 164A-B, Fig. 1 A), communicating with photon counting electronics (e.g., 198, Fig. 1 A), and organizing photon counting data into images.
  • CM 304 may be configured to automatically control at least one motorized component 310 to thereby adjust a position of one or more steering mirrors 312-314. Where each steering mirror 312-314 is controlled by an individual motorized component, CM 304 may be configured to independently, automatically control individual motorized components.
  • CM 304 may include or access data storage 306 containing data and/or images related to controlling at least one motorized component 310.
  • CM 304 may access data storage 306 containing mapped coordinate systems, profiles, settings, or configurations that may enable CM 304 to automatically control at least one motorized component 308.
  • data storage 306 may comprise previously stored photon counting data.
  • Data storage 306 may include non-transitory computer readable media, such as flash memory, random access memory, or other storage devices.
  • data storage 306 may be external to and/or or integrated with computing platform 302 and/or CM 304.
  • computing platform 302 and/or CM 304 may include one or more interfaces 308.
  • one or more interfaces 308 may include remote controls used to operate steering mirrors 312-314 either wirelessly or with wired contact.
  • a CM user (not shown) may be able to interact with one or more interfaces 308 to remotely control movement of steering mirrors 312-314 via at least one motorized component 310.
  • a CM user may be an automated system or may be controlled or controllable by a human user to operate steering mirrors 312-314.
  • interface 308 may include a joystick or other control implement for a CM user to manipulate. Through manipulation of interface(s) 308 steering mirrors 312-314 may be adjusted into the directed positions.
  • the CM user may interact with one or more interfaces 308 in order to provide instructions to CM 304 to remotely control movement of steering mirrors 312-314 via at least one motorized component 310.
  • At least one motorized component 310 can comprise a servomotor, voice coil, piezo, galvanometer, etc., configured to manipulate, adjust, move and/or reposition components of steering device 300.
  • at least one motorized component 310 can comprise two separate servomotors that can be configured to independently adjust steering mirrors 312, 314.
  • At least one motorized component 310 can be integrated with or configured separately from steering mirrors 312, 314.
  • steering device 300 can be configured such that each steering mirror is independently repositionable and adjustable.
  • motorized component 310 can be configured to adjust steering mirrors 312, 314 along XY axes at least, approximately 20 degrees or more, to move a deflection angle of the first and second pulses.
  • Method 400 can be accomplished at a two-photon excitation imaging system, such as system 100, including a source (e.g., 110) for producing light pulses, a steering device (e.g., 300) comprising first movable steering mirror, a second movable steering mirror, and at least one component in communication with the first movable steering mirror and the second movable steering mirror for independently repositioning the first and/or second movable steering mirror in XY axes.
  • a source e.g., 110
  • a steering device e.g., 300
  • method 400 is in no way limited to system 100.
  • a first pulse of light and a second pulse of light are provided.
  • a light source 110 can be configured to generate a light pulse that can be split by PBS cube 134B into a first pulse of light and a second pulse of light.
  • first steering mirror 160 may be provided on a first pathway 114 so that the first pulse of light can travel without delay to first steering mirror 160.
  • second steering mirror 162 may be provided on a second pathway 116, a portion of which is configured as a delay path, so that the second pulse of light travels with a 6.25 ns delay to second steering mirror 162.
  • the first and second pulses of light are recombined.
  • the first and second pulses of light can be recombined at a recombination relay 170 and a PBS cube 134C.
  • the recombined first and second pulses of light are directed through an objective to a specimen.
  • the first and second pulses of light can be directed through a scan lens 190 and a tube lens 192 and be directed into a back aperture of an objective 102 to a specimen 104.
  • method 400 can comprise remotely and independently repositioning, by at least one motorized component, the first movable steering mirror and the second movable steering mirror in the XY axes.
  • at least one motorized component 310 can be used to repositioning a first movable steering mirror 160 and a second movable steering mirror 162.
  • method 400 can comprise individually repositioning, by at least one tunable lens, a focal plane for at least one of the first pathway and the second pathway.
  • at least one tunable lens can be an electrically tunable lenses (ETL) 164A, B for a focal plane for at least one of a first pathway 114 and a second pathway 116.
  • ETL electrically tunable lenses
  • At least one of the first movable steering mirror and the second movable steering mirror are configured to impart solid angle deflections on at least one of the first pulse of light and the second pulse of light.
  • solid angle deflections ⁇ - ⁇ , ⁇ 2 may be imparted on the first pulse of light and/or the second pulse of light.
  • the solid angle deflections are configured to determine central locations of two individual regions of interest (ROIs) within a field of view (FOV).
  • ROIs regions of interest
  • FOV field of view
  • solid angle deflections ⁇ - ⁇ , ⁇ 2 may be configured to determine central locations of two individual ROIs 108A, 108B within a FOV 106.
  • the FOV is of a neural area, and the two individual ROIs are generated within the neural area (see, Fig. 1 D).
  • exemplary process 400 is for illustrative purposes and that different and/or additional actions may be used. It will also be appreciated that various actions described herein may occur in a different order or sequence.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention porte sur des systèmes d'imagerie à excitation à deux photons, sur des dispositifs de direction pour systèmes d'imagerie à excitation à deux photons, et sur des procédés associés. Dans certains aspects, les dispositifs de direction peuvent comprendre un premier miroir de direction mobile, un second miroir de direction mobile, et au moins un élément en communication avec le premier miroir de direction mobile et le second miroir de direction mobile de façon à repositionner indépendamment le premier et/ou le second miroir(s) de direction mobile dans des axes XY. Les dispositifs de direction peuvent être mis en œuvre dans un système de d'imagerie à excitation à deux photons. Selon certains aspects, le système d'imagerie peut comprendre une source pour produire des impulsions lumineuses, un dispositif de direction et un relais de recombinaison. Le dispositif de direction peut être conçu de façon à diriger des première et seconde impulsions de lumière, respectivement, vers le relais de recombinaison, et vers un objectif à travers lequel les première et seconde impulsions de lumière sont dirigées à travers celui-ci vers un spécimen, après que les première et seconde impulsions de lumière ont été recombinées.
PCT/US2015/040173 2014-07-11 2015-07-13 Dispositif de direction pour systèmes d'imagerie à excitation à deux photons Ceased WO2016007954A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102020130275A1 (de) 2020-11-17 2022-05-19 Abberior Instruments Gmbh Hochauflösendes mikroskop, mikrooptisches system und detektionsanordnung

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034613A (en) * 1989-11-14 1991-07-23 Cornell Research Foundation, Inc. Two-photon laser microscopy
JP2005301065A (ja) * 2004-04-14 2005-10-27 Olympus Corp 観察装置
JP2008096778A (ja) * 2006-10-13 2008-04-24 National Institute Of Advanced Industrial & Technology 光軸自動調整機能搭載２光子レーザ顕微鏡

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5034613A (en) * 1989-11-14 1991-07-23 Cornell Research Foundation, Inc. Two-photon laser microscopy
JP2005301065A (ja) * 2004-04-14 2005-10-27 Olympus Corp 観察装置
JP2008096778A (ja) * 2006-10-13 2008-04-24 National Institute Of Advanced Industrial & Technology 光軸自動調整機能搭載２光子レーザ顕微鏡

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102020130275A1 (de) 2020-11-17 2022-05-19 Abberior Instruments Gmbh Hochauflösendes mikroskop, mikrooptisches system und detektionsanordnung

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