Existing approaches for imaging structures within a living animal's brain include imaging using ‘mini-cam’ head mounted recordings in freely moving animals and using point-scanning 2-photon (head-fixed) systems. Mini-cam images are 2D epi-fluorescence and can be blurry and challenging to interpret. And 2-photon images typically only capture a single 2D plane and not a 3D volume. 2D data is difficult to interpret, captures a limited volume of tissue, and makes cell identification challenging. The prior art approaches of capturing activity in a single 2D plane not only samples a fraction of the network, but it also precludes any analysis of interactions between cells that extend between multiple planes.
U.S. Pat. Nos. 10,061,111, 10,831,014, 10,835,111, 10,852,520, and 10,908,088, each of which is incorporated herein by reference, describe a variety of approaches for implementing Swept, Confocally-Aligned Planar Excitation (SCAPE) microscopy, which is a 3D imaging technique.
One aspect of this application is directed to a first imaging apparatus. The first imaging apparatus comprises a first set of optical components, a GRIN lens, a second set of optical components, a scanning element, and a third objective with an associated optical interface. The first set of optical components has a proximal end and a distal end. The first set of optical components includes a first objective disposed at the distal end of the first set of optical components. The GRIN lens is positioned distally beyond the first objective. The second set of optical components has a proximal end and a distal end. The second set of optical components includes a second objective disposed at the distal end of the second set of optical components.
The scanning element is disposed proximally with respect to the proximal end of the first set of optical components and proximally with respect to the proximal end of the second set of optical components. The scanning element is positioned to route a sheet of excitation light so that the sheet of excitation light will pass in a proximal to distal direction through the first set of optical components and through the GRIN lens, and project into a sample that is positioned distally beyond the GRIN lens. The sheet of excitation light is projected into the sample at an oblique angle with respect to an optical axis of the first objective, and the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element. The GRIN lens and the first set of optical components route detection light from the sample in a distal to proximal direction back to the scanning element. The scanning element is also positioned to route the detection light so that the detection light will pass through the second set of optical components in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second set of optical components. The third objective with the associated optical interface are collectively positioned to route light that arrives at the oblique intermediate image plane towards a camera, wherein the third objective and the associated optical interface are collectively configured to provide a zero working distance to maximize detection NA.
Some embodiments of the first imaging apparatus further comprise a light source that generates a beam of excitation light; at least one optical component that expands the beam of excitation light into the sheet of excitation light and directs the sheet of excitation light towards the scanning element; and the camera.
Some embodiments of the first imaging apparatus further comprise an image splitter positioned between the third objective and the camera. The image splitter is configured to direct first wavelength light to a first portion of an image sensor within the camera and to direct second wavelength light to a second portion of the image sensor.
In some embodiments of the first imaging apparatus, the second objective is an air objective and the third objective is a non-air immersion objective, and the associated optical interface comprises a fluid chamber positioned so that the oblique intermediate image plane is formed at an interface of an immersion medium of the third objective.
In some embodiments of the first imaging apparatus, the second objective is an air objective and the third objective is a non-air immersion objective, and the associated optical interface comprises a cured polymer spacer having a refractive index that matches an immersion medium of the third objective. The cured polymer spacer is affixed directly to a front surface of the third objective, and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer.
In some embodiments of the first imaging apparatus, the second objective is an air objective and the third objective is a 1.0 NA water immersion objective, and the associated optical interface comprises a cured polymer spacer having a refractive index of 1.33. The cured polymer spacer is affixed directly to a front surface of the third objective, and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. Optionally, in these embodiments, the third objective is not coverglass corrected.
In some embodiments of the first imaging apparatus, the third objective and the associated optical interface are integrated together into a single package.
Another aspect of this application is directed to a second imaging apparatus. The second imaging apparatus comprises a first set of optical components, a GRIN lens, a second set of optical components, a scanning element, a bundle of optical fibers, and a third objective. The first set of optical components has a proximal end and a distal end, and the first set of optical components includes a first objective disposed at the distal end of the first set of optical components. The GRIN lens is positioned distally beyond the first objective. The second set of optical components has a proximal end and a distal end, and the second set of optical components includes a second objective disposed at the distal end of the second set of optical components.
The scanning element is disposed proximally with respect to the proximal end of the first set of optical components and proximally with respect to the proximal end of the second set of optical components. The scanning element is positioned to route a sheet of excitation light so that the sheet of excitation light will pass in a proximal to distal direction through the first set of optical components and through the GRIN lens, and project into a sample that is positioned distally beyond the GRIN lens. The sheet of excitation light is projected into the sample at an oblique angle with respect to an optical axis of the first objective, and the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element. The GRIN lens and the first set of optical components route detection light from the sample in a distal to proximal direction back to the scanning element. The scanning element is also positioned to route the detection light so that the detection light will pass through the second set of optical components in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second set of optical components.
The bundle of optical fibers has an optical axis, a first end, and a second end, and the first end is positioned at the oblique intermediate image plane so that light from the oblique intermediate image plane enters the first end of the bundle and is directed through the bundle to the second end of the bundle. The first end is positioned to both collect light and provide image rotation. The third objective is positioned to accept light that exits the second end of bundle and route the accepted light towards a camera, and the third objective has an optical axis that is aligned with the optical axis of the bundle.
Some embodiments of the second apparatus further comprise a light source that generates a beam of excitation light; at least one optical component that expands the beam of excitation light into the sheet of excitation light and directs the sheet of excitation light towards the scanning element; and the camera.
In some embodiments of the second apparatus, the first end is beveled with respect to the optical axis of the bundle. In some embodiments of the second apparatus, the bundle of optical fibers comprises a bundle of tapered fibers that are oriented so that the diameters of the tapered fibers are largest at the second end of the bundle.
In some embodiments of the second apparatus, the first end is beveled with respect to the optical axis of the bundle, the bundle of optical fibers comprises a bundle of tapered fibers that are oriented so that the diameters of the tapered fibers are largest at the second end of the bundle, and the first end of the bundle of optical fibers has an NA of 1.0.
Some embodiments of the second apparatus further comprise a wide-field camera, a second light source, and a first beam splitter. The first beam splitter is positioned within the first set of optical components, and is configured to route illumination light from the second light source towards the first objective so that the illumination light illuminates an outer surface of a region of tissue that surrounds the GRIN lens after the GRIN lens has been embedded in subject tissue. The first beam splitter is further configured to route light that arrives from the outer surface towards the wide-field camera.
Some embodiments of the second apparatus further comprise a wide-field camera, a second light source, and a first beam splitter. The first beam splitter is positioned within the first set of optical components, and is configured to route illumination light from the second light source towards the first objective so that the illumination light illuminates an outer surface of a region of tissue that surrounds the GRIN lens after the GRIN lens has been embedded in subject tissue. The first beam splitter is further configured to route fluorescence light that arrives from the outer surface towards the wide-field camera.
Optionally, the embodiments of the previous paragraph may further comprise a second beam splitter positioned and configured to route illumination light from the second light source towards the first beam splitter and route fluorescence light arriving from the first beam splitter towards the wide-field camera.
Another aspect of this application is directed to a third imaging apparatus that comprises a first set of optical components, a second set of optical components, a scanning element, and at least one additional optical component. The first set of optical components has a proximal end and a distal end, and the first set of optical components includes a first GRIN objective disposed at the distal end of the first set of optical components. The second set of optical components has a proximal end and a distal end, and the second set of optical components includes a second objective disposed at the distal end of the second set of optical components.
The scanning element is disposed proximally with respect to the proximal end of the first set of optical components and proximally with respect to the proximal end of the second set of optical components. The scanning element is positioned to route a sheet of excitation light so that the sheet of excitation light will pass in a proximal to distal direction through the first set of optical components and project into a sample that is positioned distally beyond the first GRIN objective. The sheet of excitation light is projected into the sample at an oblique angle with respect to an optical axis of the first GRIN objective, and the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element. The first set of optical components routes detection light from the sample in a distal to proximal direction back to the scanning element. The scanning element is also positioned to route the detection light so that the detection light will pass through the second set of optical components in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second objective.
The at least one additional optical component is positioned distally beyond the oblique intermediate image plane. The at least one additional optical component is positioned and configured to (a) route light that arrives at the oblique intermediate image plane towards a camera, and (b) to correct for an angle of the oblique intermediate image plane.
Some embodiments of the third apparatus further comprise a light source that generates a beam of excitation light; and at least one optical component that expands the beam of excitation light into the sheet of excitation light and directs the sheet of excitation light towards the scanning element; and the camera.
In some embodiments of the third apparatus, the second objective is a GRIN objective. Optionally, in these embodiments, the first GRIN objective and the second objective have identical specifications.
In some embodiments of the third apparatus, the at least one additional optical component comprises a tapered bundle of optical fibers having a small end and a large end, and a polymer spacer with a refractive index of 1.33 having a front face and a rear face. The front face is positioned against the second objective and the rear face is positioned against the small end of the tapered bundle of optical fibers.
In some embodiments of the third apparatus, the at least one additional optical component comprises a bundle of optical fibers having a first end that is positioned at the oblique intermediate image plane. Optionally, in these embodiments, the first end is beveled with respect to the optical axis of the bundle of optical fibers.
In some embodiments of the third apparatus, the at least one additional optical component comprises a third objective having an optical axis that is perpendicular to the oblique intermediate image plane.
In some embodiments of the third apparatus, the at least one additional optical component comprises a third objective having an optical axis that is perpendicular to the oblique intermediate image plane. These embodiments further comprise a cured polymer spacer having a refractive index that matches an immersion medium of the third objective. The cured polymer spacer is affixed directly to a front surface of the third objective, and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. The second objective is an air objective and the third objective is a non-air immersion objective.
In some embodiments of the third apparatus, the at least one additional optical component comprises a third objective having an optical axis that is perpendicular to the oblique intermediate image plane. These embodiments further comprise a cured polymer spacer having a refractive index of 1.33. The cured polymer spacer is affixed directly to a front surface of the third objective, and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. The second objective is an air objective and the third objective is a 1.0 NA water immersion objective.
In some embodiments of the third apparatus, the at least one additional optical component comprises a third objective having an optical axis that is perpendicular to the oblique intermediate image plane, and the third objective and an associated optical interface are integrated together into a single package.
Various embodiments are described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements.
This application describes improvements to conventional SCAPE systems that incorporate a gradient index (GRIN) lens. As used herein: O1, O2, and O3 respectively refer to the first, second, and third objectives in a SCAPE system from sample to detector, and NA refers to numerical aperture.
One of optical microscopy's biggest limitations is its limited ability to image deep into scattering tissue. Imaging the deeper layers of mouse cortex at cellular resolution remains a significant problem, and cellular imaging of deep brain regions such as hippocampus, thalamus and striatum is beyond the reach of conventional non-invasive microscopy from the cortical surface.
GRIN lenses are solid glass cylinders that can relay images from their distal to proximal tip. With diameters between 200 and 2000 microns and lengths over 8 mm, they can be carefully inserted into the living brain and provide optical access to almost any brain region with minimal disruption to the function of overlying tissues. GRIN lenses can therefore overcome the penetration depth limitations of prior art cortical imaging methods caused by the scattering properties of the brain, permitting observation of a wide range of deeper brain regions including hippocampus, thalamus, striatum, orbitofrontal cortex and brainstem. The GRIN lens base embodiments described herein can capture the activity and interactions of a wide range of cell types during behavior, including cellular activity patterns in freely moving awake animals.
The embodiments described herein build upon the inventor's development of high-speed 3D swept confocally aligned planar excitation (SCAPE) microscopy to develop GRIN-SCAPE, a technology for high-speed, high-resolution, optionally multispectral 3D imaging of cellular activity through implanted GRIN lenses. GRIN-SCAPE can provide the ability to image in a wide array of brain regions beyond the cortex to capture the 3D dynamics of large populations of multiple cell-types in awake, behaving animals. GRIN-SCAPE can enable the study of circuit dynamics within structures that have widely differing cellular compositions, architectures, dynamics and functions compared to cortex.
The inventor's preliminary data confirms the feasibility of GRIN-SCAPE, and shows that 1-photon excitation is sufficient for well-resolved 3D imaging in-vivo, greatly reducing complexity and improving affordability.
When a GRIN lens is inserted into the brain or other tissue, the GRIN lens relays images of deep cells to the proximal side of the lens, as depicted in
Because GRIN lenses typically have NAs on the order of 0.5, the NAs of the objectives O1 and O2 in a SCAPE system that is being used to capture images through a GRIN lens must be reduced to a similar level, as depicted in
This application describes three approaches for overcoming this problem, and ensuring that the light that exits the second objective O2 is captured. These three approaches are referred to herein as GRIN-SCAPE-1, GRIN-SCAPE-meso, and GRIN-SCAPE-mini.
The
The
The
The GRIN lens 8 and the first set of optical components 10-14 route detection light from the sample in a distal to proximal direction back to the scanning element 50. The scanning element 50 is also positioned to route the detection light so that the detection light will pass through the second set of optical components 20-24 in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second set of optical components (i.e., to the left the second objective 20 in
In the embodiment illustrated in
The
In the illustrated embodiment, the expansion of the beam of excitation light into the sheet of excitation light is accomplished using a Plossl lens 72 and two cylindrical lenses 74, 76, and the sheet of excitation light is directed towards the scanning element 50 by a small mirror 80. But in alternative embodiments, these two functions could be implemented using different components. For example, instead of expanding the beam of excitation light into a sheet of excitation light using lenses, the expansion of the beam into the sheet could be implemented using an additional galvanometer (not shown). And if that additional galvanometer is positioned at the location of the small mirror 80 in
The illustrated embodiment in
When the second objective 20 is an air objective and the third objective 30 is a non-air immersion objective, the associated optical interface 32 can be a fluid chamber positioned so that the oblique intermediate image plane is formed at an interface of an immersion medium of the third objective 30.
Alternatively, when the second objective 20 is an air objective and the third objective 30 is a non-air immersion objective, the associated optical interface 32 can be a cured polymer spacer with a refractive index that matches an immersion medium of the third objective 30. In these embodiments, the cured polymer spacer is affixed directly to a front surface of the third objective 30, and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. For example, the associated optical interface 32 could be a cured polymer spacer having a refractive index of 1.33 that is affixed directly to a front surface of the third objective 30, positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. In these embodiments, the third objective 30 will typically not be coverglass corrected.
As yet another alternative, the third objective and the associated optical interface can be integrated together into a single package, e.g., as in the AMS-AYG v1.0 objective, which has an NA of 1.0, a working distance of 0, and an effective focal length of 5 mm.
The GRIN-SCAPE-1 approach depicted in
Multi-spectral detection is possible using the GRIN-SCAPE-1 approach, e.g., using retro-orbital delivery of dual color viruses. This may be used to leverage new labelling strategies to capture the activity of specific cell types, and interactions between multiple cell types in real time (excitatory/inhibitory neuron, astrocytes, microglia etc.). Being able to do this in any brain region (rather than just cortex) could greatly enhance our understanding of the structure—function relationships between specialized cell types in mammalian brain. If penetration depth into scattering brain is limiting, red and near infrared fluorescent indicators may be used to permit access to much larger fields of view in combination with different GRIN lenses.
We shall now digress to describe the optical theory of how the third objective 30 and the associated optical interface 32 (e.g., the cured polymer spacer in the
Expected angle-dependent, polarization-dependent reflection losses for n1 to n2, air-to-glass vs. air-to-water refractive indices are shown in
Considering O1, O2, and O3 NAs and RIs, and referring to
A benefit to moving to non-air immersion lenses as O2 could be to leverage higher NA from O1. But a 1.1 NA water lens at O1 would generate 55.7° at O2 which could be accommodated by a 40×0.95 NA air at O2 (barring WD constraints). Increasing the NA of O1 and O2 would increase resolution and throughput—but increase oblique angle, reflection losses and, in general, would decrease FOV.
Even for cases where O1 and O2 are low NA (e.g., 0.5 in air) as is the case in the
We have devised an alternative ZWD approach building on seminal work by Yang et al. entitled Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution, Nature methods, 2019; 16 (6):501-4. Yang et al demonstrated that if a 1.0 NA ZWD lens is used as O3, positioned such that the intermediate image plane exactly aligns with the front surface of the lens, almost all incident light can be accepted into the objective lens. This approach leverages the critical angle property of refraction where a beam incident at 90° will bend into a medium of higher refractive index n at an angle of asin(1/n). In water (n=1.33) this angle is 48.8° which naturally corresponds to the acceptance angle of a 1.0 NA water immersion objective lens. Although commercial ZWD objective lenses with solid glass ‘snouts’ are now being manufactured, they have limited fields of view (150/450 um) and cost $15,000/$30,000 respectively. We recently developed a versatile and low cost alternative ZWD approach that simply casts a precise ‘blob’ of optical grade 1.33 refractive index UV curable polymer onto the tip of a standard 2 mm WD 1.0 NA water immersion objective. The resulting lens provides >1 mm fields of view, while increasing system throughput and NA by a factor of 2-3× for high NA systems, and more for low NA configurations that would otherwise be intangible. This simple approach greatly improves the resolution and throughput of conventional SCAPE systems, but is a key component of our GRIN-SCAPE-1 design to enable imaging through low NA GRIN lenses.
We shall now digress further to describe a suitable technique for fabricating the cured polymer spacer and for affixing the cured polymer spacer to the third objective 30.
The spacer has an appropriate refractive index that can be attached to the front of an immersion 1.0 NA lens that is used as the third objective (O3) to convert it to a ZWD lens to maximize detection NA. This has been achieved using a 1.0 NA, 2 mm WD 20× water immersion objective lens and a UV curable polymer with 1.33 refractive index. This lens is not coverglass corrected and thus the spacer was formed as a single unit without a glass coverslip or other material at the focal plane. The material used also has low autofluorescence. Details in
Turning now to
In this third approach, the blob's front surface is cast onto a very flat mirror 95 rather than a coverglass or microscope slide. Dielectric front surface mirrors are manufactured with ultra-flat surfaces—precise to within around a quarter wavelength. It will therefore be flat to a tolerance of less than 250 nm. Not only does this make them ideal for casting an ultra-flat focal plane of the blob, but the fact that the front surface of the blob contacts a mirror is used for the alignment process as detailed below.
Referring now to
The most important steps of the approach depicted in
An excellent way to obtain precise adjustment of the position of the first solid mass 91 is to project collimated light through the objective lens 30 towards the mirror 95, while detecting collimation properties of light reflected by the mirror 95. A determination that the first solid mass 91 has arrived at the final position is made when the light reflected by the mirror 95 is precisely collimated. This can be accomplished, for example, using a shear plate.
A preferred approach for curing of the second quantity 92 of the UV curable polymer is to project UV light through the objective lens 30 into the second quantity of the UV curable polymer. Optionally, subsequent to the projecting of the UV light through the objective lens into the second quantity 92 of the UV curable polymer, additional UV light is applied to further cure the second quantity of the UV curable polymer.
After curing of the second quantity of the UV curable polymer, the mirror 95 is removed from the lower surface of the first solid mass 91 so that the objective lens 30 can be used.
The zero working distance approach can be extended beyond the example provided above. Any type of solid material (or constrained liquid) with appropriate refractive index could be used to modify existing immersion lenses. There exist many UV-curable (or otherwise curable/activatable e.g., via time, heat, radiation or chemical) compounds or glues that have precise refractive indices that could be used to cast permanent extensions to immersion lenses (not just water immersion lenses, and not necessarily 1.0 NA lenses). See, e.g., https://www.mypolymers.com/products. RI matching gels with varying viscosity and minimal evaporation are also available. See, e.g., https://www.cargille.com/optical-gels/ and https://www.thorlabs.com/thorproduct.cfm?partnumber=G608N3.
Another option is to use PDMS (refractive index ˜1.43) in combination with lenses designed for cleared tissue imaging (clarity RI ˜1.4), some of which have correction collars to permit precise matching for the refractive index of the ‘blob’ material. A precision cast spacer could provide a permanent modification to these high NA, long WD lenses to capture more light at O3.
Cover-glass corrected immersion lenses could also be used, with a glass-fronted chamber, as described herein with the space filled with liquid or, e.g., curable polymers. FEP-based front surface water chambers could be used for water immersion dipping lenses without coverglass correction. Certain other plastics or silicone materials could also be made into solid blocks or chambers to match common silicone or oil immersion refractive indices. Multi-immersion and refractive index adjustable lenses could also be employed for ease of material selection.
It is advantageous to add a protecting tube or housing around the completed modified lens, optionally with a removable cap, to protect the ‘blob’ component from dust and other environmental factors which could alter is optical properties, shape or material or optical integrity. This case could take the form of capped chambers depicted (without addition of immersion liquid) enabling adjustment of both lenses for alignment.
The benefits of this ‘blob’ approach on objective lenses such as the NA 1.0 water objective as ZWD O3 include the following: (1) It is a simple and inexpensive modification of a common ˜$6,000 objective lens (e.g., water immersion 1.0 NA lenses). (2) Using a deformable polymer material prevents damage during alignment and can be shaped to accommodate different O2 geometries. (3) The blob can be removed/replaced/refreshed as needed. (4) Large (i.e., >1 mm) fields of view are achievable, with well characterized performance of the O3 lens used. (5) Reduced surface reflection for the air:1.33 interface, compared to high NA glass. (6) The same 100% acceptance angle of light from O2 is achievable. And there is better tolerance of defocus at the image plane owing to smaller refractive index mismatch between air and water compared to air and glass. This is important because remote focus mapping of the oblique plane will generally introduce small curvature of oblique plane. Note, however, that this could be a limiting factor in depth range available for the ZWD approach.
We shall now digress yet again to describe the optical theory of how the GRIN lens 8 that is positioned below the first objective O1 10 operates.
Turning now to
Turning now to
Turning now to
One example embodiment where the GRIN relay is made from two GRIN pieces of different focusing power (equivalently, different NA) is shown. This GRIN relay has a higher-NA top half and a lower-NA bottom half, so that oblique sheet angle will decrease (i.e., be more straight with respect to the optical axis), but the sheet width will increase. The second example shows a reversed case, where the lower-NA half is on the top, so that the sheet angle will increase, while the sheet width shrinks at the output, sample-facing side.
There could also be other optical components, such as a specially designed diffractive optical element or phase mask, sandwiched between the GRIN elements, to help correct optical aberrations such as spherical aberration, or chromatic focal shift, etc. An example GRIN relay that incorporate a diffractive optical element for chromatic correction is shown.
Turning now to
Having completed our digressions, we shall now return to our original train of thought, and will describe additional ways to avoid the situation in which none of the light that exits the second objective O2 is captured by the third objective O3.
The
The
The GRIN lens 8 and the first set of optical components 10-14 route detection light from the sample in a distal to proximal direction back to the scanning element 50. The scanning element 50 is also positioned to route the detection light so that the detection light will pass through the second set of optical components 20-24 in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second set of optical components (i.e., to the left the second objective 20 in
The embodiment illustrated in
A third objective 30 is positioned to accept light that exits the second end of bundle 130 and route the accepted light towards a camera 40. The third objective can have an optical axis that is aligned with the optical axis of the portion of the bundle 130 that is being used. The optical theory of how the GRIN lens 8 operates in this
The
The first end of the bundle 130 can be beveled with respect to the optical axis of the bundle as depicted in
In some preferred implementations of the
The embodiment depicted in
In this
The light that arrives from the outer surface of the brain could be reflected light (i.e., light at the same wavelength of the second light source 85) or fluorescence light (i.e., light at a different wavelength than the second light source 85). In the latter case, the wide-field imaging of the whole cortex can be accomplished with epi-fluorescence (e.g., using jRGECO1a exciting in green and emitting in red), while obtaining a narrower view down the GRIN lens (e.g., using GCaMP exciting in blue and emitting in green).
Note that in the embodiment depicted in
The large field of view of this
The
Note that in the
The
Returning to
The
The
The scanning element 150 is positioned to route a sheet of excitation light so that the sheet of excitation light will pass in a proximal to distal direction through the first set of optical components 110-114, and project into a sample that is positioned distally beyond the first GRIN objective 110. The sheet of excitation light is projected into the sample at an oblique angle with respect to an optical axis of the first GRIN objective 110, and the sheet of excitation light is projected into the sample at a position that varies depending on an orientation of the scanning element 150.
The first set of optical components 110-114 route detection light from the sample in a distal to proximal direction back to the scanning element 150. The scanning element 150 is also positioned to route the detection light so that the detection light will pass through the second set of optical components 120-124 in a proximal to distal direction and form an oblique intermediate image plane at a position that is distally beyond the distal end of the second set of the second objective (i.e., to the left the second GRIN objective 120 in
At least one additional optical component 200-210 is positioned distally beyond the oblique intermediate image plane, and the at least one additional optical component is positioned and configured to (a) route light that arrives at the oblique intermediate image plane towards a camera (not shown), and (b) to correct for an angle of the oblique intermediate image plane.
The excitation beam in this
The pros of this embodiment include: minimal customization, except grinding the taper and customizing three cylindrical/Powell lenses 171-176; excellent O1 NA and collection efficiency; cm-level package size; how much the primary GRIN inserts into brain can be decided when mounting it. Cons of this embodiment include that the sampling density is limited by the taper 205 to be ˜2.8 μm.
A variety of approaches may be used to implement the at least one optical component. For example, in the embodiment illustrated in
In other embodiments, the at least one additional optical component comprises a bundle of optical fibers having a first end that is positioned at the oblique intermediate image plane. Optionally, in these embodiments, the first end is beveled with respect to the optical axis of the bundle of optical fibers.
In other embodiments, the at least one additional optical component comprises a third objective having an optical axis that is perpendicular to the oblique intermediate image plane. Optionally, these embodiments can further comprise a cured polymer spacer having a refractive index that matches an immersion medium of the third objective (e.g., 1.33), wherein the cured polymer spacer is affixed directly to a front surface of the third objective (e.g., a 1.0 NA water immersion objective), and the cured polymer spacer is positioned so that the oblique intermediate image plane is formed on a face of the cured polymer spacer. In these embodiments, the second objective 20 can be an air objective and the third objective can be a non-air immersion objective.
the EFL of the virtual primary objective is
the EFL of the virtual tube lens is
The total length of the GRIN objective is 8.36 mm, so the telescope is roughly a 4f-system (probably designed for telecentric fiber scan). Assuming this will be inserted into the mouse brain, we can then bridge it with a scan lens, MEMS scanner, etc., as shown in
In
One example choice is the micro plano-convex lens Edmunds #49-176 (3.0 mm in diameter), or the small achromat Edmunds #63-714 (4.0 mm in diameter), both VIS-NIR coated and 6 mm EFL.
In the detection arm, another telescope can be added to map the O1 BFL, for simplicity, to O2 BFL with 1:1 ratio, and ƒO2 set to nW·ƒO1≈0.93 mm EFL, working in air. Potential choices include a single Edmunds #43-394 of 1.0 mm EFL (although it is only a plano-convex lens), or a Plossl comprising two Edmunds #65-300 (2.0 mm EFL), or a customized high-NA micro-objective.
The BFL of O1 might not coincide with the front focal plane of the virtual T1 (as the original GRIN objective might not be exactly 4f), so its image formed by the T1-S1-S2-T2 telescope might not overlap with focal plane of T2, but we can simply shift O2 to match it.
Turning now to
For this embodiment and the
Turning now to
Turning now to
The scanner (GM) could be a MEMS mirror (working under either non-resonant or resonant mode), or a micro-polygon driven by some micro-motor, or other miniature mirror system or deflector. The dichroic mirror (DM) is used to couple the excitation sheet. It can be replaced by a micro reflective mirror, or micro-prism with reflective surface, which will occlude the collection aperture a little, but can potentially reduce the footprint.
The third objective O3 here can have zero working distance, realized via customization, or by combining it with a polymer-based “blob” to couple the intermediate image more efficiently (e.g., as described above in connection with the
To illustrate this, the two examples on the left of
To improve the collection NA of GRIN lens, one option is to combine it with a high-index half-ball lens, e.g., a 2.0-mm-diameter S-LAH79 half-ball lens from Edmunds (#90-858). The refractive index of S-LAH79 is n≈2.0, so the EFL of this half-ball lens can be estimated to be R/(n−1) 1.0 mm, then the collection NA approaches 1.0, ideally. In reality, this concept design has severe aberrations. Careful design is preferably used to optimize the performance. For example, the half-ball lens can be split into multiple pieces to gradually bend the beams, and the gradient profile of the GRIN pieces can also be tweaked to have special fourth-order profiles to minimize aberration.
A zero-WD GRIN-based high-NA composite objective is practically feasible, as described in https://doi.org/10.1117/3.934997.ch51 and Barretto, R. P., B. Messerschmidt, and M. J. Schnitzer, (2009), ‘In vivo fluorescence imaging with high-resolution microlenses’, Nat Methods, 6: 511-2., each of which is incorporated herein by reference.
When a MEMS mirror is used as the scanning element in the GRIN-SCAPE-mini embodiments, the MEMS mirror diameter needs to be big enough to match the aperture of the primary GRIN-based objective. Typical commercial products from, for example, Mirrorcle Technologies, can fabricate an integrated mirror 2.4 mm in diameter, and a bond mirror (i.e., a mirror fabricated separately and bond to the scanner later) even bigger. The entire package with a 2-mm-diameter mirror can be smaller than 2.5 cm. MEMS scanner from Mirrorcle are typically 2-axis. Single-axis scanner from Fraunhofer IPMS can be even smaller.
The O2 arm uses the same scan lens (EFL=4 mm), then an Edmunds #84-127 achromat (EFL=3.0 mm). O2 itself should be a customized air/dry objective with 0.9 mm EFL and 0.5 NA, so that the total mag from O1 to O2 will be 0.7/0.16×4/4×0.9/3.0=1.31, ensuring quasi-isotropic magnification. This customized O2 can be made from combining multiple singlets and/or doublets, or combining GRIN lens with refractive lens like shown.
Then O3 can be either 1) a beveled tapered faceplate coupled to a fiber bundle as described before, or 2) say the ˜4.4×0.7-NA O1-tube lens (i.e., the GT-MO-070-016-VISNIR-30-20) bound with a water-equivalent polymer blob to enhance collection. The latter design magnifies the intermediate image, thereby allowing dense sampling even using a fiber bundle or small CCD/CMOS directly behind O3 and tube lens. And O3 can be customized to other magnification and higher NA also to better match the collection device, and to enhance the overall collection efficiency.
The following fabrication methods can be appropriate for the embodiments described herein: Positioning of components can be facilitated by precision machined aluminum casing, or high quality 3D printed casing. Components can be glued in place, positioned by a second system such as a precision alignment translation stage or robotic arm. 2-3 points for fine adjustment may be needed to ensure alignment. If components can be fully encased in index matching UV cure polymer, stability, dust and moisture resistance could be significantly improved. Expensive components can be connectorized to the scanning head.
Finally,
Using a fiber bundle to relay GRIN-SCAPE-mini's images to a higher quality camera does not need to limit the spatial resolution of this system. First, the ZWD and/or fiber taper methods detailed above perform image rotation and would permit the resultant image to be magnified onto a connectorized flexible imaging fiber bundle whose fiber size (e.g., 3.3 micron, 0.4 NA) could be equivalent to >½ the required optical resolution of the system and would thus over-sample. This bundle could then be imaged onto the fast camera, again matching pixel sizes for minimal loss of image integrity. Moreover, SCAPE's image formation captures Y-Z projections of the sample, which can be much smaller in size than the full desired X-Y field of view, with X resolution and field of view determined by galvo scanning range and imaging speed. For example, a 600×600×200 micron volume could be sampled by a rectangular fiber bundle with 200×600 fibers for 1 micron sampling density. Such imaging fiber bundles are very affordable compared to the kinds used for high fidelity endoscopy applications requiring dense sampling over large fields of view.
Optionally, in any of the embodiments described above, adding multi-color imaging to a GRIN SCAPE system provides a major advantage to permit imaging of multiple cell types. This could incorporate strategies for image splitting, spectral unmixing and multiplexed laser illumination. Utilizing red-shifted or near infrared single-photon fluorophores, or two-photon excitation could permit deeper imaging of the tissue at the distal tip of the GRIN lens and thus a larger population of neurons. Note, however, that it may be important to consider chromatic aberration more carefully in GRIN lens systems compared to conventional SCAPE systems.
GRIN-specific improvements include the following: (a) GRIN lenses all introduce aberrations including field curvature. (b) Methods could be used to correct this curvature either in software as post-hoc analysis (after determining the geometric properties of the 3D distortion between the image volume and the object volume). This could utilize a uniform 3D phantom. Or imaging of the same structured shape with and without the GRIN lens in place. (c) Distortion could also be corrected in hardware such as by using a spatial light modulator or DMD to alter the applied and returning wavefronts. This could greatly enhance resolution and uniformity of imaging, although corrections may be needed to adjust with the scanning sheet.
The GRIN systems described herein can incorporate methods for repeatedly aligning small cameras and illuminators to the GRIN lens. These can be 3D printed and can include magnets for secure repositioning. GRIN lenses can be implanted into the brain and secured to the skull. After recovery, based mounts can be glued onto the skull while checking an image to ensure alignment. The imaging part is then removed and a cover used to protect the GRIN lens. A similar approach could be used to make alignment of the mouse under our bench-top GRIN lens systems more repeatable and reliable. A top plate aligned to the SCAPE system could be fixed in place, and made compatible with existing skull-base hardware. For fixation the animal could be clipped into the SCAPE-secured mount. Secondary fixation of the mouse via a skull mounted bar can optionally be used to minimize motion artifacts during imaging.
Head-fixed GRIN-SCAPE embodiments can: a) image awake, behaving mice with existing GRIN-implants (e.g., implants compatible with Inscopix and mini-cam); b) establish performance in phantoms and by comparing data between modalities in the same mice for validation of cell-specific activity pattern extraction; and c) explore extensions to simultaneous GRIN-SCAPE+pan-cortical wide-field, multi-GRIN imaging and parallel optogenetic manipulation.
Multi-color cell type recordings can: a) extend GRIN-SCAPE to multi-spectral recordings of functional indicators in different cell types (astrocytes, interneurons, neurons, microglia, neuronal sub-populations); and b) validate labelling strategies and algorithms for cell-type specific extraction of activity patterns during behavior.
With the growing recognition of cell type diversity throughout the brain, the embodiments described herein go beyond imaging only excitatory neurons, and can be used to capture dynamic interactions between many cell types simultaneously (e.g., inhibitory neurons and astrocytes). To capture these interactions, multi-spectral labelling and imaging with high enough speed and 3D resolution to observe activity in the entirety of cells (rather than just their soma) is helpful. The combined ability to resolve fully 3D activity in multiple cell types, at high speeds, and in any region throughout the mammalian brain in behaving animals could transform our understanding of brain circuits and the functional role of cell types in behavior.
GRIN-SCAPE can provide high fidelity, fully 3D realtime dynamic recordings (e.g., 600×600×250 micron volumes at over 10 VPS) anywhere in the awake, behaving mammalian brain.
The GRIN-SCAPE embodiments described herein provide high-speed, fully 3D, optically sectioned images at the tip of an implanted GRIN lens. This feature would permit simultaneous recordings of larger populations of cells, require less precise placement of the GRIN lens and permit imaging further away from the region of potential damage from the GRIN insertion. The 3D coordinates of each cell will be known, and should unambiguously define its identity to permit tracking of activity of the same cell from session to session (with structural validation using point-scanning 2-photon if desired). By optimizing resolution and simultaneous multi-spectral acquisition, we will add to the types of questions that can be asked using existing techniques by permitting analysis of full 3D activity throughout single cells, and/or intricate dynamic interactions between cells and cell types within a 3D volume. By pairing GRIN-SCAPE with methods capable of labelling specific cell types with different color indicators of cellular activity we will be able to capture local interactions between multiple cell types in real time behavior.
Compatibility of GRIN-SCAPE-1 with head-mounted camera based imaging could permit comparisons between freely moving and head-fixed behaviors and representations. The versatile form of GRIN-SCAPE-Meso systems will can also permit imaginative experiments that combine imaging from multiple brain regions at once to evaluate brain-wide circuits (e.g. pan-cortical wide-field imaging of sensory and motor cortices with simultaneous cellular imaging of striatum in the context of spontaneous movements). This system would also be compatible with a range of optogenetic manipulations including patterned cortical activation and inactivation, GRIN-based exposure of the deep brain region during imaging and holographic single-cell optogenetic activation or silencing during GRIN-SCAPE volumetric imaging. GRIN-SCAPE is useful in mice, as well as GCaMP contrast in the brains of weakly electric fish, Siamese fighting fish and adult salamanders.
While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.
This Application is a continuation of International Application PCT/US2022/029444, filed May 16, 2022, which claims the benefit of U.S. Provisional Applications 63/189,195 (filed May 16, 2021), 63/189,797 (filed May 18, 2021), and 63/190,110 (filed May 18, 2021), each of which is incorporated herein by reference in its entirety.
This invention was made with government support under grants NS108213, NS094296, NS104649, and CA236554 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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63189797 | May 2021 | US | |
63190110 | May 2021 | US | |
63189195 | May 2021 | US |
Number | Date | Country | |
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Parent | PCT/US2022/029444 | May 2022 | US |
Child | 18388947 | US |