Microscope with multiple illumination channels for optogenetic stimulation and fluorescence imaging

1. Field of the Invention

The present invention relates to biological microscopy, and more particularly concerns a microscope probe for in vivo or in vitro optogenetic stimulation and fluorescence imaging through multiple illumination channels.

2. Background of the Invention

When making microscopic observations of in vivo biological specimens, minimally invasive techniques are required. However, conventional microscopes feature large size microscope objectives. Large objectives cannot be implanted within a sample without causing significant damage to the sample, such as removing structures in order to facilitate access to deep layers within the sample. Less invasive fiber-optic cannulae that are typically used in optogenetics experiments are typically configured only for optical stimulation or silencing of a certain class of neurons without imaging capabilities. More recent solutions include miniaturized microscopes that can be detachably coupled to a base plate mounted on a subject, such as those disclosed in U.S. Patent Application Publications U.S. 20120062723 and U.S. 20150309295.

While the ability to perform fluorescence microscopy in in vivo and in vitro configurations is desirable, for optogenetic applications, existing systems are limited in their flexibility of performing optogenetic stimulation.

Therefore, it would be desirable to provide an optogenetic compatible fluorescence microscope.

The invention encompasses microscopes capable of performing optogenetic stimulation and fluorescence imaging on a sample having an attached optical cannula, which is a fixture on the sample having one or more optical probes focusing and/or extending into the sample for delivering light into and receiving light from regions or points within the sample. The invention also encompasses a method of performing optogenetic stimulation and fluorescence imaging on a sample. The optogenetic stimulation and fluorescence imaging can thus be synchronized so that simultaneous or sequential optogenetic stimulation and fluorescence imaging measurements are performed.

In one aspect, the microscope includes a first connector for receiving an optical illumination connection coupling light from two or more illumination sources and a second connector adapted to mechanically connect to the cannula. The second connector includes one or more optical interfaces for coupling light returning from the sample to the microscope and coupling illumination from the illumination sources to the sample. The microscope further includes an optical filter having a dual band-pass transmission spectrum characteristic for filtering the light provided by the illumination sources, so that the transmitting bands correspond to the wavelengths of the illumination sources.

In another aspect, the illumination connection couples light from one or more illumination sources, but to two different regions of the sample via the second connector having separate optical interfaces for coupling light from the at least one illumination source to the different regions of the sample.

The foregoing and other objectives, features, and advantages of the invention will be apparent from the following, more particular, description of the preferred embodiment of the invention, as illustrated in the accompanying drawings.

The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives, and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying drawings, wherein like reference numerals indicate like components, and:

FIG. 1 is a side cross-section view and FIG. 2 is a front cross-section view of an exemplary miniaturized multimodal microscope system.

FIG. 3 is a side cross-section view of another exemplary miniaturized multimodal microscope system.

FIG. 4 is a side cross-section view and FIG. 5 is a front cross-section view of yet another exemplary miniaturized multimodal microscope system.

FIG. 6 is a graph depicting optical transmission band characteristics of filters that may be used within the miniaturized multimodal microscope systems depicted herein.

FIG. 7 is a front cross-section view of still another exemplary miniaturized multimodal microscope system.

FIG. 8 is a top view and FIG. 9 is a side cross-section view of an exemplary cannula for use in the exemplary miniaturized multimodal microscope systems of FIG. 7 and FIGS. 10-12 .

FIG. 10 and FIG. 12 are front cross-section views and FIG. 11 is a side cross-section view of another exemplary miniaturized multimodal microscope system.

The systems described herein are miniaturized multimodal systems of generally less than a cubic inch in size that combine capability for optical stimulation of light sensitive ion channels (optogenetic stimulation) with capability for fluorescence imaging of fluorescent proteins for applications requiring simultaneous or sequential optical stimulation and monitoring of cell activity. The system includes an objective lens to create a magnified image of a region of the sample on an image sensor. The objective lens may be integrated in a microscope or be integrated in a cannula to which the microscope is attached. The microscope generally includes one or more filters to separate illumination light from fluorescence signal and a connection for receiving illumination light from at least two external light sources with an optical arrangement to bring illumination lights to the subject: one source for fluorescence imaging and the other for optogenetic stimulation of the region of interest. Alternatively, or in combination, the cannula and connection to the cannula may include two different illumination paths to either perform fluorescence imaging in one region and optogenetic stimulation in another region or perform both fluorescence imaging in one region and optogenetic stimulation in each region. The miniaturized system is configured to easily connect to and disconnect from the cannula and includes a specific connector adapted to provide precise optical alignment with the cannula, which may be a cannula such as those described in U.S. patent application Ser. No. 14/310,012 entitled “IMAGE RELAYING CANNULA WITH DETACHABLE SELF-ALIGNING CONNECTOR” filed on Jun. 20, 2014, the disclosure of which is incorporated herein by reference. The imaging system is designed to observe a field or fields of view smaller than 0.5 mm 2 (to reduce invasiveness) with a spatial resolution at the micron scale.

Optogenetics experiments use light to control the activity of cells in living tissues, and of particular interest is the modulation of neuronal activity in the brain. To become light sensitive, the cells under study are marked with genetically encoded light sensitive ion channels (e.g., channelrhodopsine, halorhodopsine, etc.). Fluorescence imaging of functional fluorescent proteins (e.g., a genetically encoded calcium indicator) is an optical method used to monitor the activity of cells. The systems described herein are multimodal systems that can combine simultaneous or sequential optogenetics stimulation of light sensitive ion channels with fluorescence imaging of fluorescent proteins in a miniature package of less than a cubic inch in size for applications requiring simultaneous optical stimulation and monitoring of cell activity.

To prevent cross-talk between the different optical modalities in the multimodal optical system (e.g. combination of fluorescence imaging with optogenetic stimulation), the illumination light of each modality must be spectrally and/or spatially distinct. In the systems described herein, the excitation source is generally external and interchangeable so that two or more excitation lights, i.e., illumination sources, can be coupled to the system. The illumination sources are optically coupled to the miniature microscope via a connector. In configurations having a single implant in the sample with a corresponding single optical coupling between the cannula and the microscope, two light sources are used, one for fluorescence imaging and the second for optogenetic stimulation. An included optical filter set is specifically designed to bring the two bands of illumination light (for imaging and stimulation) to the sample and collect the fluorescence signal from the cannula and direct it to an image sensor. In another configuration, the modalities are spatially separated by providing two implants in the sample to target two specific areas, and providing corresponding optical couplings between the cannula and the microscope. The implants may be a relay lens, e.g. a GRIN lens, and/or a fiber optic. The relay lens may be implanted in one area for epi-fluorescence imaging and optogenetic stimulation, and a fiber optic may be implanted for optogenetic stimulation only. Other configurations and combinations such as those disclosed herein or otherwise provided by combining elements that are shown by the present disclosure or their equivalents are contemplated and the examples chosen as illustrations should not be considered limiting. In the examples given herein, the field of view of the microscope described herein is less than 0.5 mm 2 and having a spatial resolution at the micron scale, which also differs from existing miniature microscope systems. By limiting the field of view to less than 0.5 mm 2 , the volume of the microscope can be reduced and the image quality improved over the field of view. To improve the portability and facilitate the access to a confined region of interest, the total size of the miniaturized microscope can be maintained below 1 in 3 with a weight of under 4 g.

Referring now to FIG. 1 and FIG. 2 , an example of a miniaturized microscope system for fluorescence measurement and optogenetic stimulation is shown in a side cross-section view and front cross-section view, respectively. A microscope body 1 A has a connector adapted for connection to a cannula 2 A that is attached to a sample, which is generally a biological test specimen for in vivo measurements as described in the above-incorporated U.S. patent application. The microscope, which includes microscope body 1 A, also includes an optical interface 3 A at the object plane of the optical system, an objective lens 5 A, and a dichroic filter 14 A to separate the returning image from the sample from the illumination pathway so that returning light from the sample is directed toward an image sensor 4 A, such as a CMOS sensor array. The returning light is magnified by 2×-10× between the object plane at optical interface 3 A and image sensor 4 A. A gradient index (GRIN) relay lens 12 A extends into the sample to image a region of interest and to optically excite fluorescent molecules and optically stimulate light-activated ion channels present in the sample in the region of interest. GRIN relay lens 12 A is used to relay an image from deep structures in the sample to the object plane at optical interface 3 A. GRIN relay lens 12 A is integrated in cannula 2 A and is optically aligned with an optical axis 13 of the objective lens 5 A. GRIN relay lens 12 A is not required for imaging surfaces just below cannula 2 A. An illumination pathway 7 extends from a second connector 16 A at the top of microscope body 1 A that receives an optical coupling, such as an optical fiber, from at least two illumination sources that provide light of at least two nominal wavelengths, one for exciting fluorescence in the sample and the other for optogenetically stimulating the sample. An illumination lens system 8 including a high numerical aperture aspheric lens 10 A and a plano-convex spherical lens 11 A forms a collimated beam that is filtered by an excitation filter 9 A which is then collimated at the optical interface 3 A, i.e., at the object plane of the objective lens 5 A, for introduction to GRIN relay lens 12 A in cannula 2 A along optical axis 13 after passing through dichroic filter 14 A. A separate high numerical aperture aspheric lens 10 A and plano-convex spherical lens 11 A are not required, and a single high numerical aperture aspheric lens may be used instead, but using two lenses reduces the angle of incidence of light at the surface of excitation filter 9 A and thereby improves filtering efficiency. Excitation filter 9 A includes at least two optical transmission bands corresponding to the nominal wavelengths of the two illumination sources coupled through connector 16 A, and cleans the illumination light before it is introduced to GRIN relay lens 12 A, removing light artifacts such as auto-fluorescence generation in the optical fibers coupling the illumination light to second connector 16 A. An emission filter 15 A has a transmission band that admits light at the expected fluorescence wavelength of the sample, preventing light from the illumination sources entering image sensor 4 A.

To reduce the number of optical components and thus the size of the microscope system, objective lens 5 A may be a finite conjugate objective lens that generates a magnified image of the object plane at a fixed depth within the sample, so that no tube lens is required between objective lens 5 A and image sensor 4 A. To optimize aberration correction and increase the numerical aperture of the microscope, the exemplary microscope system features high numerical aperture aspheric objective lens 10 A, which in the example has a numerical aperture between 0.3 and 0.5. To reduce the total size of the system, the focal length of objective lens 5 A and any included tube lens are chosen to produce a magnification ratio below 10× between the object plane at optical interface 3 A and image sensor 4 A. In alternative embodiments, a tube lens is inserted in an imaging pathway 6 to adjust magnification and reduce angular divergence at the surface of emission filter 15 A and thereby improve filtering efficiency. To avoid imaging artifacts due to illumination non-uniformity, the microscope system is configured to provide uniform illumination over the total field of view in the object plane at optical interface 3 A. Illumination lens system 8 is configured to image the illumination at the back focal plane of objective lens 5 A, which ensures that defects or dust present at the surface of the fiber optic that is connected to second connector 16 A are not imaged in the object plane, which is especially important when illumination sources and microscope body 1 A may be connected and disconnected many times.

Referring now to FIG. 3 , another example of a microscope system for fluorescence measurement and optogenetic stimulation is shown in a side cross-section view. The microscope system of FIG. 3 differs from the microscope system of FIGS. 1-2 in that the objective lens is integrated in a cannula 2 B and is provided by a high numerical aperture objective lens 5 B. Since the optical path differs from the microscope system of FIGS. 1-2 , a high numerical aperture aspheric lens 10 B, a plano-convex spherical lens 11 B, an excitation filter 9 B, an emission filter 15 B and an image sensor 4 B may be of different dimensions. A dichroic filter 14 B and a microscope body 1 B also generally have differing dimensions from dichroic filter 14 A of FIGS. 1-2 , but otherwise the remainder of the components integrated within microscope body 1 B are similar or identical to and perform the same functions as the components in the microscope system of FIGS. 1-2 . By including high numerical aperture objective lens 5 B in cannula 2 B, the working distance is increased up to 2.5 mm and the image field of view are increased up to 0.5 mm 2 without increasing invasiveness in the subject and no GRIN relay lens is required in the sample.

Referring now to FIG. 4 and FIG. 5 , yet another example of a microscope system for fluorescence measurement and optogenetic stimulation is shown in a side cross-section view and front cross-section view, respectively. The microscope system of FIGS. 4-5 is similar to the microscope system of FIGS. 1-2 , so only differences between them will be described in detail below. The microscope system of FIGS. 4-5 further includes an optical patch cord 17 , e.g., multi-mode optical fiber or fiber bundle that conducts the light of two different illumination wavelengths from the two illumination sources such as those described above with reference to FIGS. 1-2 . A threaded female connector 22 A mates with a male threaded connector 16 B of a microscope body 1 C. A bored recess 19 in microscope body 1 C receives a ferrule 20 that holds the terminated end of optical patch cord 17 , which contacts a hub 21 providing an optical interface receiving the illumination light, i.e., the light for fluorescence excitation combined with the light for optogenetic stimulation, which is directed to an optical interface 3 B at the object plane of an objective lens 5 C for introduction to a GRIN relay lens 12 B in a cannula 2 C. An electrical cable 23 provides the electrical interface to an image sensor 4 C which includes the control and data signals 18 A needed to read image data from image sensor 4 C, as well as power to operate image sensor 4 C. While the illustrated system includes wired connections to image sensor 4 C, in other examples the microscope system may be operated via wireless control using an integrated microcontroller and radio-frequency (RF) transceivers or optical transceivers. Further, the connections to the microscope systems may be made with rotary joints to prevent entanglement and alleviate stress that might otherwise be applied to the interface cables and fiber optics when used on freely moving specimens. A hybrid rotary joint incorporating electrical and fiber optic connections can be provided for this purpose.

Cannula 2 C, includes an orientation key 26 , which provides rotational alignment of cannula 2 C with microscope body 1 C. An interior top surface 28 of cannula 2 C is shaped to adapt cannula 2 C to accept a guiding taper 27 A around the bottom portion of microscope body 1 C and a cylindrical shoulder 24 A contacts a corresponding shoulder 25 A on microscope body 1 C to prevent movement of microscope body 1 C with respect to cannula 2 C once microscope body 1 C is secured to cannula 2 C. A precise optical alignment between objective lens 5 C and GRIN relay lens 12 B is achieved once microscope body 1 C is secured to cannula 2 C. As seen in FIG. 5 , additional components that secure microscope body 1 C to cannula 2 C are shown. A latch hook 31 A expands to latch around a shoulder 25 that defines a slot 32 A around the exterior surface of cannula 2 C and the inner surface of an extension 29 A of latch hook 31 A slides along side walls 37 of microscope body 1 C. Side walls 37 of microscope body 1 C are shaped to form an acute angle with respect to the cylindrical axis of the cannula 2 C, so that, when latch hooks 31 A are moved downwards toward cannula 2 C, side walls 37 act as guides for expanding latch hooks 31 A to release shoulder 25 of cannula 2 C. The upper end of latch hook 31 A forms a retention shoulder 30 A secured by a locking nut 36 threaded onto a male threaded portion 35 of microscope body 1 C, and which compresses a compression spring 33 A that provides for secure latching of latch hooks 31 A by pressing against the lower surface of retention shoulder 30 A and a shoulder 34 on microscope body 1 C to pull latch hooks 31 A upwards. Cannula 2 C and microscope body 1 C are connected by pressing on latch hooks 31 A toward cannula 2 C. Tightening locking nut 36 on microscope body 1 C before pressing on latch hooks 31 A, reduces the force applied on the subject. A pin may be inserted between latch hooks 31 A and cannula 2 C to release latch hooks 31 A from slot 32 A. Cannula 2 C is secured to the specimen being observed, in general, by fastening a flange 38 A of cannula 2 C to the specimen with an adhesive or with fasteners such as screws. The flange 38 A is attached to a body 40 A of cannula 2 C. To adjust the focus and the working distance of the microscope system, cannula 2 C includes a focus adjustment ring 39 A that is glued and/or screwed on the specimen via flange 38 A. In the example, body 40 A of the cannula 2 C includes a male threaded portion on an outer surface. A threaded female inner surface of focus adjustment ring 39 A mates with a threaded male portion of body 40 A and is rotated to adjust the distance between the microscope system and the specimen when focus adjustment ring 39 A is attached to the specimen.

FIG. 6 illustrates the optical band-pass characteristics of filter components in the above-described microscope systems. Excitation filter 9 includes transmission bands for two illumination bands/wavelengths 41 and 42 , which in the example correspond to the wavelength for optogenetic stimulation and fluorescent excitation, respectively. Another transmission band for an emission band/wavelength 43 corresponds to the emission spectrum of the fluorescent sample, and illustrates a suitable band-pass characteristic for emission filters 15 A, 15 B as described above. Further, if the fluorescence emission band/wavelength 43 is between the two illumination bands/wavelengths 41 , 42 , then the band-pass characteristic of dichroic filters 14 A, 14 B in the through direction can be similar (or identical) to the dual transmission band characteristic of excitation filters 9 A, 9 B. Otherwise if the emission band/wavelength 43 is of longer wavelength(s) than the two illumination bands/wavelengths 41 , 42 , then a short-pass characteristic can be used for dichroic filters 14 A, 14 B in the through direction.

Referring now to FIG. 7 , still another example of a microscope system for fluorescence measurement and optogenetic stimulation is shown in a front cross-section view. The microscope system of FIG. 7 differs from the microscope system of FIGS. 1-5 in a cannula 2 D includes two optical probes, one of which is a fiber optic implant 48 for optogenetic stimulation and one a GRIN lens 47 for fluorescence imaging. Cannula 2 D is affixed to the subject with a mounting ring 46 . Since the optical paths differ from those in the microscope systems of FIGS. 1-5 , a high numerical aperture aspheric lens 10 C, a plano-convex spherical lens 11 C, and an excitation filter 9 C will generally be of different dimensions from their corresponding components in microscope systems of FIGS. 1-5 . A dichroic filter 14 C and a microscope body 1 D also generally have differing dimensions, but otherwise the remainder of the components integrated within microscope body 1 D are similar or identical to and perform the same functions as the components in the microscope systems of FIGS. 1-5 . The fluorescence imaging portion of the microscope system of FIG. 7 images a first region 44 within the sample along a fluorescence imaging optical pathway, while the optogenetic stimulation portion stimulates the sample in a second region 45 along an optogenetic stimulation pathway. A dual fiber optical patch cord 49 includes separate fibers/bundles for the optogenetic illumination and fluorescence illumination. Fiber optic patch cord 49 is secured to microscope body 1 D by a threaded cap 22 B that is threaded onto a male threaded connector 16 C of microscope body 1 D. The optogenetic illumination is provided from a fiber terminating at the end of a first ferrule 50 A, which couples the optogenetic stimulation light to a guiding ferrule 53 of an internal optical fiber 55 that conducts light to another guiding ferrule 54 securing a termination end of internal optical fiber 55 to provide the optical interface that abuts a top end of fiber optic optogenetic stimulation implant 48 when microscope body 1 D is secured to cannula 2 D. Microscope body 1 D is secured to cannula 2 D using a releasable latch hook arrangement as described above with reference to FIGS. 4-5 that pulls cannula 2 D against a shoulder 24 B on microscope body 1 D. An interior surface 27 B of cannula 2 D is shaped to mate with the exterior lower surface of microscope body 1 D. For the fluorescence imaging channel, another fiber from dual fiber optical patch cord 49 is inserted in a channel 51 in microscope body 1 D. Optical fiber patch cord 49 terminates at the end of a second ferrule 50 B and couples the fluorescence excitation stimulation light to a hub 52 that conducts light through a lens system as illustrated in FIGS. 1-5 to provide the fluorescence excitation illumination to and receiving fluorescence emissions from an objective lens 5 D. Objective lens 5 D couples the fluorescence excitation and emissions to and from GRIN lens 47 , which couples the excitation to the sample and receives the emissions from the sample.

Referring now to FIG. 8 and FIG. 9 , details of cannula 2 D are shown in a top view and a side cross-section view, respectively. A shoulder 24 B around the top of cannula 2 D and the internal mating wall surfaces have a shape that is semi-cylindrical at each end 56 and a flattened portion 57 formed in the middle of the outline to accommodate both the optogenetic stimulation implant 48 and GRIN lens 47 . A body 40 B of cannula 2 D attaches to the specimen via a focus adjustment ring 39 B that has flattened extensions for adhering to the specimen. Focus adjustment ring 39 B slides along the body 40 B of cannula 2 D to adjust the focus of the microscope system.

Referring now to FIGS. 10-12 , another example of a miniaturized microscope system for fluorescence measurement and optogenetic stimulation is shown. FIG. 11 is a side cross-section view and FIG. 10 and FIG. 12 are front cross-section views of the microscope system. The illustrated microscope system can support up to four channels of measurement, two fluorescence imaging channels and two optogenetic stimulation channels by including a cannula having two relay lenses 12 C, 12 D such as GRIN lenses that can be used to illuminate regions at the ends of relay lenses 12 C, 12 D in the sample at two different wavelengths each, one for optogenetic stimulation and one for fluorescence excitation. Alternatively the microscope system can be used to provide two independent fluorescence imaging channels without optogenetic stimulation. Returning light from regions 58 at the bottom ends of relay lenses 12 C, 12 D and guided to the top ends of relay lenses 12 C, 12 D is captured by a pair of high numerical aperture aspheric objective lenses 5 E, 5 F within a microscope body 1 E and is directed by a pair of dichroic filters 14 D, 14 E through a pair of rhomboid prisms 63 and a pair of emission filters 15 C to a pair of image sensors 4 D. Rhomboid prisms 63 are provided to allow independent optical pathways 60 and 61 to be imaged onto image sensors 4 D separated by a fixed distance and the distance between the images can be controlled by rotating rhomboid prisms 63 . Alternatively, image sensor 4 D may be a single image sensor configured to capture both fields of view from both objective lenses 5 E, 5 F. The illumination light (both the fluorescence excitation wavelength and the optogenetic stimulation wavelength for each channel) is provided by a pair of optical fibers 59 A, 59 B included in a connection cable 62 , which also includes electrical cables 18 B for interfacing to and powering image sensors 4 D. Each channel includes an excitation filter 9 D, 9 E, which may have identical characteristics if the optogenetic stimulation and fluorescence excitation wavelengths are the same for both channels, or the two excitation filters 9 D, 9 E may differ. Both excitation filters 9 D, 9 E will generally have the dual band-pass type of characteristic described above with reference to FIG. 6 and filter the illumination provided through optical fibers 59 A, 59 B and through a pair of high numerical aperture collimation aspheric lenses 10 D, 10 E. (If the system is used for fluorescence imaging only, then the excitation filters 9 D, 9 E require only a single transmission band.) The pair of emission filters 15 C will also generally have characteristics as described above with reference to FIGS. 1-2 . A pair of plano-convex spherical lenses 11 D, 11 E directs the illumination light received from excitation filters 9 D, 9 E and focuses the light at the back focal planes of high numerical aperture aspheric objective lenses 5 E, 5 F, which provide a pair of optical interfaces at the top terminations of relay lenses 12 C, 12 D when microscope body 1 E is coupled to a cannula 2 E.

Cannula 2 E has an interior top surface shaped to adapt cannula 2 E to accept the guiding taper around the bottom portion of microscope body 1 E. Additional components that secure microscope body 1 E to cannula 2 E are shown. A shoulder on cannula 2 E contacts a corresponding shoulder on microscope body 1 E to prevent movement of microscope body 1 E with respect to cannula 2 E once microscope body 1 E is secured to cannula 2 E. A precise optical alignment between the objective lenses 5 E, 5 F and their corresponding relay lenses 12 C, 12 D is obtained once microscope body 1 E is secured to cannula 2 E. A latch hook 31 B expands to latch around a slot 32 B formed around the exterior surface of cannula 2 E and the inner surface of latch hook 31 B slides along a side wall of microscope body 1 E. The upper end of latch hook 31 B forms a retention shoulder 30 B that is loaded by a compression spring 33 B that provides for secure latching of latch hook 31 B by pressing against the lower surface of retention shoulder 30 B to pull latch hook 31 B upwards. Pulling latch hook 31 B upwards also provides a secure three-dimensional alignment of cannula 2 E with microscope body 1 E. A pin may be inserted between latch hook 31 B and cannula 2 E to release latch hook 31 B from slot 32 B.

While the invention has been particularly shown and described with reference to the preferred embodiment thereof, it will be understood by those skilled in the art that the foregoing and other changes in form, and details may be made therein without departing from the spirit and scope of the invention.