Non-invasive imaging system for imaging biological materials

Abstract

Described herein is an imaging device (100) adapted to be incorporated into a device (200) containing biological material (102). The imaging device (100) includes a sample holder (104) configured to hold a sample of the biological material (102). An input (106) is configured to receive a light beam (108). An illumination system (110) is configured to convert the light beam (108) into a two-dimensional light sheet (112) and direct the light sheet (112) onto a targeted illumination zone (114). A transport mechanism (116) is adapted to move the targeted illumination zone (114) relative to the sample holder such that the light sheet (112) illuminates the biological material (102) across the sample. The imaging system (116) is positioned to receive at least a portion of the returning light from the biological material (102) and direct the returning light onto the image sensor (120) to generate multiple images of the sample taken at different positions of the light sheet across the sample.

Description

[Technical Field]

[0001] This application relates to imaging of biological material, and in particular to imaging of live gametes and / or embryos.

[0002] Embodiments of the present invention are particularly adapted for performing non-invasive light sheet fluorescence microscopy on live gametes or embryos, although it will be appreciated that the present invention is applicable to a wider range of applications and to other uses, such as non-fluorescence imaging. [Background technology]

[0003] Assisted reproductive technologies (ART) have evolved over the past 40 years. The success rate of ART can be improved by transferring multiple embryos. However, this technique comes with additional costs and complications related to the increased risk of multiple pregnancies.

[0004] More recently, selecting a single embryo with the highest likelihood of yielding a live birth has become an alternative strategy to improve the success rate of assisted reproduction. Current embryo selection techniques have limited success and / or may cause damage to the embryo itself.

[0005] Therefore, there is a strong need for alternative methods that can accurately assess embryo quality and directly guide the selection process without any potential adverse effects on embryo integrity.

[0006] Selecting the most suitable embryo for implantation is crucial for the success rate of assisted reproductive technologies and the health of the offspring. In addition to morphological assessment using light microscopy, one promising alternative is non-invasive imaging of live embryos to demonstrate metabolic performance. Embryonic metabolism plays a crucial role during early development, as significant metabolic changes occur during the first few days after fertilization.

[0007] However, assessment of the mitochondrial metabolic state of embryos has only been achieved by state-of-the-art fluorescence microscopy, which is costly and challenging, thus limiting its potential deployment in fertility clinics. For example, microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM) and hyperspectral microscopy, have been used to assess embryo viability. However, both FLIM and hyperspectral microscopy are expensive and highly complex to operate, and laser excitation can result in phototoxicity that may impair embryo viability.

[0008] Any discussion of background art throughout this specification should in no way be taken as an admission that such art is widely known or forms part of the common general knowledge in the field. Summary of the Invention

[0009] According to a first aspect of the present invention, there is provided an imaging device adapted to be incorporated into a device for containing biological material, the imaging device including: a sample holder configured to hold a sample of the biological material; an input for receiving a light beam; an illumination system configured to transform the light beam into a two-dimensional light sheet and direct the light sheet onto a target illumination zone; a transport mechanism adapted to move the target illumination zone relative to the sample holder such that the light sheet passes across the sample to illuminate the biological material; and an imaging system arranged to receive at least a portion of the returned light from the biological material and direct the returned light onto an image sensor to generate a plurality of images of the sample taken at different positions of the light sheet across the sample.

[0010] In some embodiments, the biological material comprises one or more gametes or embryos, and the apparatus for culturing the biological material comprises an incubator for culturing the one or more gametes or embryos.

[0011] In some embodiments, the light returned from the biological material comprises fluorescent light emanating from the biological material, hi some embodiments, the light returned from the biological material comprises light emitted by autofluorescence from one or more gametes or embryos.

[0012] In some embodiments, the transport mechanism includes a first actuator adapted to selectively move one or more microlenses in the illumination system such that the target illumination zone moves across the sample holder. In some embodiments, the transport mechanism includes a second actuator adapted to move one or more microlenses in the imaging system in conjunction with the first actuator. The first and / or second actuators can include motorized stages. In some embodiments, the first and second actuators include a single motorized stage configured to move the illumination system and the imaging system together.

[0013] In some embodiments, the sample holder comprises a microfluidic channel and the transport mechanism comprises a microfluidic system configured to move the sample along the microfluidic channel through the illumination zone such that one or more gametes or embryos pass through the light sheet.

[0014] In some embodiments, the transport mechanism includes an actuator configured to move the sample holder so that the one or more gametes or embryos pass through the light sheet.

[0015] In some embodiments, the illumination system and the imaging system are formed in an integral structure.

[0016] In some embodiments, one or more gametes or embryos are unstained.

[0017] In some embodiments, the imaging system has a numerical aperture of 1 or greater.

[0018] In some embodiments, the input comprises an optical fiber.

[0019] In some embodiments, the illumination system is configured to generate the light sheet in a substantially horizontal plane, while in other embodiments the illumination system is configured to generate the light sheet in a substantially vertical plane.

[0020] In some embodiments, the illumination system includes a single cylindrical microlens.

[0021] In some embodiments, the light beam has a wavelength in the range of 400 nm to 850 nm.

[0022] Preferably, the illumination and imaging systems are micro-optical systems formed from components smaller than conventional tabletop optical systems.

[0023] In some embodiments, the imaging system is adapted to generate one or more multispectral images of the sample over a number of different wavelength ranges.

[0024] According to a second aspect of the present invention, there is provided a method of imaging biological material disposed in a device containing the biological material, the method comprising: receiving a light beam from an input; arranging an illumination system to transform the light beam into a two-dimensional light sheet and direct the light sheet onto a target illumination zone; moving the target illumination zone relative to a sample holder holding a sample of the biological material such that the light sheet passes across the sample to illuminate the biological material; and arranging an imaging system to receive at least a portion of the return light from the biological material and direct the return light onto an image sensor to generate a plurality of images of the biological material taken at different positions of the light sheet across the sample.

[0025] Exemplary embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a schematic side view of an imaging device for imaging biological material incorporated into a device containing the biological material. [Figure 2] 1 is a schematic system diagram of an apparatus for containing biological material, including an imaging device. [Figure 3] 2 is a schematic perspective view of the first embodiment of the imaging device of FIG. 1 as seen from above. [Figure 4] 1. FIG. 4 is a schematic perspective view of a second embodiment of the imaging device of FIG. 1, as seen from above. [Figure 5] FIG. 2 is a schematic plan view of a third embodiment of the imaging device of FIG. [Figure 6] FIG. 6 is an enlarged view of the illumination area of ​​the third embodiment of FIG. 5. [Figure 7] 1 is a flow chart showing the main steps of a method for imaging biological material when the biological material is in a device that contains the biological material, such as an incubator. [Figure 8](a) Color photograph showing the setup of the polydimethylsiloxane (PDMS) device. (b) Schematic of the optofluidic device concept showing the attachment of an IVF pipette tip at the inlet of a microchannel. (c) Micrograph of the device showing three two-cell mouse embryos moving from the IVF pipette tip to the microchannel and panel. (d) Micrograph of the device showing two-cell mouse embryos passing through a light sheet. [Figure 9] (a) NAD(P)H autofluorescence images of different cross sections of a two-cell embryo acquired using the optofluidic device of Figure 8. The sequence shows cross sections of a mouse embryo every 6.6 μm (a subset of cross sections collected every 0.45 μm). (b) Three-dimensional image of the NAD(P)H signal reconstructed using the entire image sequence, showing the spatial distribution of NAD(P)H in blastomere 1 and blastomere 2. (c) Maximum internal projection of the entire image sequence. [Figure 10] (a) Schematic of the scanning area of ​​the imaging device in Figure 8. The embryo moves at a constant velocity through the microchannel and intersects with the light sheet. The white lines indicate the flow lines of the microfluidic channel. (b) An experimental image of the superimposed focused laser beam shows the location where the light sheet forms in the enlarged image of the microchannel in the imaging device in Figure 8. The light sheet at the focal point is 1.8 μm thick (FWHM in the y-axis) and 75 μm high (FWHM in the z-axis), and therefore the area of ​​major intensity is 135 μm². (c) A heat map displaying the exposure dose as a function of flow velocity and laser power. Doses above 50 J cm² are indicated by dots. Asterisks indicate the optimal exposure doses used in this experiment, labeled as high dose (16 J cm²) and low dose (8 J cm²). [Figure 11](a) Comparison of the signal-to-noise ratio (SNR) of raw NAD(P)H fluorescence signals between a maximum intensity projection (MIP) image obtained using a confocal fluorescence microscope and an optofluidic device. (b) MIP image captured at a high dose output (16 J cm-2). (c) MIP image captured at a low dose output (8 J cm-2). (d) Comparison of the intensity profile of line L1 shown in (a)-(c) with the intensity profile of the background (bg line). (e) Comparison of the intensity profile of line L2 shown in (a)-(c) with the intensity profile of the background (bg line). (f) Comparison of the intensity profile of line L3 shown in (a)-(c) with the intensity profile of the background (bg line). (g) Comparison of the intensity profile of line L4 shown in (a)-(c) with the intensity profile of the background (bg line). (h) Comparison of the intensity profile of line L5 shown in (a)-(c) with the intensity profile of the background (bg line). [Figure 12] (a) shows a reconstructed 3D image of a blastocyst-stage mouse embryo (control sample) cultured without inhibitor treatment using the microfluidic system shown in Figures 5 and 6. (b) shows a reconstructed 3D image of an early blastocyst-stage mouse embryo (inhibitor sample) cultured with inhibitor (FK866) treatment using the microfluidic system shown in Figures 5 and 6. (c) shows a plot of the intensity distribution of each image in a stack recorded from the autofluorescence signal of a blastocyst-stage embryo without inhibitor treatment (upper curve) and a plot of the intensity distribution of each image in a stack recorded from the autofluorescence signal of an embryo with inhibitor treatment (lower curve). (d) shows a boxplot of the intensity distribution of the control sample and the inhibitor sample. [Figure 13] For the high-dose experiment (16 Jcm-2, n=30), the total number of embryos that reached the blastocyst stage in each condition (control, sham treatment, illuminated) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] Embodiments of the present invention are particularly adapted to imaging biological material in the form of gametes or embryos in a non-invasive environment to enable information to be obtained about the metabolic and genetic integrity of the gametes / embryos, although it will be appreciated that the present invention is also applicable in a broader context to imaging other types of biological material.

[0028] (System Overview) 1, there is shown an imaging device 100 adapted to be incorporated into an apparatus 200 that houses biological material 102. The apparatus 200 is preferably a benchtop or portable incubator apparatus adapted to preserve, hold, and culture the biological material 102 under controlled conditions to prevent damage to the biological material 102. By way of example, if the biological material 102 includes gametes or embryos, the apparatus 200 may be an incubator apparatus configured to incubate or culture the gametes or embryos at a temperature of about 37° C. and under about 5% CO2.

[0029] 2 , device 200 includes various other components in addition to imaging device 100, such as a controller 202, a processor 204, a memory 206, and input / output 208. Controller 202 is adapted to control various elements of device 200, such as the temperature, climate, and movement of elements within imaging device 100, as described below. Processor 204 is adapted to process images captured by imaging device 100, such that a three-dimensional image can be generated from multiple two-dimensional images. Memory 206 is adapted to store data, including image data from imaging device 100 and other data related to culturing biological material. Device 200 also includes input / output components in the form of a user interface (e.g., a touchscreen display), a network port, a power cable, and a wireless network controller (e.g., a Wi-Fi device) for communicating with external devices.

[0030] 1 , the imaging device 100 includes a sample holder 104 configured to hold a sample of biological material 102. The sample holder 104 may be in the form of a microwell, cuvette, or capillary that is either sealed to define an internal sealed environment or has one or more openings that allow the biological material 102 to be at least partially exposed to the environment within the device 200.

[0031] The imaging device 100 also includes an input 106 that receives the light beam 108. The input 106 may be an optical fiber or a fiber optic connector adapted to accept an optical fiber. The optical fiber or other input is adapted to generate or propagate light from a light source, such as a laser, to generate the light beam 108. The light source may have a single narrow linewidth with a center wavelength or may include a broad range of wavelengths. In some embodiments, the light source may include a tunable laser or multiple light sources with different spectral profiles.

[0032] The laser light source preferably emits electromagnetic radiation in the wavelength range of 400 nm to 850 nm, although radiation emissions around 405 nm and 468 nm have been found to be particularly advantageous for non-invasively illuminating embryos to initiate autofluorescence.

[0033] By way of example, a suitable laser operating at 405 nm is a Fabry-Perot fiber-coupled laser source (Thorlabs, Inc., New Jersey, USA, part number: S3FC405), which can be connected to the input 106 in the form of a single-mode optical fiber (Thorlabs, Inc., New Jersey, USA, part number: P1-405B-FC).

[0034] Illumination micro-optics 110 is configured to convert light beam 108 into a thin sheet of light 112 and direct the light sheet 112 toward a target illumination zone 114. Optical system 110 is also referred to as a "micro-optics" because it includes components with dimensions smaller than typical tabletop optics. This includes components such as microlenses and microprisms, which typically have physical dimensions in the range of a few millimeters. However, it should be understood that larger components can also be used in micro-optics 110, in which case micro-optics 110 can be referred to as a conventional imaging system.

[0035] The light sheet 112 is formed by focusing the light beam in only one dimension with a cylindrical lens or similar optical element, producing a highly elliptical light beam profile. The light sheet 112 has a thin, focused axial thickness (typically on the order of nanometers or microns) and is used to illuminate a thin slice of the sample.

[0036] The transport mechanism 116 is adapted to move the target illumination zone 114 relative to the sample holder 104 so that the light sheet 112 passes across the sample and illuminates the biological material. The term "relatively" is used to mean that the target illumination zone 114 and / or the sample holder 104 are movable relative to one another. In the embodiment shown in FIG. 1 , the target illumination zone 114 moves while the sample holder 104 and the biological material 102 are held stationary. This has the advantage of reducing the chance of the biological material 102 being damaged during movement. In other embodiments, the sample holder 104 moves while the target illumination zone 114 is held stationary.

[0037] The imaging micro-optics 118 are positioned to receive at least a portion of the light returning from the biological material and direct the returning light onto the image sensor 120 to generate multiple images of the sample. These images are obtained at different positions of the light sheet moving across the sample. Depending on the biological material 102 being imaged and the particular application, the returning light may represent reflected light, backscattered light, fluorescence, or autofluorescence from the sample. The optical system 118 is also referred to as "micro-optics" because it includes components with dimensions smaller than typical benchtop optical systems. This includes components such as microlenses and microprisms, which typically have physical dimensions in the range of a few millimeters. However, it should be understood that components with larger dimensions may also be used in the micro-optics 118.

[0038] Referring to FIG. 3, a first embodiment of the imaging system 100A is shown. Corresponding features from the imaging system 100 of FIG. 1 are designated with like reference numerals. In the imaging system 100A, the transport mechanism 116 includes an actuator (not shown) configured to selectively adjust the position of a motion stage 122. The actuator may include a mechanical or motorized device, such as a screw-type actuator, or may include a piezoelectric device. The motion stage 122 is adapted to selectively vertically move the cylindrical microlenses 124 within the illumination micro-optics 110, along with the imaging micro-optics 118, the input section 106, and the image sensor 120. This causes the target illumination zone 114 and the light sheet 112 to move vertically across the sample holder 104 and the biological sample 102. The light sheet 112 generated by the cylindrical microlenses 124 is substantially horizontally flat so as to illuminate a horizontal slice of the biological sample 102. The vertical thickness of the light sheet 112 preferably ranges from a few hundred nanometers to a few microns. During this movement, at each vertical position of the motion stage 122, a portion of the returning light from the biological sample 102 is guided along an imaging optical path through the imaging micro-optics 118 and imaged onto the sensor array 126 of the image sensor 120. In other embodiments, the light sheet 112 generated by the cylindrical microlenses 124 is substantially vertically flat so that it can illuminate a vertical slice of the biological sample 102.

[0039] In imaging system 100A, imaging micro-optics 118 includes a series of lenses 118A-118E, similar to a microscope objective, for shaping the returning light and focusing it onto sensor array 126. However, it should be understood that imaging micro-optics may include other numbers and configurations of optical elements, such as lenses, mirrors, and prisms. Furthermore, in imaging system 100A, illumination micro-optics 110, imaging micro-optics 118, and input section 106 are each mounted on a motion stage 122 and can move vertically together when the position of motion stage 122 is adjusted. Motion stage 122 can be controlled by incubator controller 202 (see FIG. 2 ) of apparatus 200 or by a separate controller. Vertical movement of motion stage 122 facilitates imaging of horizontal slices of biological material 102 at each stage position.

[0040] The resulting stack of two-dimensional images can be combined by processor 204 to generate one or more three-dimensional images of the biological material, such as a fluorescence image in the case of an autofluorescent material. The thickness of light sheet 112 and the relative velocity of sample holder 104 with respect to light sheet 112 determine, at least in part, the resolution of the resulting images. Other factors, such as the numerical aperture of the detection objective lens, may also dictate the image resolution.

[0041] In some embodiments, multiple light sources or tunable light sources can be coupled via input 106 to obtain multispectral images by making multiple passes of light sheet 112 through biological material 102 during a single imaging session. Alternatively, multiple wavelengths of light can be simultaneously superimposed on illumination zone 114 at any one time, allowing a multispectral image to be generated from a single pass of light sheet 112 through biological material 102.

[0042] In imaging system 100A, the illumination optical path, including input 106 and cylindrical microlens 124, is positioned orthogonally to the imaging optical path, including imaging micro-optics 118 and image sensor 120. This is a configuration to improve the signal-to-noise ratio in a light sheet fluorescence microscope (LSFM) system. If biological material 102 contains one or more gametes or embryos, illumination with a wavelength of, for example, 405 nm can induce autofluorescence, and a portion of the light from this process can be directed through imaging micro-optics 118 and imaged by image sensor 120 to generate a fluorescence image of the sample.

[0043] In imaging system 100A, input section 106, illumination cylindrical micro-lens 124 and imaging micro-optics 118, and image sensor 120 each move in conjunction with motion stage 122, although it should be understood that other configurations are possible. In some embodiments, only a subset of these components move during imaging. By way of example, in one embodiment, the transport mechanism includes an actuator adapted to move illumination micro-optics 110 and / or one or more micro-lenses in input section 106 without moving imaging micro-optics 118 or image sensor 120. In other embodiments, two separate actuators are used, one to selectively move illumination micro-optics 110 and input section 106, and the other to selectively move imaging micro-optics 118 and image sensor 120.

[0044] FIG. 4 illustrates another embodiment of imaging system 100B, which operates similarly to imaging system 100A, but with its components oriented vertically. In particular, input 106 is positioned substantially vertically and directs light beam 108 vertically upward through cylindrical illumination microlens 124 to generate a substantially vertical light sheet 112. Motion stage 122 is configured for horizontal sliding movement, thereby allowing input 106, microlens 124, imaging micro-optics 118, and image sensor 120 (each mounted on motion stage 122) to move relative to one another. This horizontal movement allows substantially vertical light sheet 112 to be scanned sequentially across multiple sample holders in the form of microwells 104A-104C, each containing a respective biological sample in the form of an embryo 102A-102C. This configuration of imaging system 100B allows multiple biological samples to be imaged without manual intervention by an operator.

[0045] In a further embodiment (not shown), the transport mechanism 116 includes one or more actuators for moving the sample holder so that the biological material 102 (e.g., one or more gametes or embryos) passes horizontally through the light sheet 112.

[0046] 5 and 6, another embodiment of the imaging system 100C is shown that incorporates a microfluidic channel for moving the biological material 102 while the illumination micro-optics 110 and the imaging micro-optics 118 remain fixed. In this embodiment, the sample holder 104 includes a microfluidic channel 130, and the transport mechanism 116 includes a microfluidic system 132. The microfluidic system 132 is configured to move the fluid sample along the microfluidic channel 130 from an input 134, through the illumination zone 114, to an output 136, thereby causing one or more gametes or embryos to pass through the light sheet 112.

[0047] 6 shows a close-up view of imaging system 100C near illumination zone 114. Exemplary dimensions and characteristics are shown. The microchannel has a width of approximately 120 μm, and cylindrical microlenses 124 generate a light sheet of 1.8 μm to 3 μm at the center of microchannel 130. In this embodiment, the light sheet has a thickness of 114 μm.

[0048] The illumination micro-optics system 110 includes a single cylindrical microlens 124. As shown, the microchannel 130 includes a corner 138 where imaging occurs. The corner 138 is designed with a sharp, protruding corner configuration to avoid optical aberrations due to index reflections miss matching. Furthermore, the inlet 134 and outlet 134 are oriented horizontally to allow for the integration of an IVF micropipette tip into the port for continuous sample tracking and safe sample recovery as the sample travels through the microchannel 130.

[0049] 5 and 6, the components forming the illumination micro-optics 110, the imaging micro-optics 118, and the microfluidic system 132 can be etched as a single unit from a single substrate material. The integrated micro-optical components are pre-aligned with the microfluidic channel 130 used to deliver the sample. The micro-optical components are cast directly in polydimethylsiloxane (PDMS).

[0050] Micro-optical components typically cause high aberrations and low numerical apertures. The imaging system 100C overcomes these challenges by combining micro-optical elements with a microfluidic system that allows samples to be manipulated in a self-aligned fashion without the need for moving parts or alignment, while maintaining the distance between all components in the microscopic range. The imaging system is efficient enough to also image unstained samples, which generate autofluorescence signals orders of magnitude lower than stained samples.

[0051] In the embodiment described above, both the illumination micro-optics 110 and the imaging micro-optics 118 are formed from a monolithic structure, such as a PDMS substrate.

[0052] In each of the above embodiments, the imaging micro-optics 118 is capable of generating a numerical aperture of 1 or greater, allowing for efficient coupling for imaging unstained gametes or embryos. In some embodiments, the micro-optics structure is monolithic, self-aligning, and capable of generating a light sheet narrow enough to be usable with objectives with NAs of 1.05 or greater.

[0053] Referring to FIG. 7 , the above-described system and device are adapted to perform a method 700 for imaging biological material 102 contained in a storage device. The method includes, in step 701, receiving a light beam from input 106. In step 702, illumination micro-optics 110 are configured to convert light beam 108 into a two-dimensional light sheet 112 and direct light sheet 112 onto a targeted illumination zone 114. In step 703, the targeted illumination zone 114 is moved relative to a sample holder 104 holding a sample of the biological material 102 so that light sheet 112 passes across the sample, illuminating the biological material 102. In step 704, imaging micro-optics 118 are configured to receive at least a portion of the returned light from the biological material and direct the returned light onto image sensor 120 to generate multiple images of the biological material 102 taken at different positions of light sheet 112 across the sample. [Example]

[0054] Example - Microfluidic Embryo Delivery An embodiment of the present invention is described below, which uses a microfluidic system to move two-cell stage mouse embryos through an imaging system in a manner similar to that shown in Figures 5 and 6. An imaging system 800 is shown schematically in Figure 8. Panel (a) is a color photograph showing the device configuration made from PDMS, panel (b) is a conceptual diagram of the optofluidic device showing the attachment of an IVF pipette tip at the inlet of a microchannel, panel (c) is a micrograph of the device showing three two-cell stage mouse embryos moving from the IVF pipette tip into the microchannel, and panel (d) is a micrograph of the device showing two-cell stage mouse embryos passing through a light sheet.

[0055] The imaging device 800 is a scalable, high-performance optofluidic device that can capture 3D images of nicotinamide adenine dinucleotide phosphate (NAD(P)H) signals in live, early-stage mouse embryos using LSFM. This optofluidic technique provides a high signal-to-noise ratio (SNR) by using a low light dose at an excitation wavelength of 405 nm. The device 800 provides a well-designed fluidic environment for safe handling of mouse embryos as they enter and exit the light sheet generated on the chip at the center of the microchannel. The non-invasiveness of this method is demonstrated by assessing the viability and development of illuminated embryos compared with non-illuminated embryos. This optofluidic method offers a promising opportunity to analyze embryo quality in real time without inducing phototoxicity or embryo damage, potentially achieving improved reproductive outcomes in fertility clinics.

[0056] The imager 800 and Petri dish are attached to a Peltier module at the outlet to maintain both the imager and the Petri dish at approximately 37°C. A heating incandescent lamp (not shown in Figure 8) was also implemented to maintain the system at 37°C. Imaging of NAD(P)H is performed via LSFM, as shown in Figure 8(b). Light sheet formation is performed on-chip using microlenses and optical fibers, and recording of the fluorescence signal is performed off-chip using an objective lens.

[0057] The optical system of the imaging device 800 was designed to acquire luminescence for NAD(P)H measurement using a detection objective lens with a numerical aperture of 1.05 (Olympus Corporation, Tokyo, Japan, part number: UPLSAPO30XS). This objective lens was equipped with a bandpass filter for blue fluorescent protein [430-490 nm] (Thorlabs, New Jersey, USA, part number: MF460-60), an infinity-corrected tube lens (Thorlabs, New Jersey, USA, part number: TTL180-A), and a CMOS camera (Basler AG, Ahrensburg, Germany, product number: acA1920-155um - Basler ace). The optical system was mounted on an XYZ translation stage (Thorlabs, NJ, USA, part number: T1220D) positioned using a rail system (Qioptiq, Rhyl, UK, X95 Profile System) on an XYZ translation platform (Thorlabs, NJ, USA, part number: T1220D). The camera sensor was set with horizontal and vertical binning factors of 2, resulting in a final pixel size of 0.39 μm for the optical detection system.

[0058] A top-view observation system was used to position the optical fiber within the device and identify the mouse embryo's position as it moved through the device. The optical system consisted of a dry, long-working-distance 5x objective (Thorlabs, NJ, USA, part number MY5X-802), a 160 mm fixed tube lens (EHD Imaging GmbH, Damme, Germany, part number FT160), an LED light source (EHD Imaging GmbH, Damme, Germany, part number IL100), and a CMOS camera (Basler AG, Ahrensburg, Germany, part number acA1920-155um - Basler ace). A fluorescence filter (Thorlabs, NJ, USA, part number MF535-22) was placed within the LED light source to block optical noise from entering the detection objective. The optical system was assembled on another XYZ stage (Thorlabs, New Jersey, USA, part number: T1220D), which in turn was mounted on a similar rail system (Qioptiq, Rhyl, UK, X95 Profile System) mounted on the same optical table used in Section 4.1.1 of this paper. The final pixel size of this top-view microscope was 1.465 μm.

[0059] The optofluidic device was fabricated from PDMS by single-step UV lithography. This created smooth, mirror-like, nearly vertical internal sidewalls, enabling the device to handle live, two-cell stage mouse embryos for the purpose of acquiring 3D images of the autofluorescent NAD(P)H signal. The design of the imaging device 800 was adapted from our previous work (see Reference 3) to safely image early-stage mouse embryos. Specifically, the inlet and outlet were redesigned to accommodate in vitro fertilization (IVF) pipette tips (see Figures 8(a) and (b)) integrated into the PDMS for easy sample handling. This feature allowed for continuous tracking of the embryo's position within the chip with a top-view camera and also facilitated its recovery after imaging (see Figures 8(c) and 8(d)). Furthermore, the system was maintained at 37°C throughout the imaging period to provide a physiologically relevant environment. After imaging, all embryos were recovered and assessed for viability, developmental status, and quality.

[0060] A low-pressure syringe pump (Cetoni, Kolbussen, Germany, product number NEM-B101-03A) was used in low-flow mode with a 500-μL glass syringe with a PEEK tubing connector (SETonic GmbH, Ilmenau, Germany, part number 3010236). To connect an IVF pipette tip (MXL3-125, The Stripper®, CooperSurgical Fertility Solutions, Denmark) to the syringe for embryo loading, a 0.5 mm ID, 1.3 mm OD rubber tubing adapter (Gecko Optical Scientific Equipment, Western Australia, Australia, part number 3100504) was connected to a 0.012-inch ID, 0.030-inch OD PTFE tubing (John Morris Group, Victoria, Australia, product number 06417-11) to assemble the pipette tip and syringe. An integrated incandescent heating lamp (Philips, industrial infrared heating incandescent lamp PAR38 IR100W 240V Red E27) was carefully positioned to maintain the temperature at 37°C to keep the medium in the syringe warm.

[0061] To load embryos from tissue culture plates into the optofluidic device, a pipette tip was attached to a syringe, and the flow rate was controlled by a syringe pump. The pipette was manually placed on the culture plate, and embryos were carefully selected using a USB microscope (ViTiny®, Microlinks Technology Corp., Taiwan, part number: UM12).

[0062] NAD(P)H autofluorescence excitation was achieved by exposure to a 405 nm light sheet. A 405 nm Fabry-Perot fiber-coupled laser source (Thorlabs, NJ, USA, part number: S3FC405) was connected to a single-mode optical fiber (Thorlabs, NJ, USA, part number: P1-405B-FC). The second end of the optical fiber was cleaved using a fiber cleaver (Thorlabs, NJ, USA, part number: XL411). To carefully position the cleaved fiber tip into the optofluidic device, the fiber was placed in a tapered V-groove fiber holder (Thorlabs, NJ, USA, part number: HFV002) on a three-axis manual stage (Thorlabs, NJ, USA, part number: MAX313D / M).

[0063] A 405 nm laser beam was emitted from an optical fiber and focused by a set of cylindrical microlenses to generate a light sheet spanning the entire channel width, such that the laser power was distributed across each cross section of the light sheet (the yz plane in Figure 10(b)).

[0064] The dimensions of the light sheet are 1.8 μm thick (FWHM in the y-axis) and 75 μm high (FWHM in the z-axis), so the area of ​​the main light intensity region is 135 μm 2 (See Figure 10(b)). Using this, the laser density distribution in the light sheet was calculated for different laser powers, resulting in the laser dose to which the embryo would be exposed as it traversed the light sheet at different velocities. The heat map in Figure 10(c) shows the exposure dose as a function of laser power and embryo velocity. To minimize the possibility of photodamage, a laser power of 50 J cm was used. -2 Two doses (one-third and one-sixth) significantly lower than the standard dose were selected, yet still achieved high signal-to-noise ratios (SNRs) and high-quality imaging. These were used to estimate embryo movement speeds of 30 μm s -1 The dose was fixed at 16 J / cm. -2 (High dose) 0.36mW, dose 8Jcm -2This was achieved by changing only the laser power so that it was 0.18 mW for (low dose).

[0065] Avoiding light in the ultraviolet region (100–400 nm) reduces the risk of embryo damage during illumination, but reduces the efficiency of fluorescence excitation at the optimal excitation wavelength of 340 nm. To overcome this drawback, a high-NA silicone immersion microscope objective (NA = 1.05) was used to increase the amount of captured fluorescence. Furthermore, the objective's 800 μm working distance requires the microfluidic channels to be positioned close to the edge of the chip, but these placements were carefully designed to avoid silicone oil dripping. Furthermore, silicone immersion oil (refractive index RI = 1.40) was used to reduce spherical aberration by filling the refractive index mismatch between the cells (intracellular RI = 1.38) and the PDMS (RI = 1.41 at a 5:1 PDMS ratio).

[0066] Because high-NA objectives capture light at a wide angle, the geometry of the microchannel corners where imaging takes place was designed with sharp protrusions (see Figure 8(d)) to avoid lateral changes in refractive index before the light is focused, thereby preventing optical aberrations. To avoid affecting the quality of the acquired images, a factor to consider is that when loading multiple embryos, each embryo needs to be spaced at least 200 μm (the length of two embryos). Otherwise, the upstream embryo (t3 in Figure 8(d)) will cause aberrations with respect to the next embryo (t2 in Figure 8(d)). This is because the fluorescence emitted from embryo t2 passes through the upstream embryo t3 before its image is captured.

[0067] These improvements combined resulted in high-contrast fluorescent images of the embryo, as shown in Figure 9. The embryo was transported through the light sheet by flowing fluid along the channel, while images were recorded, resulting in a stack of cross-sectional images. By using a top-view camera (see Figure 8(a)) to record the speed at which the embryo passed through the light sheet, the collected image stacks could be used to reconstruct a volumetric image.

[0068] Compared to prior art systems, the imaging device 800 configuration in Figure 8 allows for more versatile recording of microscope-grade image quality (i.e., high spatial resolution and high SNR) when using high-NA objectives (NA>1). This is because the physical constraints imposed by the orthogonal geometry are eliminated, allowing the use of objectives with a wider range of working distances. Single-objective LSFM is achieved by eliminating the excitation objective and using a micromirror to reflect and focus the light sheet at the center of the microchannel.

[0069] When a two-cell embryo passes through the light sheet (see Figure 8(c)), biomolecules in its two blastomeres are excited by photons from a 405 nm laser light source, causing autofluorescence signals to be emitted from NADH and NADPH in its mitochondria and cytoplasm (see Figure 9). However, because the signal intensity of NADH is several times stronger than that of NADPH, we follow the convention that this fluorescence signal comes only from NAD(P)H biomolecules. The NAD(P)H autofluorescence signal originates from two different sources in the embryo. The blurred areas in Figure 9(c) belong to cytoplasmic NAD(P)H, while the clear areas belong to mitochondrial NAD(P)H. Because NAD(P)H is highly concentrated in mitochondria compared to the cytoplasm, the autofluorescence signal intensity of mitochondrial NAD(P)H is greater.

[0070] To reduce the risk of phototoxicity, 3D images of two-cell embryos were acquired in less than 2 seconds. The specimen was then scanned through a light sheet in a microchannel for approximately 30 μm seconds. -1 Crossing at a constant speed of 0.01-0.02 μL min -1 The embryos were transported at a low flow rate. If the flow rate fluctuates, for example due to the presence of air bubbles, axial sampling is affected. The optimal velocity (30 μm s -1 ) results in a 5% reduction in the number of cross-sectional images, but the impact on image quality is negligible.

[0071] The thickness of the cross-sectional image is defined by the thickness of the light sheet, and the axial resolution depends only on the NA of the detection objective. In the optofluidic system described herein, the light sheet thickness is 1.8 μm at FWHM, resulting in an axial resolution of 1.04 μm (NA of the detection objective = 1.05). It is known that a light sheet thicker than the axial resolution reduces image contrast but leads to improved axial resolution. In this case, the theoretical axial resolution was improved by only 20%. This resulted in high-contrast fluorescence images every 0.45 μm at 66.67 frames per second. Importantly, by operating the system at this speed and frame rate, undersampling in 3D imaging of mouse embryos was avoided.

[0072] The full-stack image generated by a two-cell embryo was converted into a volumetric image (see Figure 9(b)). Notably, NAD(P)H is present in high concentrations in the mitochondria and cytoplasm, and 3D imaging of the NAD(P)H signal revealed normal spatial distribution of mitochondria among the embryonic blastomeres, as demonstrated by similar techniques such as FLIM. Furthermore, recognizing the live spatial distribution of NAD(P)H within the embryo during embryo development allows us to further understand its relationship with conventional embryo morphology and distinguish between normal and abnormal mitochondrial distribution at various stages of early embryonic development. Incorporating NAD(P)H signal as a determinant in clinical workflows may open new avenues for selecting the highest-quality embryos based on their metabolic activity and increasing the chances of clinical pregnancy and live birth in IVF patients.

[0073] The tradeoff of using a low excitation dose is a lower intensity image. Therefore, the image quality was evaluated by SNR, which was compared with NAD(P)H images obtained using confocal microscopy. Maximum intensity projection (MIP) images are shown in Figure 11(a)–(c). To compare image quality, intensity profiles were acquired on five lines (L1–L5) evenly distributed throughout the embryo image. These intensity profiles were compared with the background (bg) intensity profile in each image (Figure 4d–h). SNR was calculated by dividing the difference in mean intensity between the line profiles and the background profile by the standard deviation.

[0074] On average, the SNR at low power for the imager 800 was 24.5 times higher than that obtained with confocal microscopy (CFM) (see FIG. 11(i) , p<0.00001; t-test). Meanwhile, at high power, the SNR was 34 times higher (see FIG. 4(i) , p<0.00001; t-test). These results demonstrate that the imager 800 is capable of detecting NAD(P)H autofluorescence signals even when exciting below doses known to cause damage, with SNR and overall image quality superior to images obtained using conventional confocal microscopy.

[0075] In one experiment, a total of 34 embryos were cultured from the 2-cell stage to the blastocyst stage in an off-chip incubator for 3.5 days. Half of the embryos were treated with FK866 to inhibit metabolic activity, which resulted in a 47% reduction in autofluorescence signal compared to untreated embryos. The results of this experiment are shown in Figure 12. Figure 12(a) is a reconstructed 3D image of a blastocyst-stage mouse embryo cultured without inhibitor treatment (control sample). This 3D image shows the spatial distribution of NAD(P)H and was reconstructed using the full image sequence.

[0076] Figure 12(b) shows a reconstructed 3D image of an early blastocyst stage mouse embryo cultured with inhibitor (FK866) treatment (inhibitor sample). This 3D image shows the spatial distribution of NAD(P)H and was reconstructed using the entire image sequence.

[0077] Figure 12(c) shows a plot of the intensity distribution for each image in a stack (60 images in total) recorded from the autofluorescence signal of blastocyst-stage embryos without inhibitor treatment (upper curve) and with inhibitor treatment (lower curve). The bold line represents the mean intensity, and the gray band indicates the range of intensity values ​​for each sample. The control group showed a 47% higher NAD(P)H autofluorescence signal than the corresponding inhibitor-treated sample.

[0078] Figure 12(d) shows the boxplot of the intensity distribution of the control and inhibitor samples, and the difference is statistically significant (p<0.0001; t-test). The results in Figure 12 demonstrate that the use of the optofluidic device described above is effective for imaging NAD(P)H autofluorescence and assessing the metabolic activity of embryos.

[0079] Finally, embryo survival was investigated. Embryos exposed at low and high dose output settings were collected and cultured to the blastocyst stage to analyze survival, development, and quality after exposure. Figure 13 shows the results for the high dose setting (16 Jcm). -2 The total number of embryos that reached the blastocyst stage in each condition (control, sham, illuminated) is shown for one experiment (n=30).

[0080] To the best of our knowledge, no optofluidic device has been developed to date that allows live mouse embryos to be safely manipulated, loaded into the device, imaged, and then retrieved to assess embryo viability.

[0081] The above invention is adapted for use in non-invasively monitoring live gametes and early embryos, specifically by generating and detecting embryonic autofluorescence without damaging the embryo.

[0082] The system is simple and compact, can be integrated into a conventional IVF incubator, and is capable of imaging embryos / gametes layer by layer in a time-lapse manner. At the same time, compared to conventional macro-optical imaging devices, the disclosed micro-imaging configuration potentially reduces the overall system cost by an order of magnitude.

[0083] (References) Below is a list of references, the contents of which are incorporated herein by cross-reference. 1. Sanchez T, Zhang M, Needleman D, Seli E: “Metabolic imaging via fluorescence lifetime imaging microscopy for egg and embryo assessment”;Fertil Steril 111, 212-218(2019). 2. McLennan HJ, Saini A, Dunning KR, Thompson JG: “Oocyte and embryo evaluation by AI and multi-spectral auto-fluorescence imaging: Livestock embryology needs to catch-up to clinical practice”;Theriogenology 150, 255-262(2020). 3. Vargas-Ordaz EJ, et al.: “Three-dimensional imaging on a chip using optofluidics light-sheet fluorescence microscopy”;Lab Chip 21, 2945-2954(2021). 4. Memeo R, et al.: “Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip”;J Biophotonics 14, e202000396(2021). 5. Sala F, et al.: “High-throughput 3D imaging of single cells with light-sheet fluorescence microscopy on chip”;Biomed Opt Express 11, 4397-4407(2020).

[0084] (interpretation) Unless otherwise specified, and as will be apparent from the discussion that follows, discussions throughout this specification using terms such as "processing," "operating," "calculating," "determining," "analyzing," and the like, are understood to refer to operations and / or processes of a computer or computing system or equivalent electronic computing device that manipulates and / or transforms data expressed as physical quantities, such as electronic quantities, into other data also expressed as physical quantities.

[0085] Similarly, the terms "controller" or "processor" can refer to any device or part of a device that processes electronic data from, for example, registers and / or memory and converts the electronic data into other electronic data that can be stored, for example, in registers and / or memory. A "computer" or "calculating machine" or "computing platform" can include one or more processors.

[0086] References throughout this specification to "one embodiment," "some embodiments," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment," "some embodiments," or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure.

[0087] As used herein, unless otherwise specified, the use of ordinal adjectives such as "first," "second," "third," etc. to describe a common object merely indicates that different instances of a similar object are being referred to and does not imply that the objects so described must be in a particular order in time, space, position, or in any other way.

[0088] In the following claims, as well as in the description herein, the terms "comprising," "consisting of," and "including" are all open terms, meaning the inclusion of at least the elements / features that follow, but not the exclusion of others. Thus, when used in a claim, the term "comprising" should not be interpreted as being limited to the means, elements, or steps listed thereafter. For example, the scope of the expression "a device comprising A and B" should not be limited to a device consisting only of elements A and B. As used herein, the terms "comprises," "includes," or "having" are also open terms, meaning the inclusion of at least the elements / features that follow, but not the exclusion of others. Thus, "comprising" is synonymous with "comprising" and has a similar meaning.

[0089] In the foregoing description of exemplary embodiments of the present disclosure, it should be understood that various features of the disclosure may be grouped together in a single embodiment, drawing, or description for the purpose of brevity and to facilitate an understanding of one or more various inventive aspects. However, this manner of disclosure is not to be interpreted as reflecting an intention that the claims require more features than are expressly recited in each claim. Rather, as reflected in the following claims, inventive aspects lie in fewer than all features of a single foregoing disclosed embodiment. Thus, the claims following the Detailed Description are expressly incorporated into this Detailed Description, with each claim standing on its own as a separate and individual embodiment of the present disclosure.

[0090] Furthermore, although some embodiments described herein may include some features included in other embodiments and not others, it is understood that combinations of features from different embodiments are within the scope of the disclosure and form different embodiments as would be understood by one of ordinary skill in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0091] In the description set forth herein, numerous specific details are presented. However, it is understood that embodiments of the present disclosure may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been described in detail in order to avoid obscuring an understanding of the present description.

[0092] The embodiments described herein are intended to cover any modifications or variations of the invention. While the invention has been described and illustrated with reference to specific exemplary embodiments, it will be appreciated that those skilled in the art will readily envision additional embodiments that fall within the scope of the invention.

Claims

1. 1. An imaging device adapted to be incorporated into a device containing biological material, comprising: a sample holder configured to hold a sample of biological material; an input for receiving a light beam; an illumination system configured to transform the light beam into a two-dimensional light sheet and direct the light sheet onto a target illumination zone; a transport mechanism adapted to move the target illumination zone relative to the sample holder so that the light sheet passes across the sample to illuminate the biological material; an imaging system arranged to receive at least a portion of the returned light from the biological material and direct the returned light onto an image sensor to generate a plurality of images of the sample taken at different positions of the light sheet across the sample; an imaging device comprising:

2. 10. The imaging device of claim 1, wherein the biological material comprises one or more gametes or embryos, and the device for culturing the biological material comprises an incubator for culturing the one or more gametes or embryos.

3. The imaging device according to claim 1 , wherein the return light from the biological material includes fluorescence emitted from the biological material.

4. The imaging device of claim 1 , wherein the return light from the biological material includes light emitted by autofluorescence from the one or more gametes or embryos.

5. 5. The imaging device of claim 1, wherein the transport mechanism includes a first actuator adapted to selectively move one or more microlenses in the illumination system such that the target illumination zone moves across the sample holder.

6. The imaging device of claim 5 , wherein the transport mechanism includes a second actuator adapted to move one or more microlenses in the imaging system in conjunction with the first actuator.

7. 7. The imaging device according to claim 5, wherein the first and / or second actuator includes a motorized stage.

8. The imaging device of claim 7 , wherein the first and second actuators include a single motorized stage configured to move the illumination system and the imaging system together.

9. 2. The imaging device of claim 1, wherein the sample holder includes a microfluidic channel, and the transport mechanism includes a microfluidic system configured to move the sample along the microfluidic channel through the targeted illumination zone so that the one or more gametes or embryos pass through the light sheet.

10. The imaging device of claim 1 , wherein the transport mechanism includes an actuator configured to move the sample holder so that the one or more gametes or embryos pass through the light sheet.

11. 11. The imaging device according to claim 1, wherein the illumination system and the imaging system are formed as an integrated structure.

12. The imaging device of claim 2 , wherein the one or more gametes or embryos are unstained.

13. 13. The imaging device according to claim 1, wherein the imaging system has a numerical aperture of 1 or more.

14. The imaging device according to any one of claims 1 to 13, wherein the input section includes an optical fiber.

15. The imaging device of any one of claims 1 to 14, wherein the illumination system is configured to generate the light sheet in a substantially horizontal plane.

16. The imaging device of any one of claims 1 to 15, wherein the illumination system is configured to generate the light sheet in a substantially vertical plane.

17. The imaging device of any one of claims 1 to 16, wherein the illumination system includes a single cylindrical microlens.

18. The imaging device according to any one of claims 1 to 17, wherein the light beam has a wavelength in the range of 400 nm to 850 nm.

19. An imaging apparatus according to any preceding claim, wherein the imaging system is adapted to generate one or more multispectral images of a sample across a number of different wavelength ranges.

20. 20. The imaging device according to claim 1, wherein the imaging system and the illumination system are micro-optical systems.

21. 1. A method for imaging biological material disposed within a device containing the biological material, comprising: receiving a light beam from an input; positioning an illumination system to transform the light beam into a two-dimensional light sheet and direct the light sheet onto a target illumination zone; moving the target illumination zone relative to a sample holder holding a sample of the biological material such that the light sheet passes across the sample to illuminate the biological material; positioning an imaging system to receive at least a portion of the returned light from the biological material and direct the returned light onto an image sensor to generate a plurality of images of the biological material taken at different positions of the light sheet across the sample; A method comprising:

Images