WO2016007954A1

WO2016007954A1 - Steering devices for two-photon excitation imaging systems

Info

Publication number: WO2016007954A1
Application number: PCT/US2015/040173
Authority: WO
Inventors: Spencer L. SMITH; Jeffrey STIRMAN; Michael KUDENOV
Original assignee: University of North Carolina at Chapel Hill; North Carolina State University
Current assignee: University of North Carolina at Chapel Hill; North Carolina State University
Priority date: 2014-07-11
Filing date: 2015-07-13
Publication date: 2016-01-14
Anticipated expiration: 2017-01-11

Abstract

Two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods are disclosed herein. In some aspects, the steering devices can include 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. The steering devices can be implemented in a two-photon excitation imaging system. In some aspects, the imaging system can include a source for producing light pulses, a steering device, and a recombination relay. The steering device can be configured to direct 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.

Description

STEERING DEVICES FOR TWO-PHOTON EXCITATION IMAGING SYSTEMS

DESCRIPTION

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application Serial No. 62/023,564 filed July 1 1 , 2014, which is herein incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number A12-1051 -001 awarded by the Human Frontier Science Program. The government has certain rights to this invention.

TECHNICAL FIELD

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.

BACKGROUND

Ensemble neuronal activity is of key interest in system neuroscience, among other disciplines, to understand sensory coding, motor output, and cognitive function. Measuring neuronal activity in populations of neurons in vivo is technically challenging, due to the densely packed neuropil and sensitive neuroanatomy that is best probed with minimally invasive approaches. However, multiphoton imaging can overcome these technical challenges.

For example, 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. Furthermore, 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.

Although multiphoton imaging has proven to be a powerful tool to study ensemble activity in local populations of neurons in neocortex, conventional approaches are limited to a full field of view (FOV) of approximately 500 μιτι - 700 μιτι. This limitation precludes the simultaneous examination over a wide FOV or areas large enough to encompass multiple regions of interest (ROIs) within a FOV. While discrete functional areas of mammalian neocortex work in concert to process sensory input and guide adaptive behavior, little is known about how cortical areas interact. Thus, in order to progress further in neuroscience a cellular-level view into ongoing neural activity across extended cortical networks is needed.

As a result, there is a need for two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods that feature a wide FOV to enable high-speed imaging in spatially separate ROIs. SUMMARY

In accordance with this disclosure, two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods are provided. In particular, 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).

In some aspects, a steering device for a two-photo excitation imaging system is provided. For example, the device 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.

In other aspects, a two-photon excitation imaging system is provided. For example, the 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. In other aspects, a two-photon excitation imaging method is provided. For example, 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.

Although some of the aspects of the subject matter disclosed herein have been stated hereinabove, and which are achieved in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying drawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the present subject matter will be more readily understood from the following detailed description which should be read in conjunction with the accompanying drawings that are given merely by way of explanatory and non-limiting example, and in which:

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;

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; and

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.

DETAILED DESCRIPTION

The present subject matter provides two-photon excitation imaging systems, steering devices for two-photon excitation imaging systems, and related methods. In one aspect, 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).

In one aspect, the present subject matter provides a novel two-photon excitation imaging system with a field of view greater than 3000 μιτι, resulting in an imaging area over 18-fold greater than that of conventional multiphoton imaging (> 7 square mm), while still achieving cellular resolution. To support high speed imaging in two discrete ROIs within this FOV simultaneously, multiplexed excitation and high-speed photon counting can be used. As used herein, "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. Firstly, 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. Secondly, 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. Thirdly, 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. Furthermore, 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. For example, for large overview imaging with two depths, both imaging pathways can be set to a maximum FOV, and set to different image depths (e.g., range is approximately 100 μιτι). Alternatively, for example, to image a full field faster, the two ROIs can be smaller and spatially separated. . Moreover, 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. Secondly, 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. To simultaneously image multiple cortical regions that are beyond the FOV, or to access subcortical brain regions, for example, 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.

Notably, 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. However, one of skill in the art can readily understand that 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.

Now referring to Figure 1 A, a schematic of an exemplary two-photon excitation imaging system, generally designated 100, is illustrated. In some aspects, 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. In some aspects, where 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. Alternately, custom objectives, such as a 0.8 numerical aperture (NA) objective 102 (see Figs. 2A-2B) can be utilized in system 100. Additionally, a working distance (e.g., approximately eight mm) can ensure that the Trepan2p microscope can be compatible with 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. For example, 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. However, the present subject matter can be readily extended into 2ⁿ pulses, where n = 1 , 2, 3... Whether or not a number of pulses can be extended depends on whether an interval between any laser pulses (across all multiplexed pulses) is longer than a fluorescence lifetime of a fluorophore imaged.

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). Alternatively, synchronization output from light source 110 can also be used to synchronize photon-counting electronics.

Additionally, in some aspects, overall power of system 100 can be attenuated via a power attenuation mechanism, generally designated 120. 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.

In some aspects, 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. Thus, 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. For example, pulse expander 140 can be disposed after second half-wave plate 132B in order to expand a size of the light pulses. In some aspects, a second pulse expander (not shown) can be utilized in system 100 after pulse recombination to overfill a back aperture of objective 102.

In some aspects, 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. For example, as illustrated in Figure 1 A, 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. For example, the first pulse can be configured with p- polarization, while 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. For example, where it is desired to delay second pulse compared to first pulse, 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. Alternatively, second pathway 116 can be shorter in distance than first pathway 114.

As illustrated in Figure 1 A, 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. By comparison, at least a portion of 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. In the delay path of second pathway 116, 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. In some aspects, there can be more or less mirrors depending on a length of the predetermined delay required. As illustrated in Figure 1 A, mirrors 152A-152E are spaced apart such that the second pulse is delayed by approximately 6.25 ns due to traveling approximately 1 .87 meters farther than the first pulse in the first pathway 114 (6.25 ns ^* c = 1 .87 m). After traveling 1 .87 m, the second pathway 116 can pass to second steering mirror 162.

Steering mirrors 160, 162 can each be separately and independently repositionable and/or movable relative to one another in an XY axis. As used herein, '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. As a result, steering mirrors 160, 162can 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. For example, by adjusting a deflection angle of one or more of the first and second pulses, steering mirrors 160, 162 can define smaller ROIs within a larger FOV. In some aspects, 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. In some aspects, 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. For example, steering mirrors 160, 162 can comprise a servomotor configured to communicate with remote controls (see, Fig. 3). In other examples, a voice coil, piezo, galvanometer, etc., may be used to move steering mirrors 160, 162. Conversely, in other aspects, 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. Where 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. For example, repositioning (e.g., tilt-tip) first steering mirror 160 can result in changing a deflection angle Ω-ι of the first pulse along the XY axes, while independent repositioning (e.g., tilt-tip) of second steering mirror 162 can result in independently changing a deflection angle Ω₂ of the second pulse along the XY axes prior to recombination. Deflection angles Ω_{1 ;} Ω₂ can determine the central locations (e.g., Χι, Υι ; 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.

In some aspects, 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. Accordingly, 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. For example, 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, while 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. As an alternative to an electro-optical lens, a single angle translator, for example, 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.

In some aspects, 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., Ω_1; Ω₂≠ 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. For example, 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.

In the above described multiplexing scheme, 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. Thus, when each of the first and second pulses are delivered to PBS cube 134C, 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.

After recombination at PBS cube 134C, 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. In some aspects, as illustrated in Figure 1 B, 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. In this manner, 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 Ω_{1 ;} Ω₂.

In some aspects and as illustrated in Figure 1 B, 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. For example, each relay unit 184A-B can comprise a 50 mm achromatic lens and a 100 mm positive meniscus lens. Notably, however, 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.

In some aspects, 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 μιτι. Therefore, each path can be fully independently positionable in the XY axes (e.g., range of approximately 3500 μιτι) and Z-axis (e.g., range of approximately 500 μιτι). This allows simultaneously scanning two different ROIs within a full FOV.

In some aspects, 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. For example, 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. In some aspects, a scan angle realized at the back aperture of objective 102 can be made smaller by an inverse factor of beam expansion. Thus, 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. In some aspects and as illustrated in Figure 1 C, 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). 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. Within 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. In some aspects, ROIs 108A and 108B can be repositioned within FOV 106 during a scanning session by repositioning steering mirrors 160, 162. For example, repositioning at least one of steering mirrors 160, 162 can result in changing a deflection angle Ω-ι 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 Ω₂ of the second pulse along the XY axis prior to recombination. Angles Ω-ι, Ω₂ can determine the central locations (e.g., Xi, Yi ; X2, Y2) of individual ROIs 108A, 108B within FOV 106. Additionally, as illustrated in Figure 1 C, a focal plane parallel to an imaging plane is represented. In some aspects, ETLs 164A, 164B can be used to individually adjust or reposition the focal plane of pathways 114, 116 along the Z-axis.

Referring now to Figure 1 D, an exemplary embodiment of FOV 106 is illustrated. In Figure 1 D, FOV 106 encompasses a neural area of specimen 104. Here a primary visual cortex V1 surrounded by higher visual areas (e.g., posteromedial, anteromedial, anterior, rostrolateral, anterolateral, lateromedial, laterointermediate) is completely encompassed within 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. Within FOV 106, first ROI 108A can be positioned in one portion of primary visual cortex V1 , while second ROI 108B can be positioned in one portion of the anteromedial area. Notably, repositioning of steering mirrors 160 and/or 162 can result in a central location of ROIs 108A and/or 108B changing. Thus, within a same imaging session, multiple ROIs within one or more FOVs can be scanned, without moving system 100 or specimen 104.

Referring back to Figure 1 A, during operation of the two-photon excitation imaging system, such as system 100, pulses from both imaging pathways 114, 116 can overfill a back aperture of objective 102. Overall, in such a configuration, system 100 can be configured to transmit approximately 13% of entering power. However, 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.

In some aspects, 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. By synchronizing PMT 198 with the laser pulses, 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. In some aspects, custom software (see, 304, Fig. 3) can control steering mirrors 160, 162, communicate with PMT 198, and can organize photon counting data into images. In some aspects, the pulsing of light source 1 10 can be measured using a photodiode (not shown) to determine which fluorescence signals came from which pathway.

Referring now to Figure 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.

As illustrated in Figure 2A, a ratio TL_FL/SL_FL results in a factor by which an excitation beam is expanded. An inverse of this factor is the factor by which a scan angle θι is decreased to result in θ₂. To determine a width of the FOV an algorithm, such as, 2 * OBJFL ^* tan(0₂) 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.

A schematic of one embodiment of objective 206 is illustrated in Figure 2B. In some aspects, a standard commercial objective can be utilized in system 200, while in other aspects a custom objective can be used. For example, 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. In some aspects, 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. For example, where a 0.80 NA objective (e.g., 102) is utilized in system 100 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. Thus, 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. Furthermore, 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.

Referring now to Figure 3, a detailed schematic of an exemplary steering device, generally designated 300, is illustrated. 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., Ω-ι, Q₂) 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.

In some aspects, 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. For example, computing platform 302 may control at least one motorized component 310, which in turn, may adjust steering mirrors 312-314 of system 316. In some aspects, 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. In some aspects, computing platform 302 may be a single node or may be distributed across multiple computing platforms or nodes. Thus, when configured as described herein, 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. For example, 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.

In some aspects, CM 304 may include or access data storage 306 containing data and/or images related to controlling at least one motorized component 310. For example, 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. Where CM 304 is configured for additional functionality, 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. In some aspects, data storage 306 may be external to and/or or integrated with computing platform 302 and/or CM 304.

In some aspects, computing platform 302 and/or CM 304 may include one or more interfaces 308. For example, 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. In some aspects, a CM user may be an automated system or may be controlled or controllable by a human user to operate steering mirrors 312-314. For example, 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. In other aspects, 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. For example, 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. In some aspects, there can be more than two steering mirrors of steering device 300. However, regardless of a number of steering mirrors, steering device 300 can be configured such that each steering mirror is independently repositionable and adjustable. Thus, where a CM user manipulates interfaces 308 of computing platform 302 and/or CM 304, 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.

Referring now to Figure 4, a flow chart depicting an exemplary method, generally designated 400, for two-photon excitation imaging is illustrated. 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. However, one having ordinary skill in the art will understand that method 400 is in no way limited to system 100.

In block 402, a first pulse of light and a second pulse of light are provided. For example, 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.

In block 404, the first pulse of light is directed, without delay, along a first pathway to a first movable steering mirror, while the second pulse of light is directed along a second pathway to a second movable steering mirror. For example, 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. By comparison, for example, 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.

In block 406, the first and second pulses of light are recombined. For example, the first and second pulses of light can be recombined at a recombination relay 170 and a PBS cube 134C.

In block 408, the recombined first and second pulses of light are directed through an objective to a specimen. For example, 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.

In some aspects, 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. For example, at least one motorized component 310 can be used to repositioning a first movable steering mirror 160 and a second movable steering mirror 162.

In some aspects, 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. For example, 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.

In some aspects, 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. For example, solid angle deflections Ω-ι, Ω₂ may be imparted on the first pulse of light and/or the second pulse of light. In some aspects, the solid angle deflections are configured to determine central locations of two individual regions of interest (ROIs) within a field of view (FOV). For example, solid angle deflections Ω-ι, Ω₂ may be configured to determine central locations of two individual ROIs 108A, 108B within a FOV 106. In some aspects, the FOV is of a neural area, and the two individual ROIs are generated within the neural area (see, Fig. 1 D).

It will be appreciated that 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.

The present subject matter can be embodied in other forms without departure from the spirit and essential characteristics thereof. The embodiments described therefore are to be considered in all respects as illustrative and not restrictive. Although the present subject matter has been described in terms of certain preferred embodiments, other embodiments that are apparent to those of ordinary skill in the art are also within the scope of the present subject matter.

Claims

1 . A steering device for a two-photon excitation imaging system, the steering device comprising:

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.

2. The steering device of claim 1 , further comprising a computing platform including at least one hardware processor and a memory, and an interface in communication with the computing platform.

3. The steering device of claim 2, wherein the interface comprises remote controls configured for remotely and independently repositioning the first and/or second movable steering mirrors via at least one motorized component.

4. The steering device of claim 1 , wherein 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 a first pulse of light and a second pulse of light.

5. The steering device of claim 4, wherein the solid angle deflections are configured to determine central locations of two individual regions of interest (ROIs) within a field of view (FOV).

6. The steering device of claim 1 , wherein the first movable steering mirror is disposed in a first pathway along which a first pulse of light is directed, without delay, to the first movable steering mirror, and wherein the second movable steering mirror is disposed in a second pathway along which a second pulse of light is directed, with a delay, to the second movable steering mirror.

7. The steering device of claim 6, 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 a recombination relay.

8. The steering device of claim 7, wherein the recombination relay is configured to direct the first and second pulses of light to an afocal relay of a galvanometer scanner.

9. The steering device of claim 1 , wherein the first movable steering mirror and the second movable steering mirror each comprise a semi-diameter of approximately 3.0 mm.

10. A two-photon excitation imaging system comprising:

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; 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.

1 1 . The system of claim 10, wherein the first movable steering mirror and the second movable steering mirror are configured to be remotely and independently repositioned in the XY axes via at least one motorized component.

12. The system of claim 10, wherein 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.

13. The system of claim 12, wherein the solid angle deflections are configured to determine central locations of two individual regions of interest (ROIs) within a field of view (FOV).

14. The system of claim 13, wherein the FOV is of a neural area, and the two individual ROIs are generated within the neural area.

15. The system of claim 10, further comprising at least one tunable lens configured to individually reposition a focal plane for at least one of the first pathway and the second pathway.

16. The system of claim 10, wherein at least a portion of the second pathway is configured as a delay path, so that the second pulse of light travels with a 6.25 ns delay to the second movable steering mirror.

17. A two-photon excitation imaging method comprising: 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.

18. The method of claim 17, further comprising 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.

19. The method of claim 17, wherein 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.

20. The method of claim 19, wherein the solid angle deflections are configured to determine central locations of two individual regions of interest (ROIs) within a field of view (FOV).

21 . The method of claim 20, wherein the FOV is of a neural area, and the two individual ROIs are generated within the neural area. The method of claim 17, further comprising individually repositioning, by at least one tunable lens, a focal plane for at least one of the first pathway and the second pathway.

The method of claim 17, wherein directing the second pulse of light to the second movable steering mirror comprises directing the second pulse of light to at least a portion of the second pathway configured as a delay path, so that the second pulse of light travels with a 6.25 ns delay to the second movable steering mirror.