Catadioptric Microscope

Abstract

The optical microscope apparatus includes a sample interrogation system configured to interrogate a sample location, and a collection system configured to collect light output from the sample as a result of being interrogated by the sample interrogation system, the collection system including a mirror disposed along an imaging axis passing through the sample location, and an optical lens system including a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Application No. 62 / 939,380, entitled CATADIOPTRIC MICROSCOPY, filed November 22, 2019, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to optical or light microscopy, including catadioptric designs. [Background technology]

[0003] Light microscopy is a technique used to interrogate biological samples with high spatiotemporal resolution. Light microscopes are used throughout the life sciences and their design and function can vary significantly. Light microscopes contain an objective lens that focuses light to form an image of the sample. Summary of the Invention

[0004] In some general aspects, the imaging device comprises a mirror positioned along an imaging axis that passes through a sample location within the interrogation volume, an optical lens system comprising a plurality of optical lenses positioned along the imaging axis, at least one of which is a multiplet optical lens, and a detection system outside the interrogation volume and configured to detect light emanating from the sample location and collected by the mirror and the optical lens system.

[0005] Embodiments may include one or more of the following features: For example, the optical lenses of the optical lens system may be arranged such that across any optical surface, all light rays in normal operation have a maximum exit angle in air within the range of 35° to 40° relative to the lens surface normal.

[0006] The detection system is capable of imaging the light emitted from the sample location at the diffraction limit of the numerical aperture of the light detection system.

[0007] The imaging device may also include a sample device configured to maintain the sample at a sample position within the interrogation volume.

[0008] The multiple optical lens may be a doublet or a triplet. The multiple optical lenses may be multiple optical lenses.

[0009] The mirrors and optical lens system can form a light collection device that is diffraction limited and has a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter.

[0010] The monocentric mirror and optical lens system may form a light collector that is diffraction-limited, and a working distance between the sample location and an element or mirror of the optical lens system may be at least 20 millimeters, and each of the optical lenses of the mirror and optical lens system may be spherical. The mirror and optical lens system may form a light collector with a numerical aperture of at least 0.8, at least 0.9, or at least 1.0 for a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter for light having a wavelength in the range of 400 to 800 nanometers emanating from the sample location. The mirror and optical lens system may form a light collector that is diffraction-limited for light having a wavelength in the range of 500 to 800 nanometers with an optical transmission efficiency of 81 to 90%. The mirror and optical lens system may form a light collector that is achromatic simultaneously over a wavelength range of 500 to 700 nanometers, a wavelength range of 700 to 800 nanometers, or a wavelength range of 450 to 500 nanometers. The mirrors and optical lens system can form a light collection device that is diffraction limited and has an etendue of at least 100 square millimeters. The mirrors and optical lens system can form a light collection device configured to reduce field dependent aberrations to less than 0.09 waves of double root mean square wavefront error.

[0011] The optical lens system may comprise a plurality of singlet optical lenses, a plurality of doublet optical lenses, and at least one triplet optical lens.

[0012] The mirror may be monocentric with the image of the surface at the sample location, and the maximum angle of incidence of the chief ray on the optical surface of the mirror over the entire field of view may be 2°, 3°, or 4°.

[0013] The optical lens system may comprise a plurality of multiplet lenses on an opposite side of the sample position from the mirror, and at least one singlet lens on a side of the sample position between the sample position and the mirror.

[0014] The detection system can detect light emitted from the sample location without making assumptions about the light.

[0015] Each of the optical lenses and mirrors of the optical lens system can be spherical. By offsetting the axial position of one or more optical lenses of the optical lens system, aberrations caused by changes in the refractive index of the sample at the sample position can be adjusted.

[0016] The mirror and optical lens system may comprise a light collector configured to provide an optically accessible sample location along a direction perpendicular to the imaging axis. The light collector may have respective working distances and curvatures of optical lenses located on either side of a sample at the sample location that provide optical access to the sample location with a numerical aperture to a surface of the sample at the sample location of at least 0.4, at least 0.5, or at least 0.6.

[0017] In another general aspect, an imaging device is configured to image a sample. The imaging device includes a mirror arranged along an imaging axis that passes through a sample position, and an optical lens system including a plurality of optical lenses arranged along the imaging axis, at least one of which is a multiplet optical lens. The mirror and the optical lenses of the optical lens system are located on either side of the sample position along the imaging axis.

[0018] Embodiments may include one or more of the following features. For example, the optical lenses of the optical lens system may be arranged such that light has a maximum divergence angle across any optical surface in the range of 35° to 40° in air.

[0019] The multiple optical lens may be a doublet or triplet lens. The multiple optical lenses may be multiple optical lenses.

[0020] The mirror and optical lens system may constitute a light collector that is diffraction limited and has a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter. The mirror and optical lens system may constitute a diffraction limited light collector, and a working distance between the sample position and any element of the optical lens system or the mirror may be at least 20 millimeters (mm), and each of the optical lenses of the mirror and optical lens system may be spherical. The mirror and optical lens system may constitute a light collector that has a numerical aperture of at least 0.8, at least 0.9, or at least 1.0 for a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter for light having a wavelength in the range of 400 to 800 nanometers emanating from the sample position. The mirror and optical lens system may constitute a light collector that is diffraction limited for light having a wavelength in the range of 500 to 800 nanometers with an optical transmission efficiency of 81 to 90%. The mirrors and optical lens system can form a collector that is achromatic over the wavelength range of 500 to 700 nanometers, the wavelength range of 700 to 800 nanometers, or the wavelength range of 450 to 500 nanometers. The mirrors and optical lens system can form a collector that is diffraction limited and has an etendue of at least 100 square millimeters.

[0021] The optical lens system may comprise a plurality of singlet optical lenses, a plurality of doublet optical lenses, and at least one triplet optical lens.

[0022] The mirror may be monocentric with the image of the surface at the sample location, and the maximum angle of incidence of the chief ray on the optical surface of the mirror over the entire field of view may be 2°, 3°, or 4°.

[0023] The mirrors and optical lens system can form a light collection arrangement configured to reduce field dependent aberrations to less than 0.09 waves of double-mean-square wavefront error.

[0024] The optical lens system may include a plurality of multiplet lenses on an opposite side of the sample position from the mirror, and at least one singlet lens on a side of the sample position between the sample position and the mirror.

[0025] Each of the optical lenses and mirrors of the optical lens system may be spherical.

[0026] By offsetting the axial position of one or more optical lenses in the optical lens system, aberrations caused by changes in the refractive index of the sample at the sample position can be adjusted.

[0027] The mirror and optical lens system may comprise a light collector configured to provide an optically accessible sample location along a direction perpendicular to the imaging axis. The light collector may have respective working distances and curvatures of optical lenses located on either side of a sample at the sample location that provide optical access to the sample location with a numerical aperture to a surface of the sample at the sample location of at least 0.4, at least 0.5, or at least 0.6.

[0028] In another general aspect, a detection device is configured to image a sample. The detection device comprises a mirror disposed along an imaging axis passing through a sample location, an optical lens system comprising a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens, and a sample device configured to define an interrogation volume and to contain a sample at a sample location within the interrogation volume. The sample device includes an immersion fluid at least partially contained by one or more optical lenses of the optical lens system. The mirror and the optical lenses of the optical lens system are located on either side of the sample location.

[0029] Embodiments may include one or more of the following features. For example, the immersion fluid may have a refractive index between 1.0 and 1.7. The immersion fluid and the sample disposed at the sample position may have the same refractive index.

[0030] The optical lenses of the optical lens system may be arranged so that light has a maximum divergence angle in air across any optical surface that is in the range of 35° to 40°.

[0031] The multiple optical lens may be a doublet or triplet lens. The multiple optical lenses may be multiple optical lenses.

[0032] The mirror and optical lens system may constitute a light collector that is diffraction limited and has a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter. The mirror and optical lens system may constitute a light collector that is diffraction limited, a working distance between the sample position and any element of the optical lens system or the mirror may be at least 20 millimeters (mm), and each of the optical lenses of the mirror and optical lens system may be spherical.

[0033] The mirror and optical lens system may form a light collector having a numerical aperture of at least 0.8, at least 0.9, or at least 1.0 for a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter for light having a wavelength in the range of 400-800 nanometers emanating from the sample location. The mirror and optical lens system may form a light collector that is diffraction-limited for light having a wavelength in the range of 500-800 nanometers with an optical transmission efficiency of 81-90%. The mirror and optical lens system may form a light collector that is achromatic over the wavelength range of 500-700 nanometers, the wavelength range of 700-800 nanometers, or the wavelength range of 450-500 nanometers.

[0034] The mirrors and optical lens system are diffraction limited and can provide a light collector with an etendue of at least 100 square millimeters.

[0035] The optical lens system may comprise a plurality of singlet optical lenses, a plurality of doublet optical lenses, and at least one triplet optical lens.

[0036] The mirror may be monocentric with the image of the surface at the sample location, and the maximum angle of incidence of the chief ray on the optical surface of the mirror over the entire field of view may be 2°, 3°, or 4°.

[0037] The mirrors and optical lens system can form a light collection arrangement configured to reduce field dependent aberrations to less than 0.09 waves of double-mean-square wavefront error.

[0038] The optical lens system may include a plurality of multiplet lenses on an opposite side of the sample position from the mirror, and at least one singlet lens on a side of the sample position between the sample position and the mirror.

[0039] Each of the optical lenses and mirrors of the optical lens system may be spherical.

[0040] By offsetting the axial position of one or more optical lenses in the optical lens system, aberrations caused by changes in the refractive index of the sample at the sample position can be adjusted.

[0041] The sample device may further comprise one or more translation and rotation stages configured to translate and / or rotate the sample at the sample location.

[0042] The mirror and optical lens system may comprise a light collector configured to provide an optically accessible sample location along a direction perpendicular to the imaging axis. The light collector may have respective working distances and curvatures of optical lenses located on either side of a sample at the sample location that provide optical access to the sample location with a numerical aperture to a surface of the sample at the sample location of at least 0.4, at least 0.5, or at least 0.6.

[0043] In another general aspect, an optical microscope apparatus includes a sample interrogation system configured to interrogate a sample location, and a collection system configured to collect light output from the sample as interrogated by the sample interrogation system, the collection system including a mirror disposed along an imaging axis that passes through the sample location, and an optical lens system including a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens.

[0044] The embodiment may include one or more of the following features. For example, the sample interrogation system may generate a plurality of excitation light beams directed to the sample location. The sample interrogation system may be an optical interrogation system configured to generate one or more light beams directed to the sample location. The one or more light beams generated by the optical interrogation system may be directed to the sample location by a mirror. The one or more light beams generated by the optical interrogation system may be directed to the sample location along a direction perpendicular to the imaging axis without interacting with the mirror. The optical interrogation system may have respective working distances and curvatures of optical lenses located on either side of the sample at the sample location that provide optical access to the sample location with a numerical aperture of at least 0.4, at least 0.5, or at least 0.6 relative to a surface of the sample at the sample location.

[0045] The optical microscope may also include a detection system configured to receive the collected light from the collection system. The detection system may acquire data at a rate of at least 1.0×10 per second. 10 The detection system can image samples with a volume at the sample location greater than 400 cubic millimeters with a spatial resolution of 0.3 microns by 0.3 microns by 0.5 microns in less than 120 minutes using Nyquist sampling.

[0046] The collection system can be configured to collect light from a sample located at the sample position, the sample having a refractive index between 1.0 and 1.7.

[0047] The light collection system may be configured to collect light from a sample at the sample location having a physical volume greater than 400 cubic millimeters or a surface area greater than 400 square millimeters.

[0048] The optical microscope apparatus may also include a control system in communication with the sample interrogation system and the light collection system and configured to adjust electrical and optical properties of the sample interrogation system and the light collection system. The optical microscope apparatus may include a detection system configured to receive collected light from the light collection system. The control system may be in communication with the detection system and may be configured to form an image of the sample from the collected light from the light collection system as the sample is interrogated by the sample interrogation system.

[0049] Each of the optical lenses and mirrors of the optical lens system may be spherical.

[0050] By offsetting the axial position of one or more optical lenses of the optical lens system from the imaging axis, aberrations caused by changes in the refractive index of the sample at the sample position can be adjusted.

[0051] The imager provides a diffraction-limited achromatic design (further facilitating high-resolution optical microscopy). It also works with immersion media, i.e., the type of media in which biological samples used in microscopy should typically be maintained. The imager incorporates one mirror and provides a catadioptric system with a very large numerical aperture and field size while still providing the degrees of freedom required for achromatization to a large extent (important for fluorescence imaging). The imager is a catadioptric system in which the majority of the optical power is provided by mirror elements that are at or near monocentric with the image plane and the optics stop, i.e., the chief rays are nearly perpendicular to the surfaces of the mirror elements. The chief rays are a set of imaging rays from all points in the object that intersect the optics stop at their centers, as discussed in more detail below. This serves to dramatically reduce field-dependent aberrations that would otherwise accompany refractive surfaces operating at such large etendues and optical powers. The imager also includes non-monocentric lens elements that can correct chromatic aberrations and flatten the image field. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 is an optical block diagram of an imaging device that is catadioptric and includes a collection device that includes at least one reflective optical element. [Diagram 2] FIG. 1 is an optical block diagram of a refractive concentrator, which is an example of a fully monocentric imaging system. [Diagram 3] FIG. 2 is an optical block diagram of an embodiment comprising the imaging device, sample device and interrogation system of FIG. [Figure 4] FIG. 2 is a block diagram of an optical microscope including the imaging device of FIG. 1 and an embodiment of a control system and interrogation system. [Diagram 5] FIG. 5 is a block diagram of an embodiment of the interrogation system of FIG. 4 comprising an optical device and optical configuration for generating and scanning a beam array of multiple curved asymmetric Bessel-like excitation (CABLE) beams. [Figure 6] FIG. 2 is an optical block diagram of an embodiment of the imaging device of FIG. [Figure 7] FIG. 2 is an optical block diagram of one embodiment of the light collection device of FIG. 1. [Figure 8] FIG. 2 is an optical block diagram of an embodiment comprising the imaging device and interrogation system of FIG. [Figure 9A] FIG. 9 is a schematic diagram illustrating optical features of an embodiment in which the interrogation system of FIG. 8 generates one or more light beams directed to a sample location by reflective optical elements. [Figure 9B] FIG. 9 is a schematic diagram illustrating optical features of an embodiment in which the interrogation system of FIG. 8 generates one or more light beams directed at a sample location without interacting with reflective optical elements. [Figure 10] FIG. 2 is an optical block diagram of one embodiment of the light collection device of FIG. 1 with an optical lens system including six lenses positioned relative to the sample position. [Figure 11A] 1 is a table showing the simulated bandpass filter cutoff wavelengths used and the peak emission wavelengths of each fluorophore that may be present in the sample at the sample position. [Figure 11B] 1 is a table showing values ​​of refractive index and Abbe number for immersion fluids of different compositions with sample positions. [Figure 12A] FIG. 13 is a graph of the Strehl ratio calculated under different imaging conditions (where colors correspond to different field positions: blue / light blue—0 mm, green—3.4 mm, red—4.9 mm, pink—6 mm). [Figure 12B] 12B is a table showing conditions corresponding to the configuration numbers in FIG. 12A. [Figure 13A] 13 is a graph of the Strehl ratio evaluation over a wider wavelength range. [Figure 13B] 13B is a table showing conditions corresponding to the configuration numbers in FIG. 13A. [Figure 14A] FIG. 1 is a perspective view of an embodiment of a sample device configured to maintain a sample at a sample position within an interrogation volume defined in a chamber of an imaging device, the sample device comprising a sample holder to which the sample is secured, the sample holder holding the sample being external to the chamber. [Figure 14B] FIG. 14B is a perspective view of the sample device of FIG. 14A, in which a sample holder containing a sample is inserted into the chamber. [Figure 15] FIG. 6 is an optical block diagram of one embodiment of the interrogation system of FIG. 5 designed to generate or create multiple curved light sheets that are probes that are directed at a sample at a sample position. [Figure 16A] FIG. 16 is an optical block diagram of one embodiment of a CABLE beam generating system of the interrogation system of FIG. [Figure 16B] FIG. 16B is an optical block diagram illustrating one embodiment of a phase pattern applied to a spatial light modulator in the CABLE beam generating system of FIG. 16A. [Figure 17] FIG. 16 is an optical block diagram of one embodiment of a beam multiplexing system of the interrogation system of FIG. [Figure 18A] FIG. 16 is an optical block diagram of one embodiment of a beam steering system of the interrogation system of FIG. [Figure 18B] FIG. 18B is a perspective view of the beam steering system of FIG. 18A. [Figure 18C] FIG. 18C is a perspective view of an embodiment of a beam steering subsystem of the beam steering system of FIG. 18B. [Figure 19] FIG. 16 is an optical block diagram of one embodiment of an illumination device of the interrogation system of FIG. [Figure 20] 2 is an optical block diagram of an embodiment of the imaging device of FIG. 1 showing an embodiment of a detection system. [Figure 21] 5 is a flow chart of a procedure carried out by the optical microscope of FIG. 4 using any one of the imaging devices of FIGS. 1, 3, 4, 6, 7, 8 and / or the interrogation system of FIG. 4. [Figure 22] The optical surface labels are given in FIG. 23, with the positive axis direction toward the right, being a table of one embodiment of an optical prescription. [Figure 23] FIG. 23 is a simplified optical block diagram of the light collection device of FIG. 10 showing the labels of the table of FIG. 22. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Referring to FIG. 1, an imaging device 100 is shown. The imaging device 100 comprises a catadioptric light collector 101, i.e. the collector 101 comprises at least one refractive optical element and at least one reflective optical element arranged along an imaging axis IA passing through a sample location 110 in an interrogation volume 115. The collector 101 thus collects and shapes light using a catadioptric optical configuration. The imaging axis IA extends along the Z-axis of a Cartesian coordinate system X, Y, Z. The imaging device 100 can be designed to be rotationally symmetric about the Z-axis and extend in each of the X, Y, and Z directions. The sample location 110 is configured to accommodate a sample to be imaged by the imaging device 100. The sample can have an extent along one or more of the X, Y, and Z directions.

[0054] The reflective optical element is a mirror 105 disposed along the imaging axis IA on a first side Z1 of the interrogation volume 115. In the following embodiment, the mirror 105 has positive optical power and is converging, and therefore has a reflective surface that curves towards the sample location 110 as it extends laterally or radially from the imaging axis IA. The majority (e.g., most) of the collector's positive optical power is provided by the mirror 105. The first side Z1 of the interrogation volume 115 is the side that extends away from the interrogation volume 115 along the positive Z-axis.

[0055] The refractive optical element is an optical lens system 120 that optically interacts with the sample location 110 and the mirror 105. The optical lens system 120 may have one or more components on a first side Z1 of the interrogation volume 115 and one or more components on a second side Z2 of the interrogation volume 115, the side extending away from the interrogation volume 115 along the negative Z-axis. The mirror 105 and the optical lens system 120 form a light collection arrangement 101 that is diffraction limited and has a field of view of at least 8 millimeters (mm), at least 10 mm, or at least 12 mm in diameter.

[0056] In some embodiments, the working distance between the sample position 110 and any element of the optical lens system 120 or the mirror 105 is at least 20 mm. In some embodiments, the mirror 105 and each of the optical lenses in the optical lens system 120 are spherical. Also, an aspheric surface is not required for the light collector 101.

[0057] In various embodiments, the light collector 101 has a numerical aperture (NA) of at least 0.8, at least 0.9, or at least 1.0 for a field of view (FOV) of at least 8 mm, at least 10 mm, or at least 12 mm in diameter for light having wavelengths in the range of 400-800 nanometers (nm) emanating from the sample location 110. In some embodiments, the light collector 101 is diffraction limited for light having wavelengths in the range of 500-800 nm with an optical transmission efficiency of 81-90%.

[0058] The optical lens system 120 includes a plurality of optical lenses arranged along the imaging axis IA on one or more of the first and second sides Z1, Z2. At least one of the optical lenses of the optical lens system 120 is a multiple optical lens 121. Also, there may be more than one multiple optical lens 121 in the optical lens system 120. Each multiple optical lens 121 may be a doublet lens or a triplet lens. For example, the optical lens system 120 may include a plurality of singlet optical lenses, a plurality of doublet optical lenses, and at least one triplet optical lens, as discussed in more detail below. The optical lenses in the optical lens system 120 may be arranged such that across any optical surface in the lens system 120, all light rays during normal operation have a maximum exit angle in air within a range of 30° to 45° or 35° to 40° relative to the surface normal of each lens.

[0059] One or more of the optical lenses of optical lens system 120 and mirror 105 may be movable and / or adjustable along imaging axis IA. This axial movement of mirror 105 and the lenses in optical lens system 120 may be achieved using a high precision recirculating ball bearing raceway.

[0060] The imaging device 100 also comprises a detection system 125 that is external to the interrogation volume 115. The detection system 125 is configured to detect light 126 emanating from the sample location 110 and collected by the collection device 101, i.e., the mirror 105 and the optical lens system 120. The light 126 may be fluorescent light emanating from a sample at the sample location 110. The detection system 125 is configured to image the light 126 emanating from the sample at the sample location 110 at the diffraction limit of the numerical aperture of the collection device 101.

[0061] The imaging device 100 allows high-resolution imaging of large volumes within a sample, and further allows new types of experiments and observations to be performed on the sample. For example, the imaging device 100 allows high-speed imaging of large chemically cleared or enlarged tissues and organs with subcellular resolution and without the need for physical cutting (e.g., high-resolution structural imaging of an entire mouse brain), whole-brain live imaging of neuronal activity in large model organisms (such as mice), or simultaneous imaging at the single-cell level of groups of freely behaving and interacting animals (e.g., simultaneous whole-brain imaging across all individuals in a group of interacting larval zebrafish or fruit flies).

[0062] The imaging device 100 includes a catadioptric light collector 101 as described herein for light-based image formation, thus avoiding the fundamental limitations of traditional objective lenses. Specifically, a mirror 105 replaces the traditional objective lens. Unlike traditional objective lenses, the mirror 105 can provide both high spatial resolution (by providing a high numerical aperture) and access to a large field of view. Thus, the light collector 101 achieves high performance for both parameters simultaneously. This is because the scaling laws of optical aberrations and errors have less impact on the light collector 101 as the etendue increases.

[0063] Traditional scaling constraints arising from optical aberrations of traditional objective lenses can be overcome in the light collection device 101, allowing high numerical apertures over a much larger field of view than ever before. For example, the imaging device 100 can provide diffraction-limited performance from 500 to 715 nanometers (nm) with a numerical aperture of 1.0, a field of view of 12 millimeters (mm), a working distance of 25 mm, and an optical transmission efficiency of 81-90% when the sample interrogation volume 115 is water. Compared to a state-of-the-art optical system such as a meso lens with a numerical aperture of 0.47 and a field of view of 6 mm, the imaging device 100 improves optical throughput (quantified as the number of resolvable elements simultaneously transmitted by the device 100) by a factor of 18.

[0064] Additionally, because the imaging device 100 is not limited by the speed of one or more cameras in the detection system 125, and the imaging device 100 has such a large field of view, it also provides the opportunity for dramatically speeding up the imaging of large samples through multiplexing the detection process, for example, using a camera array that parallelizes image acquisition across the entire field of view. 10 voxels, possibly 1.4×10 per second 10 An embodiment of an imaging device 100 using a camera array consisting of ten sCMOS cameras, providing an imaging speed of 100x1000, is discussed below with respect to FIGS.

[0065] As mentioned above, the mirror 105 provides a very large portion of the positive optical power of the light collection device 101. The mirror 105 may be monocentric, i.e., it is arranged monocentrically or nearly monocentrically with the surface of the object (i.e., sample or specimen) being imaged at the sample position 110 and the optical aperture SS. In other words, the mirror 105 is field symmetric, meaning that a set of rays emanating from a field point of the object (sample) interacts with the mirror in the same way as a set of rays coming from other field points, or the mirror 105 may be nearly field symmetric and exhibit small deviations from this condition. The optical aperture SS may be defined as an aperture stop or lens ring in the light collection device 101 that physically limits the solid angle of the rays (from the light 126) that pass through the device 101 from an on-axis object point. The optical aperture SS therefore limits the brightness of the image formed from the light 126 to the detection system 125. The optical aperture SS may be an aperture stop or may be on the surface of one of the lenses in the optical lens system 120.

[0066] In general, a fully monocentric optical system is a system in which all optical surfaces, including the object surface and the image surface, share a common center of curvature located at the optical system stop. The optical concentrator 101 may be a fully monocentric or nearly monocentric optical system. By way of consideration and comparison, an example of a fully monocentric imaging system using a refractive optical concentrator 202 is shown in FIG. 2. Unlike the optical concentrator 101 in FIG. 1, the refractive optical concentrator 202 only comprises diffractive optical elements R1, R2 disposed between the object space OS and the imaging space IS. Due to the symmetry of the optical system with respect to the field variables, which is consistent with the fact that the chief ray CR of the optical system is normally incident on every optical surface (of each of the optical elements R1, R2), there is no field-dependent aberration of the optical system. Such an optical system may have only spherical and axial chromatic aberrations, and the size of the field itself is not limited in the usual way by field-dependent aberrations, but by cosine foreshortening of the optical system stop, which gives an effective width of zero at a 90 degree field angle, or by overlapping of optical surfaces on either side of the stop, which deviates from being hemispherical.

[0067] One important measure of an optical system is its etendue. For conventional optical systems, such as a microscope (in which the imaging device 100 may be incorporated), the etendue is equal to the area of ​​the field of view (FOV) times the square of the numerical aperture (NA) available in that FOV. Light can propagate through an optical system with large aberrations, contributing to simple transmission of light energy or imaging that is not diffraction-limited. However, in optical microscopy, where the fundamental goal is to obtain a high-quality, high-resolution image of a sample (at the sample position 110), diffraction-limited imaging may be important and / or required, so that only FOVs and NAs that can be transmitted with correspondingly low aberrations are considered when calculating the etendue. If we assume that the imaging device 100 is diffraction-limited and the etendue in question is the diffraction-limited etendue, the etendue of the imaging device 100 is proportional to the number of individually resolvable pixels (recels) that can be transmitted through the device 100 for a given wavelength of light.

[0068] The detection system 125 uses one or more digital image sensors (or cameras). Each sensor, or sensor array, can have more pixels than the human eye and the supported resolution of conventional objective lenses. It can therefore be advantageous to develop a light collection device 101 (and imager 100) with a higher etendue to match the level of resolution desired for imaging a particular sample in the detection system 125. The imager 100 is designed to provide a higher etendue than is obtainable with conventional microscope objective lenses.

[0069] Also, designing an optical system in which all aberrations are kept to a small fraction of the wavelength of light 126 becomes increasingly difficult as the etendue becomes larger. In microscope objectives that are refractive (utilizing only light refraction at the lens surfaces) and support the large numerical apertures required for high-resolution imaging, only lens surfaces that satisfy certain narrow conditions can be utilized for the high-power tip elements (typically the first through fourth positive meniscus lenses that contribute most of the lens's positive power). For example, one condition is the aplanatic condition, and another is the concentric condition. The aplanatic condition (where the ratio of the slope of the marginal ray to the refractive index around the ray is constant across the lens surface) allows for large optical surface powers without introducing spherical, coma, or astigmatism, but this is only possible if the object surface is immersed in the same refractive index as the lens material. The concentric condition (where the angle of incidence of the marginal ray at the surface is zero) does not introduce spherical or coma aberrations, but also no optical power. In high NA refractive microscope objectives, one or more aplanatic or nearly aplanatic surfaces are used in the tip, and usually one or more nearly concentric surfaces are also used in the tip. These conditions are met at etendue levels above those of typical refractive objectives, e.g., an etendue of 6.2 mm. 2 However, this is at the expense and inconvenience of a significant physical increase in lens size and an increase in the number of elements.

[0070] Imager 100 offers an alternative, efficient high etendue optical design strategy by achieving a more dramatic increase in etendue without the need for cumbersome lens sizes and numbers.

[0071] 3, in some embodiments, imaging device 100 is an imaging device 300 that additionally includes a sample device 330 and an interrogation system 340, each of which will now be described.

[0072] The sample apparatus 330 is configured to maintain the sample 335 at the sample position 110 within the interrogation volume 115. The sample apparatus 330 may comprise a holding device and one or more translation and / or rotation stages (such as the motion stage 1439 in FIGS. 14A and 14B ) that allow translation and / or rotation, respectively, of the sample 335 (by translating and rotating the holding device within or out of the interrogation volume 115). The holding device holds the sample 335 at the sample position 110 within the interrogation volume 115. The holding device may include an immersion fluid surrounding the sample 335 or a device that contains the sample 335 and allows the sample 335 to be imaged from various angles. An embodiment of the sample apparatus 330 is described with reference to FIGS. 14A and 14B .

[0073] The interrogation system 340 is specifically configured to interrogate the sample location 110 while the sample 335 is placed at the sample location 110. In particular, the interrogation system 340 acts on and interacts with the sample 335 to cause the sample 335 to output light 126 that is collected by the light collection device 101 and then detected or sensed in the detection system 125. Thus, the interrogation system 340 may generate one or more probes, such as excitation optical or light beams, that are directed at the sample location. For example, wide-field illumination, light sheet illumination, or (multi-)point scanning may be utilized in the interrogation system 340. An embodiment using light sheet illumination is discussed below with reference to Figures 15-19. An embodiment of the interrogation system 340 is described with reference to Figures 9A, 9B, 14A, 14B, and 15-19.

[0074] 4, the imaging device 100 may be implemented as an imaging device 400 within an optical microscope 450. The optical microscope 450 comprises a control system 455, an interrogation system 440, and the imaging device 400. The interrogation system 440 comprises an optical device 442 and an optical arrangement 444 that generate one or more probes 440p that are directed toward the sample location 110 within the imaging device 400.

[0075] The imaging device 400 collects the fluorescent light (via light collection device 101), directs the light to a camera in the detection system 125, and translates the sample for volumetric imaging (using a sample positioning subsystem in the sample device 330). The detection system 125 can include a filter wheel array FA (as shown in FIGS. 14A and 14B ) consisting of multiple filter wheels, each housing multiple (e.g., six) fluorescent filters, and a camera array consisting of multiple cameras (e.g., sCMOS cameras such as C14120-20P (Hamamatsu, Japan)) for fast parallel imaging of the sample at the sample position 110. The number of filter wheels in the filter wheel array matches the number of cameras in the camera array.

[0076] The control system 455 includes a main controller 456, control electronics 457, and an image acquisition module 458. The control system operates all of the optical, electrical, and mechanical components of the optical microscope 450, including the light assembly 442 and optical configuration 444, as well as the components in the imaging device 400. For example, the control system 455 can be configured to control the spatial light modulator 1660 ( FIG. 16A ), the galvanometer scanner GS ( FIG. 18C ), the translation and rotation stages in the optical configuration 442, and the filter wheel and camera in the imaging device 400.

[0077] One or more of these components of the main controller 456 may include hardware such as one or more output devices (e.g., a monitor or printer), one or more user input interfaces such as a keyboard, mouse, touch display, or microphone, one or more processing units including specialized workstations for performing specific tasks, memory (e.g., random access memory, read-only memory, virtual memory, etc.), and one or more storage devices such as hard disk drives, solid state drives, or optical disks. The processing units may be stand-alone processors or may be sub-computers such as their own workstations. The control system 455 may have a distributed architecture where some functions are assigned or located on one computer while other functions are assigned or located on another computer.

[0078] The master controller 456 coordinates all of the electrical and optical components of the optical microscope 450. The master controller 456 may include a computer running LabVIEW control software that coordinates and controls the aspects of the optical microscope 450.

[0079] In some embodiments, the master controller 456 is a server that runs an operating system (such as Windows 10) and LabVIEW microscope control software. The LabVIEW software coordinates all components of the optical microscope 450 to execute the imaging workflow and ensure synchronous image acquisition. More specifically, the master controller 456 sends control commands to the control electronics 457 to generate appropriate drive and trigger signals for the light device 442 and optical arrangement 442 of the interrogation system 440 and the imager 400. The master controller 456 also sends commands (e.g., over Ethernet) to the image acquisition nodes in the image acquisition module 458 to set parameters associated with the cameras in the detection system 125 and to start and / or stop image acquisition in the detection system 125.

[0080] The image acquisition module 458 is configured to execute an imaging workflow including acquiring and storing image data from the detection system 125. An output from the image acquisition module 458 is provided to the master controller 456, and communication between the image acquisition module 458 and the master controller 456 can be wired or wireless. The image acquisition module 458 can operate with multiple image acquisition nodes, each of which runs LabVIEW image acquisition software and is connected to one camera in the imaging device 100 (e.g., in the detection system 125) by a CoaXPress cable for image acquisition. For example, as discussed below with reference to Figs. 15-19, the detection system 125 can include ten imaging subsystems or detectors, in which case the image acquisition module 458 can operate with ten image acquisition nodes. All image acquisition nodes can also be connected to a master control computer (of the master controller 456) by Ethernet communication to receive image acquisition commands from the master control computer of the master controller 456.

[0081] The image acquisition node is responsible for operating each dedicated camera (in the detection system 125) and acquiring and temporarily storing data according to instructions given by the master controller 456. More specifically, the image acquisition node will receive commands from the master controller 456 to set camera parameters including exposure time, area mode, area of ​​interest, etc., and will configure each connected camera accordingly. The image acquisition node will also start the acquisition process after receiving a command from the master controller 456 to start image acquisition, and will configure the cameras to wait for a start trigger signal. When the cameras receive a start trigger signal from the control electronics 457, they will synchronously start image acquisition. The image acquisition node can also receive a command to abort an ongoing acquisition, and will immediately stop acquisition when this command is received.

[0082] The control electronics 457 is configured to generate and synchronize control signals for the individual components in the interrogation system 440 and the imaging device 400, including, but not limited to, the galvanometer scanners, filter wheels, translation stages, and cameras. The control electronics 457 comprises a housing that connects to the main controller 456, several analog input and output cards, and a serial interface card all mounted within the housing.

[0083] The control electronics 457 can be implemented in an enclosure (e.g., National Instruments PXIe-1078) connected to the main controller 456 by a remote controller card (e.g., National Instruments PCIe-8381) installed in one of the PCIe slots of the main controller 456. Three data acquisition cards can be installed in the enclosure: two PXIe-6738 acquisition cards (National Instruments) providing 64 analog output (AO) channels and one PXIe-6363 acquisition card (National Instruments) providing 32 analog input (AI) channels. Operated by the main controller 456, the PXIe-6738 acquisition cards generate analog voltage signals to drive the galvanometer scanners GSa, GSb in the beam steering system 547 (FIG. 5) and modulate the laser intensity of the probe 836 (FIG. 8). The PXIe-6738 acquisition card also generates TTL or LVCMOS signals to synchronize the camera, stage and filter wheel, and to enable / disable lasers within the interrogation system 440. One serial interface card (National Instruments PXIe8430 / 8) can be mounted within the enclosure to provide eight RS-232 communication ports used to control devices including the light fixture 442 and motion stages.

[0084] As described above, the interrogation system 440 includes an optical arrangement 442 and an optical configuration 444 that generate one or more probes 440p that are directed toward the sample location 110 in the imaging device 400. The field of view of the illumination optics in the interrogation system 440 matches the field of view of the detection optics in the imaging device 400, so that any portion that can be imaged by the detection system 125 can be illuminated by the interrogation system 440.

[0085] 5, interrogation system 440 is implemented as interrogation system 540 comprising optical device 442 and optical configuration 544. Interrogation system 540 generates and scans a beam array of multiple (e.g., 10) curved asymmetric Bessel-like excitation (CABLE) beams. Optical configuration 544 comprises CABLE beam generation system 545, beam multiplexing system 546, beam steering system 547, and illumination configuration 548 (which may include custom tube lenses and / or objective lenses). In other embodiments, it may be possible to generate a scanned laser light sheet from the output of optical device 442 using a Gaussian beam.

[0086] Features relating to the imaging device 400 are discussed next, followed by a discussion of the interrogation system 540.

[0087] Referring to FIG. 6, an embodiment 600 of the imaging device 100 is shown. The imaging device 600 comprises an optical lens system 620 including multiple optical lenses. In this embodiment, the optical lens system 620 includes only two optical lenses 620_1 and 621_1. In other embodiments, the optical lens system 620 can include more than two optical lenses. The optical lenses are arranged along the imaging axis IA. At least one of the optical lenses is a multiplet optical lens. In this embodiment, the lens 621_1 is depicted as a multiplet lens. There may be more than one multiplet lens in the optical lens system 620. The imaging device 600 also comprises a detection system 125 that is outside the interrogation volume 115. The detection system 125 is configured to detect light 626 emanating from the sample position 110 and collected by the mirror 105 and the optical lens system 620. Some rays of the light 626 are shown in dashed lines for reference.

[0088] The optical lenses of the optical lens system 620 are arranged such that across all optical surfaces, the maximum exit angle in air of all light rays 626 in normal operation is in the range of 35°-40° with respect to the lens surface normal, where the exit angle of the light rays 626 is the angle that the light rays 626 make with the surface normal of the optical lens (e.g., optical lenses 620_1 and 621_1) as they exit the optical lens. The multiplet optical lens 621_1 can be a doublet lens or a triplet lens.

[0089] The detection system 125 images the light 626 emitted from the sample location 110 at the diffraction limit of the numerical aperture of the imaging device 100. The mirror 105 and optical lens system 620 form a light collection device that is diffraction limited and has a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter.

[0090] The working distance between the sample position 110 and any element of the optical lens system 620 or the mirror 105 is at least 20 millimeters (mm), and the mirror 105 and each of the optical lenses 620_1, 621_1 of the optical lens system 620 are spherical. This means that the surface that interacts with the light 626 at each of the mirror 105 and the optical lenses 620_1, 621_1 has a spherical shape. The working distance can be considered as the effective distance from the nearest optical element to the nearest surface of the sample when the sample is in focus.

[0091] The collector 601 has a numerical aperture of at least 0.8, at least 0.9, or at least 1.0 for a field of view of at least 8 millimeters, at least 10 millimeters, or at least 12 millimeters in diameter for light 626 emanating from the sample location 110 having a wavelength in the range of 400-800 nanometers. The collector 601 is diffraction limited for light having a wavelength in the range of 500-800 nanometers with an optical transmission efficiency of 81-90%. The collector 601 may be achromatic simultaneously over the wavelength range of 500-700 nanometers, the wavelength range of 700-800 nanometers, or the wavelength range of 450-500 nanometers. The collector 601 may have an etendue of at least 100 square millimeters.

[0092] Mirror 105 is a curved mirror having an optically reflective surface 105S that interacts with light 626 emitted from a sample at sample location 110. Mirror 105 may be monocentric or nearly monocentric with an image of the surface at sample location 110. The maximum angle of incidence of the chief ray (discussed with respect to FIG. 2 and also shown in FIG. 10) on the optical surface of mirror 105 in the full field of view is 2°, 3°, or 4°.

[0093] The condenser 601 is configured to reduce field dependent aberrations to less than 0.2, 0.1, 0.09, or 0.07 waves of double root mean square wavefront error.

[0094] The detection system 125 detects the light 626 emitted from the sample location 110 without making any assumptions about the light 626. The detection system 125 can include multiple detection channels, each specialized to detect a particular wavelength range to allow for multi-color detection.

[0095] The axial position of one or more of the optical lenses 620_1, 621_1 of the optical lens system 620 can be offset from other optical lenses and components of the light collection device 601 to adjust for aberrations caused by, for example, changes in the refractive index of the immersion fluid in the interrogation volume 115 or the sample at the sample location 110. The axial position of the optical lenses is offset by adjusting the position of the optical lenses along the imaging axis IA. In this way, the imaging device 600 is configured to adapt the microscope to a new sample at the start of a new experiment. For example, the immersion fluid held in the interrogation volume 115 (e.g., immersion fluid 718 or immersion fluid 1418) may have a slightly different chemical composition during one experiment relative to another. For example, the concentration of one of the components in the immersion fluid may change, which in turn changes the optical properties of the immersion fluid, changing the optical path of the light 626. To compensate for this change in the optical path, the lenses can be moved accordingly. As another example, the sample 335 may change from experiment to experiment, or the sample 335 itself may change along its volume in a single experiment. Optical properties may also change as a function of time; that is, for example, if some water in the immersion fluid evaporates, this may increase the concentration of some chemical components in the immersion fluid (which may increase the refractive index over time). The control system 455 can monitor these changes during the experiment and compensate for the changes during imaging.

[0096] The collector 601 is configured to provide an optically accessible sample position 110 along a direction perpendicular to the imaging axis IA, for example the collector 601 has a sufficiently large working distance on either side of the sample position 110 and the optical surfaces facing the sample position 110 (the optical surfaces of the optical lenses 620_1, 621_1 facing the sample position) have a sufficiently small curvature to allow optical access to the sample position 110 with a numerical aperture of at least 0.4, at least 0.5, or at least 0.6.

[0097] One or more of the optical elements of the focusing device 601 may be disposed on translation and / or rotation stages TRS1, TRS2, TRS3 controlled by control electronics 457 of a control system 455 to adjust / translate / rotate the position of such components.

[0098] In some embodiments, as shown in FIGS. 7 and 10, mirror 105 and the optical lenses of optical lens system 620 can be located on either side of sample position 110 along imaging axis IA.

[0099] Referring to FIG. 7, a light collection device 701, which is part of an imaging device 700, is configured to image a sample 735 located at a sample location 110 by collecting light 726 emanating from the sample 735 and directing the collected light towards a detection system 125. The light collection device 701 comprises a mirror 105 arranged along an imaging axis IA passing through the sample location 110. The light collection device 701 comprises an optical lens system 720. The light collection device 701 also comprises a sample device 730 configured to define an interrogation volume 731 and to accommodate the sample 735 at the sample location 110 within the interrogation volume 731. The sample device 730 includes an immersion fluid 718 at least partially enclosed by one or more optical lenses 720_1 of the optical lens system 720. The immersion fluid 718 may have a refractive index between 1.0 and 1.7. The immersion fluid 718 and the sample 735 disposed at the sample position 110 may have the same refractive index.

[0100] 8, an imaging device 800 (which is part of an optical microscope) comprises an interrogation system 840 configured to interrogate a sample location 110. The interrogation system 840 may be configured to generate a number of excitation light (optical) beams directed towards the sample location 110. These excitation light beams serve to excite fluorophores or other molecules within a sample 835 at the sample location 110, which emit light 826 that is collected by the imaging device 800. To this end, the imaging device 800 comprises a light collection device 801 configured to collect light 826 output from the sample 835 at the sample location 110, which is a result of the sample 835 being interrogated by one or more probes 836 from the interrogation system 840. The light collection device 801, as discussed above, comprises a mirror 105 arranged along an imaging axis IA passing through the sample position 110, and an optical lens system 820 having a number of optical lenses 820_1, 821_1 arranged along the imaging axis IA.

[0101] In some embodiments, the interrogation system 840 is an optical interrogation system configured to generate one or more light beams directed toward the sample location 110 as a probe 836 .

[0102] 9A, one or more light beams 836 generated by the interrogation system 840 are directed by a mirror 105 to a sample 835 at a sample location 110. For example, the light beam 836 may be directed to the sample location 110 after reflecting off an optically reflective surface 105S of the mirror 105. Interaction of the light beam 836 with the sample 835 causes the sample 835 to emit fluorescent light 826, which is collected by a light collection device 801.

[0103] 9B, one or more light beams 836 generated by the interrogation system 840 are directed toward the sample 835 at the sample location 110 along a direction that is not parallel (e.g., perpendicular) to the imaging axis IA. In these embodiments, the light beams 836 travel to the sample location 110 without interacting with the mirror 105. Interaction of the light beams 836 with the sample 835 causes fluorescent light 826 to be emitted from the sample 835, which is collected by the light collection device 801.

[0104] 8, the imaging device 800 may also include a detection system 825 configured to receive collected light 826 from the light collection device 801, such as the detection system 125 shown in FIG. 1. The detection system 825 may acquire data at a rate of at least 1×10 per second. 10 The detection system 825 can image a sample 835 at the sample location 110 having a volume of greater than 100, 200, 300, or 400 cubic millimeters with a spatial resolution of equal to or better than 0.3 microns by 0.3 microns by 0.5 microns in a time of less than 120 minutes. The detection system 825 can use Nyquist sampling.

[0105] The control system 455 can be in communication with the interrogation system 840 and the light collector 801. The control system 455 is configured to adjust electrical and optical properties of the interrogation system 840 and the light collector 801. The control system 455 can also be in communication with the detection system 825 and can be configured to form an image of the sample from the light 826 collected by the light collector 801 as the sample 835 is interrogated by the interrogation system 840.

[0106] 10, in another embodiment, a light collector 1001 is configured to image a sample 1035 disposed at a sample position 1010. The light collector 1001 includes an optical lens system 1020 having a plurality of lenses. The optical lens system 1020 includes a plurality of multiplet lenses on a side of the sample position 1010 opposite the mirror 1005 with respect to the sample position 1010, and at least one singlet lens on a side of the sample position 1010 between the sample position 1010 and the mirror 1005. The optical lens system 1020 includes six lenses 1020_1, 1020_2, 1021_1, 1021_2, 1021_3, and 1021_4, where the lenses 1021_1, 1021_2, 1021_3, and 1021_4 are multiplet lenses.

[0107] Lens 1020_1 (which is a singlet) is located on the side of sample position 1010 between sample position 1010 and mirror 1005. Lens 1021_1 is a doublet lens on the opposite side of sample position 1010 from lens 1020_1. Lenses 1020_1 and 1021_1 define interrogation volume 1015. Lens 1021_2 is a triplet lens, lens 1021_3 is a doublet lens, and lens 1021_4 is a triplet lens. Lens 1020_2 (which is the last lens in collection device 1001 on the path to detection system 125) is a singlet.

[0108] The light collection device 1001 also includes a chamber 1017 that defines an interrogation volume 1015. A sample 1035 is received at a sample position 1010 within the interrogation volume 1015. In some embodiments, one or more walls 1019 of the chamber 1017 hold an immersion fluid 1018 within the interrogation volume 1015. The immersion fluid 1018 may also be partially contained by lenses 1020_1 and 1021_1 of the optical lens system 1020. A flexible seal 1031 is formed between the chamber wall 1019 and the lens 1021_1 that allows some relative movement between the chamber wall 1019 and the lens 1021_1. The immersion fluid 1018 may have a refractive index between 1.0 and 1.7. The immersion fluid 1018 and the sample 1035 disposed at the sample position 1010 may have the same refractive index.

[0109] Concentrator 1001 may be implemented in imager 100 of Figure 1. In such a configuration, imager 100 is nominally diffraction-limited and achromatic over the wavelength range of 500 to 715 nanometers (nm), with its diffraction-limited numerical aperture of 1.0 and FOV of 12 mm in diameter, resulting in a focal length of 113 mm. 2 Using the nominal design (i.e., no performance loss due to manufacturing tolerances), the number of individually resolvable pixels at the Abbe resolution limit is 2.2 × 10 at 510 nm. 9 There will be one.

[0110] In some embodiments, the collector 1001 is a custom designed NA 1.0 mirror-based objective lens with a 12 mm field of view and a 25 mm working distance. The collector 1001 can be designed for imaging media and samples with refractive indices between 1.33 and 1.34. The large field of view provides two advantages: (1) the ability to image large samples 1035 up to 12 mm x 12 mm x 25 mm without requiring physical cutting or lateral translation of the sample, and (2) the ability to simultaneously image different portions of the sample 1035 using multiple cameras to improve imaging speed. The imager 100 (implemented with any of the collectors 101, 601, 701, 801, 1001) can capture up to 1.4 x 10 images per second.10 It can be imaged at voxel rates, and as a result a sample with physical dimensions of 12 mm × 8 mm × 6 mm, corresponding to the average size of a mouse brain, is imaged in just 2 hours using Nyquist sampling with a spatial resolution of 0.3 μm × 0.3 μm × 0.5 μm.

[0111] The imaging device 100 in which the light collection device 101, 601, 701, 801 or 1001 is implemented is a high numerical aperture finite conjugate imaging system that transmits light from a probe image plane in a chamber 1017 (defined in the device 1001 by the chamber walls 1019 and the lenses 1020_1 and 1021_1) filled with an immersion fluid 1018 to a distant detection image plane in the detection system 125. The probe image plane in the chamber 1017 is determined by the geometry of the one or more light beams 836 and is determined by the shape of the focal plane geometry. The probe image plane in the chamber 1017 may be curved in the case of using CABLE beams to generate the one or more light beams 836 as described below with reference to Figures 15-19. The detection image plane in the detection system 125 is one or more image planes in detector space that are conjugate to the focal plane at the sample position 110. The detection image plane may be perfectly flat or curved in other embodiments. An array of camera chips can be used to record the image at the image plane, or if the image plane is curved, a curved array of flat camera chips can be used.

[0112] In some embodiments, the immersion fluid 1018 is water or water with various salts and buffers, the NA is 1.0, the field of view on the sample side is 12 mm in diameter, and the magnification is 35x, resulting in an image plane with a diameter of 420 mm. Upon leaving the sample 1035, the light collected and imaged by the collector 1001 first traverses the lens 1020_1 that forms one of the walls of the immersion fluid chamber, and then traverses a small air gap before hitting the concave mirror 1005, which provides most of the positive optical power of the collector 1001 and is nearly monocentric with the optical aperture SS. This near monocentricity ensures that the chief ray CR of the collected light 1026 has an angle of incidence close to zero on the mirror 1005, so that all field-dependent aberrations resulting from the mirror 1005 are naturally small.

[0113] After reflecting off the mirror 1005, the imaged light 1026 again traverses the lens 1020_1 and re-enters the interrogation volume 1015. Passing backwards, some of the light 1026 is occluded or strays (in the case of a thin or highly transparent sample 1035) by the sample 1035. However, due to the large beam size of the light 1026 passing through the interrogation volume 1015, the amount of light 1026 occluded by the sample 1035 is small. For example, in some embodiments, less than 5%, less than 3%, less than 2%, or about 1.4% of the light that would otherwise be imaged is occluded by the sample 1035.

[0114] The imaged light 1026 then enters lens 1021_1, which forms the other wall of the immersion chamber. After lens 1021_1, the light traverses four additional lenses 1021_2, 1021_3, 1021_4, and 1020_2, which function to correct wavefront and chromatic aberrations already incurred by the imaged light 1026. After exiting lens 1020_2, the light 1026 then travels towards the image plane of a detection system 125 within the imaging device 100.

[0115] The presence of the immersion fluid chamber 1017 and the need to achromatize the collector 1001 may preclude the existence of a completely monocentric imaging solution, which generally seems to lack the degrees of freedom required to eliminate axial color, as there are few positive power imaging solutions using immersion liquid. The small deviations from monocentrism present in the mirror 1005, the larger deviations in the lens 1020_1, and the large deviations in the immersion surface IS of the lens 1021_1 require corrective actions of the lenses 1021_2, 1021_3, 1021_4, 1020_2, and the small deviations from monocentrism seen in the mirror 1005 are themselves the result of optimizing all of the optical systems together simultaneously for aberration minimization, which is usually done during optical design.

[0116] The optical aperture SS in the light collection device 1001 is located at the surface of the lens 1021_2. In other embodiments, a variable size aperture may be located near this location. An accessory window / filter 1022 is located near the optical aperture SS. The window 1022 may be used as an interference or absorption filter for unwanted light (such as excitation light in fluorescence microscopy).

[0117] As shown in FIG. 3, in some embodiments, after exiting the lens 1020_2, the light 126 passes through an environmental window 322. The detection system 125 is very sensitive to the refractive index of the immersion fluid 1018 due to the long path length of the imaging light through the immersion fluid 1018. Also, fluids such as water tend to experience a fairly large variation in refractive index with temperature. Therefore, the temperature and temperature gradient of the immersion fluid 1018 can be controlled to require that the optical components in the detection system 125 be housed in a temperature and humidity regulated chamber (not shown in FIG. 3). The environmental window 322 separates the interior of this temperature and humidity regulated chamber from the outside (outside the lab space).

[0118] A second consequence of the sensitivity of the detection system 125 to the refractive index of the immersion fluid 1018 is that small changes in the composition of the immersion fluid 1018 can have a non-trivial effect on the imaging performance in the detection system 125. It is desirable to allow for some variability in the immersion fluid 1018, since different sample preparations require different immersion compositions for optimal clearing and imaging performance. For example, different preparations of expanded tissue may be optimally imaged in the presence of different amounts of phosphate or saline sodium citrate buffers, which may increase the refractive index of water from 1.333 (d-line, 587 nm) to 1.343, and this variation may be large depending on the design of the detection system 125. To accommodate these variations in the design, the mirror 1005, lenses 1021_1 and 1021_2 (set), and lens 1021_3 are configured to move axially (along the imaging axis IA) a small amount depending on the composition of the immersion fluid 1018, allowing for the recovery of diffraction-limited performance through small changes in aberration balance.

[0119] One imaging criterion that can be used to optimize the optical design of the light collection device 1001 is the root mean square optical path difference (RMS OPD) for several different variables. The first variable is the field position. The second variable is the fluorophore variable. Specifically, color performance is evaluated separately for the weighted bandwidths of four different exemplary fluorophores: eGFP, mOrange, mCherry, and mPlum. Figure 11A shows the simulated bandpass filter cutoff wavelengths used and the peak emission wavelength of each fluorophore.

[0120] The third variable is the composition of the immersion fluid 1018, whose performance was evaluated using the refractive index of different compositions of the immersion fluid 1018, such as water, phosphate buffered saline (1xPBS), and two different concentrations of saline sodium citrate buffer (2xSSC and 5xSSC). The refractive index of these immersion fluids 1018 can be measured at multiple (e.g., 13) wavelengths within a wavelength range (e.g., wavelength range 435-715 nm) using a custom-built refractometer with an accuracy of better than 0.0001. This data can be input into optical design software Zemax OpticStudio to use these materials in optical designs. Figure 11B shows the refractive index and Abbe number values ​​of these buffers at the d-line. A final evaluation of the nominally constructed optical system performance can be performed by calculating the Huygens-Strehl ratio under several imaging conditions using Zemax OpticStudio. Figures 12A and 12B show the results of the nominal optical system design. All Strehl ratios perform at or above the usual 0.8 cutoff that defines diffraction-limited performance, except for the condition using eGFP and 5xSSC imaging medium with a 6 mm field of view.

[0121] The axial positions of mirror 1005, lenses 1021_1 and 1021_2, and lens 1021_3 can be moved between imaging media, but not between different fluorophores. That is, diffraction-limited simultaneous imaging with multiple fluorophores is possible, assuming multiple excitation beams and cameras are color-multiplexed and used simultaneously. During optimization, different fluorophores are evaluated separately, but the light collection device 1001 can work well over a larger wavelength range that is equally weighted. Figures 13A and 13B show the results under these wavelength conditions. Finally, although no optimization was performed for wavelengths below 500 nm, it was found that performance of the collector 1001 at or near the diffraction limit is possible for fluorophores with emission wavelengths up to 435 nm, as long as the compensators of the immersion fluid 1018 (mirror 1005, lenses 1021_1 and 1021_2, and lens 1021_3) can be moved to accommodate these wavelengths (relative to their positions at wavelengths of 500-715 nm), and a somewhat smaller bandpass width can be used (20-40 nm).

[0122] 14A and 14B, an embodiment 1430 of the sample device 330 is shown. The sample device 1430 is configured to maintain a sample 1435 at a sample position 1410 within an interrogation volume 1415 defined within a chamber 1417. The sample device 1430 may comprise a sample holder 1437 to which the sample is fixed. The sample 1435 may be a biological sample or a chemically fixed, cleared and / or enlarged sample and is disposed on or within the sample holder 1437. The interior of the chamber 1417 is filled with an immersion fluid 1418. The immersion fluid 1418 may have a refractive index between 1.33 and 1.34 and / or may match the refractive index of the sample 1435.

[0123] The sample device 1430 also comprises a positioning system 1438 to which the sample holder 1437 is fixed. The positioning system 143·BR>W comprises a motion (e.g. translational and / or rotational) stage 1439 fixed to the sample holder 1437 and configured to move the sample holder 1430 along a direction parallel to the X-axis. The motion stage 1439 may also be configured to move the sample holder 1437 along one or more of the Y-axis and Z-axis or along a direction in the YZ plane and / or to rotate the sample holder 1437 about any of the X-axis, Y-axis or Z-axis. The positioning system 1438 (and motion stage 1439) may be in communication with the control system 455 to control the insertion of the sample holder 1437 (and sample 1435) into the immersion fluid 1418 and to position the sample 1435 relative to the imaging focal plane of the collection device 101.

[0124] The chamber 1417 is designed with an optically transparent port that allows light to pass between the interrogation volume 1415 and the exterior of the chamber 1417. The port is located on a first side Z1 relative to the sample position 1410, adjacent to a mirror 1405 (which is an embodiment of mirror 105). The port is located on a second side Z2 relative to the sample position 1410, adjacent to a portion 1420p of an optical lens system 1420 located on the second side Z2.

[0125] An interrogation port 1433 is located on the side of the chamber 1417. The interrogation port 1433 provides an optical path for one or more light beams 836 generated by the interrogation system 840 that are directed toward a sample 1435 at the sample location 1410 along a direction that is non-parallel (e.g., perpendicular) to the imaging axis IA. In these embodiments, the light beams 836 travel to the sample location 1410 without interacting with the mirror 1405.

[0126] In an embodiment discussed below in which the one or more light beams 836 are a set of ten curved asymmetric Bessel-like excitation (CABLE) light beams IBo, the interrogation port 1433 is large enough to allow optical access of the CABLE beam IBo to the sample 1435.

[0127] 5, the interrogation system 540 comprises the light device 442 and the optical configuration 544 comprising a CABLE beam generation system 545, a beam multiplexing system 546, a beam steering system 547, and an illumination configuration 548 (which may include custom tube lenses and / or objective lenses). An embodiment 1540 of the interrogation system 540 will now be discussed with respect to FIG. 15. The interrogation system 1540 is designed to generate or create multiple (e.g., 10) curved light sheets that are probes 836 that are directed at the sample at the sample location 110.

[0128] The interrogation system 1540 generates a curved asymmetric Bessel-like excitation (CABLE) beam, multiplexes a single beam into ten beams arranged along a single axis, directs the ten beams to a sample at the sample location 110, and scans the beam across the sample. The interrogation system 1540 includes an optical arrangement 1542 that includes light sources that output laser light at various wavelengths and optics at the outputs of the light sources that expand the light beams generated by the light sources. The interrogation system 1540 includes a CABLE beam generation system 1545, a beam multiplexing system 1546, a beam steering system 1547, and an illumination arrangement 1548 that includes optics (such as a tube lens and an objective lens) for directing the beams to a sample at the sample location 110. Each of these features of the interrogation system 1540 is discussed next.

[0129] The light source in the light device 1542 may be a high power laser system (e.g., HP Lightengine manufactured by Omicron-Laserage, Germany). The laser system comprises a number (e.g., six) of individual high power laser units, each with a different wavelength. For example, the wavelengths may be 488 nm, 532 nm, 560 nm, 592 nm, 631 nm, and 670 nm, respectively. The output laser power may be, for example, 500 mW for the 488 nm laser and 1000 mW for all other lasers. The intensity of each laser beam may be modulated by its associated acousto-optic modulator (AOM). The light beams from the six lasers are combined using a dichroic mirror so that all laser beams exit the laser system from the same exit port. The output light beam from the laser system may be coupled into a high power optical fiber. After exiting the optical fiber, the beam is collimated and expanded by a collimator and beam expander before being provided to the CABLE beam generating system 1545.

[0130] A CABLE beam can be generated by CABLE beam generating system 1545. The CABLE beam has a curved trajectory that matches the curvature of the focal plane of the objective lens of collection device 101 (determined by mirror 105). The CABLE beam is very long (700 μm) and very thin (0.6 μm) (at the central peak of the CABLE beam), allowing high resolution imaging while fully utilizing the imaging area in detection system 125 (e.g., sCMOS cameras that can be used in detection system 125 have large chip sizes).

[0131] The Bessel-like light sheet illumination scheme may have drawbacks: the side lobes of the Bessel-like beam may generate significant fluorescence background that significantly reduces the image contrast. To solve this problem, a method for generating an asymmetric Bessel-like beam with suppressed side lobes along a specific direction is implemented as discussed next.

[0132] Referring to FIG. 16A, the CABLE beam generating system 1545 includes a spatial light modulator 1660, a pair of achromatic lenses (first lens 1661 and second lens 1662) in a 4-f system, a ring aperture 1663 in the front focal plane of the first lens 1661, and a mirror 1664 that redirects light from the spatial light modulator 1660 to the first lens 1661. The spatial light modulator 1660 can be a phase-only spatial light modulator (such as Meadowlark's SLM MSP1920-0635-HSP8). A phase pattern is applied by the spatial light modulator 1660. A laser beam is directed to the spatial light modulator 1660 and diffracted (by mirror 1664) onto a path to the first lens 1661. The light is then filtered by the ring aperture 1663, which transmits light only inside the ring shape. The light then forms the CABLE beam after passing through a second lens 1662 .

[0133] Specifically, referring to FIG. 16B, the phase pattern applied to the spatial light modulator 1660 acts as a mask 1660m consisting of two opposing triangles with tip angle θ applied to the phase pattern generating a curved Bessel-like beam. After applying the mask 1660m, the light reflected from the masked area of ​​the spatial light modulator 1660 is focused by the first lens 1661 to a single spot 1663s at the position of the aperture 1663, while the diffracted light originating from the unmasked area of ​​the spatial light modulator 1660 is focused by the first lens 1661 to a ring pattern 1663r. The ring aperture 1663 blocks the light from the masked area of ​​the spatial light modulator 1660 and transmits the light from the unmasked area of ​​the spatial light modulator 1660. After passing through the second lens 1662, the light forms a CABLE beam with directionally suppressed side lobes.

[0134] Referring to FIG. 17, the beam multiplexing system 1546 is comprised of an achromatic half-wave plate 1765, a polarization sensitive beam splitter (PBS) 1766, a number of non-polarizing 50:50 beam splitters (one of which is labeled 1767), and a number of mirrors (one of which is labeled 1768). These optical components are arranged to split an illumination beam IB into ten illumination beams IBo (where o is an integer between 1 and 10) that are equal in intensity and equally spaced along a row. Furthermore, by having each beam travel the same distance through glass and air, the wavefronts of the beams IBo lie in the same plane when they exit the multiplexing system.

[0135] As shown in FIG. 17, the polarized laser beam IB first passes through an achromatic half-wave plate 1765, followed by a PBS 1766. By rotating the half-wave plate 1765, the intensity of the two output beams produced by the PBS 1766 can be matched. After the beam is split into two by the PBS 1766, three mirrors 1768 are placed around the PBS 1766 to direct both beams in a forward direction while keeping the optical path lengths the same. Each beam then enters the next splitting stage. For the upper beam resulting from the first splitting stage (into the plane of the paper in FIG. 17), a 50:50 beam splitter 1767 is used to split the beam into two beams of equal intensity. Three mirrors 1768 are placed around the beam splitter 1767 to direct the beams in a forward direction while keeping the optical path lengths of all beams the same. The split beams then proceed to the next two splitting stages using similar optical architectures. The beam splitter 1767 and mirror 1768 of the final stage are smaller than those of the previous stages so that the distance between the beams can be maintained. This geometry allows the creation of many illumination beams (10 in this case) within the field of view of the interrogation system 440 and the imaging device 400. Specifically, the illumination beams are simultaneously within the field of view of the objective lenses of the interrogation system 440 and the imaging device 400 (e.g. mirror 105 and illumination arrangement 1548). For the downward beam resulting from the first split stage (into the page of FIG. 17), the mirror 1768 and beam splitter 1767 are positioned such that the beam travels the same distance through glass and air as its counterpart from the top of the splitting system.

[0136] 18A and 18B, the beam steering system 1547 is comprised of ten beam steering subsystems 1870-o, where o is an integer between 1 and 10. Each beam steering subsystem 1870-o receives a respective illumination beam IBo from the beam multiplexing system 1546. The array of subsystems 1870-o is used to scan the ten beams IBo and position the beams IBo on a curved surface that matches the curvature of the focal plane of the objective lens.

[0137] Referring to FIG. 18C, each subsystem 1870-o is composed of two lenses 1871a, 1871b, and two galvanometer scanners, a first two-axis galvanometer scanner GSa and a second two-axis galvanometer scanner GSb. The first two-axis galvanometer scanner GSa includes a galvanometer scanner GSax (arranged along the x-axis) and a galvanometer scanner GSay (arranged along the y-axis), and the second galvanometer scanner GSb includes a galvanometer scanner GSbx (arranged along the x-axis) and a galvanometer scanner GSby (arranged along the y-axis). Each two-axis galvanometer scanner GS can be, for example, a model 6SD12380 or 6SD12381 from Cambridge Technology. The illumination beam IBo is first pivoted by the first two-axis galvanometer scanner GSa. The first lens 1871a is disposed behind the first two-axis galvanometer scanner GSa such that the back focal plane of the first lens 1871a is located at the geometric center between the x- and y-scan mirrors of the first two-axis galvanometer scanner GSa. The second two-axis galvanometer scanner GSb is disposed so that the front focal plane of the first lens 1871a coincides with the center of the y-scan mirror of the second two-axis galvanometer scanner GSb. The second lens 1871b is disposed behind the second two-axis galvanometer scanner GSb such that the back focal plane of the second lens 1871b coincides with the center of the y-scan mirror of the second two-axis galvanometer scanner GSb. Thus, the second lens 1871b converts the pivoting motion of the beam provided by the second two-axis galvanometer scanner GSb into lateral and axial beam shifts.

[0138] 18A and 18B, ten identical beam steering subsystems 1870-o are arranged side by side. The relative spacing between the subsystems 1870-o can be less than 5 centimeters, for example about 25 millimeters (mm), to construct a complete beam steering system 1547 for steering ten illumination beams IBo. To ensure that all ten beams IBo are located on the curved focal plane of the detection objective lens (i.e., mirror 105), the x-scan mirror of each second two-axis galvanometer scanner GSb of each beam steering subsystem 1870-o rotates to a specific angle that shifts the beam IBo upward by a distance required to place each beam on the curved focal plane.

[0139] 19, the illumination configuration 1548 includes a custom tube lens 1973 and a custom interrogation objective lens 1974 that cooperate to image the ten illumination beams IBo onto a sample at the sample position 110. The output of the illumination configuration 1548 is one or more probes (or one or more light beams) 836 (e.g., as shown in FIGS. 9A and 9B). The interrogation objective lens 1974 is designed to provide diffraction-limited performance within the ring aperture 1663r associated with the CABLE beam. The interrogation objective lens 1974 may have an NA of 0.4, a field of view of 12 mm, and a working distance of 90 mm in some embodiments.

[0140] Referring to FIG. 20, in some embodiments, the image field (in the detection system 125) can be divided into sub-FOVs (FOV-o, where o is an integer between 1 and 10) and imaged by multiple digital imaging sensors. The detection system 2025 of FIG. 20 comprises ten cameras 2075-o (where o is an integer between 1 and 10) that span from one end of the FOV to the other along a plane or line in the x dimension, with the total width of all imaging chips being slightly greater than one-third of the width of the entire FOV. Each of the cameras 2075-o images a field of view illuminated by one CABLE beam IBo, and each camera 2075-o has a corresponding emission filter 2076-o (where o is an integer between 1 and 10). Each emission filter 2076-o is rotated differently from the others such that the angle of incidence of light across each sub-FOV is minimized or reduced as much as possible on each filter 2076-o.

[0141] In three dimensions, the camera array 2075-o can be divided into three sections using folding mirrors to provide adequate sub-FOV spacing when using commercially available cameras with a large ratio of housing width to imaging chip width.

[0142] 20, the total optically available detection FOV covered by the imaging sensors of cameras 2075o may be a portion of the total FOV of the detection system 2025. For example, the total available detection FOV from all cameras 2075o may be 2-5% of the total FOV of the detection system 2025. Different illumination schemes and / or more economically and efficiently (possibly custom) packaged imaging sensors may enable use of significantly more of the available detection FOV.

[0143] Referring again to Figures 14A and 14B, the optical microscope 450 and imaging device 100 can be used to image a wide variety of samples or specimens 1435. Samples that are relatively transparent and have a refractive index between 1.33 and 1.34 can generally provide very good image quality. An example of a biological sample in this region is a magnified biological tissue (using an expansion microscopy protocol), such as a magnified section of a mouse brain. After magnification, the brain or brain section is mounted on a sample holder 1437. In some embodiments, the interrogation volume 1415 is filled with a transparent aqueous solution (which is the immersion fluid 1418) suitable for imaging the magnified sample 1435. The sample 1435 is lowered into the immersion fluid 1418 and positioned relative to the imaging focal plane of the mirror 1405 using an x-translation stage.

[0144] Before acquiring the images, referring again to FIG. 4, the optical microscope 450 can be adjusted to optimally cooperate with the sample 1435 and the immersion fluid 1418 by optimizing the position and angle of the probe beam 836 relative to the sample position 1410 (FIGS. 9A and 9B) and by matching the optics to the exact refractive index of the immersion fluid 1418 in the imaging chamber 1417. To optimize the position and angle of the probe beam 836, images can be acquired by the detection system 125 and quality metrics associated with these images can be calculated and evaluated. Based on these measurements, optimal offset voltages can be calculated and applied to the pivot and scan galvanometer scanners GSa, GSb. This process can be repeated until the image quality is deemed optimal (or within an acceptable range of values). Similarly, images can be acquired by the detection system 125 to match the refractive index of the immersion fluid 1418 in the imaging chamber 1417 and quality metrics associated with these images can be calculated and evaluated. Based on these measurements, optimal offset positions are calculated and applied to the translation stages attached to the lens groups (lenses) of optical lens system 120. This process can be repeated until the image quality is deemed optimal (or within an acceptable range).

[0145] 21, procedure 2180 may be performed using imaging devices 100, 300, 400, 600, 700, 800 and / or interrogation system 440 with optical microscope 450 of FIG.

[0146] When the optical microscope 450 is powered on, an initialization step may be performed on the main controller 456 to establish communication between the main controller 456 and other electronic components in the microscope, including, for example, the spatial light modulator 1660 (FIG. 16A), the translation and / or rotation stage (such as the motion stage 1439 or the translation / rotation stages TRS1, TRS2, TRS3), and the filter wheel (such as may be used with the filter array FA and located in the imaging device 100). The translation and / or rotation stage and the filter wheel / filter array FA may initially be set to a home position during the initialization step. An initialization step is also performed on the data acquisition nodes to establish communication between these nodes and their associated cameras. One or more cameras in the detection system 125 are initialized with default acquisition parameters. After initialization, the optical microscope 450 is ready for operation.

[0147] Step 2180 describes the image acquisition workflow of the optical microscope 450 based on an embodiment of the interrogation system 1540. The entire sample 735, 835, 1035, 1435 can be imaged by repeated acquisition of sub-volumes within the sample. The size of each sub-volume can be determined by the size of the camera chip and the magnification of the detection optics in the imaging device 100. In one particular embodiment, the size of each sub-volume is 408 μm×723 μm×Σ μm. The parameter Σ is flexible (within the working distance of the detection optics) and is determined by the user. Ten cameras (as shown in the embodiment of FIG. 20) can be used to acquire ten sub-volumes simultaneously.

[0148] To start image acquisition, the specifications of the optical microscope 450 are set (2181). For example, the required laser line or color is activated in the interrogation system 1540, the corresponding emission filter is rotated into position by the filter wheel FA, and the spatial light modulator 1660 is updated with the corresponding phase pattern. A laser beam with the desired excitation wavelength is turned on. The collimated laser light is expanded and converted into a CABLE beam IBo by the CABLE beam generation system 1545. The curvature of the CABLE beam IBo is adjusted to match the curvature of the focal plane of the detection objective (mirror 105). The IBo beam is then split by the beam multiplexing system 1546 into ten beams that are matched in intensity and arranged along a row. The split beams are positioned on a curved surface with a curvature that matches the curvature of the focal plane of the detection objective (mirror 105) by the beam steering system 1547. The beam IBo is guided into the sample and scanned in the curved focal plane to generate ten curved light sheets.

[0149] The ten illumination beams IBo / 836 are moved to their respective starting positions (2182), and then the beams IBo / 836 are scanned at a repetition rate of 120 Hz (matched to the frame rate of the camera in the detection system 125) using a sawtooth input waveform. Meanwhile, the sample is translated at a constant speed using a z-stage, and images are acquired simultaneously (2183). For example, the emitted fluorescence is collected by the detection objective 105 and filtered by appropriate fluorescence filters 2076-o in front of each camera 2075-o to eliminate the laser light (light from the probe 836) and keep only the fluorescence. The cameras are operated in a light sheet mode, so that the line propagation speed of the active area scanned across the camera chip matches the scanning speed of the illumination beams IBo. The images captured by the cameras 2075-o are sent to the corresponding image acquisition node for storage and post-processing.

[0150] If multi-color imaging is desired (2184), the specifications are adjusted (2185). Specifically, the currently active laser line or color is disabled, the next required laser line or color is activated, and one or more filter wheels are rotated to switch the filters to correspond to the active laser line. The phase pattern in the spatial light modulator 1660 is updated accordingly, and the same set of sub-volumes is imaged again (2183). Once the imaging of the ten sub-volumes is complete, the sample 735, 835, 1035, 1435 is translated (2186) along x, y, or z to image the next set of ten sub-volumes (2183). This procedure continues (2187) until the entire volume of the sample 735, 835, 1035, 1435 has been imaged. If multiple viewpoint imaging is desired (2188), the sample 735, 835, 1035, 1435 is rotated by a particular angle using the rotation stage (2189) and the imaging process is repeated in step 2182.

[0151] FIG. 22 provides an example optical prescription with the optical surface labels shown in FIG. 23, with the positive axis direction toward the right side of the page.

[0152] In volumetric image acquisition, the sample is translated at a constant speed by the z-stage. In multi-color imaging, the active laser beam is switched to a different color by deactivating the first laser beam and activating a laser unit emitting a different wavelength, the corresponding fluorescence filter is selected by the filter wheel, and the phase pattern in the spatial light modulator 1660 is adjusted accordingly (FIG. 16A). The same sample volume is then imaged again using a second laser wavelength by translating the z-stage. Alternatively, the laser beam can be switched from one image plane to another, such that the sample volume is scanned only once along the z-axis. Optionally, a rotation stage is used to rotate the sample in different orientations to facilitate multi-view image acquisition by re-imaging the sample volume after the acquisition of one view of the sample volume is completed.

Claims

1. 1. An imaging device for imaging a sample, comprising: a mirror disposed on a first side of a sample location within an interrogation volume along an imaging axis, the imaging axis passing through the sample location; and an optical lens system comprising a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens, the optical lens system having one or more optical lenses disposed on both the first and second sides of the sample location within the interrogation volume, the second side being opposite the first side; An imaging device comprising:

2. 2. The imaging device of claim 1, wherein the optical lenses of the optical lens system are arranged such that light has a maximum divergence angle in the range of 35° to 40° in air across any optical surface.

3. 1. A detection device for imaging a sample, comprising: a mirror disposed on a first side of a sample location within an interrogation volume along an imaging axis, the imaging axis passing through the sample location; and an optical lens system comprising a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens, the optical lens system having one or more optical lenses disposed on both the first and second sides of the sample location within the interrogation volume, the second side being opposite the first side; a sample device defining an interrogation volume and containing a sample at the sample location within the interrogation volume, the sample device including an immersion fluid at least partially contained by one or more optical lenses of the optical lens system; A detection device comprising:

4. The detection apparatus of claim 3 , wherein the immersion fluid has a refractive index between 1.0 and 1.

7.

5. The detection apparatus of claim 3 , wherein the immersion fluid and the sample disposed at the sample location have the same refractive index.

6. The detection apparatus of claim 3 , wherein the sample device further comprises one or more translation and rotation stages for translating and / or rotating a sample at the sample location.

7. a sample interrogation system for determining the location of the sample; a light collection system for collecting light output from a sample as interrogated by the sample interrogation system, a mirror disposed within a sample interrogation volume on a first side of the sample location along an imaging axis, the imaging axis passing through the sample location; and an optical lens system comprising a plurality of optical lenses disposed along the imaging axis, at least one of which is a multiplet optical lens, the optical lens system having one or more optical lenses disposed on both the first side and a second side of the sample location within the sample interrogation volume, the second side being opposite the first side; a light collecting system having An optical microscope apparatus comprising:

8. The optical microscope apparatus of claim 7 , wherein the sample interrogation system is an optical interrogation system that generates one or more light beams directed at the sample location.

9. The optical microscope apparatus of claim 8 , wherein the one or more light beams generated by the optical interrogation system are directed to the sample location by the mirror.

10. The optical microscope apparatus of claim 8 , wherein the one or more light beams generated by the optical interrogation system are directed to the sample location along a direction perpendicular to the imaging axis without interacting with the mirror.

11. 11. The optical microscope apparatus of claim 10, wherein the optical interrogation system has working distances and curvatures of the optical lenses located on either side of the sample at the sample location that provide optical access to the sample location with a numerical aperture to a surface of the sample at the sample location of at least 0.4, at least 0.5, or at least 0.

6.

12. The optical microscope apparatus of claim 7 , further comprising a detection system that receives the collected light from the collection system.

13. The detection system acquires data at a rate of at least 1.0×10 per second. １０ The optical microscope apparatus of claim 12 which is a voxel.

14. 13. The optical microscope apparatus of claim 12, wherein the detection system uses Nyquist sampling to image a sample at the sample location having a volume greater than 400 cubic millimeters in a time less than 120 minutes with a spatial resolution of 0.3 microns by 0.3 microns by 0.5 microns.

15. 8. The optical microscope apparatus of claim 7, wherein the light collection system collects light from a sample at the sample location having a refractive index between 1.0 and 1.

7.

16. 8. The optical microscope apparatus of claim 7, wherein the light collection system collects light from a sample at the sample location having a physical volume greater than 400 cubic millimeters or a surface area greater than 400 square millimeters.

17. 8. The optical microscope apparatus of claim 7, further comprising a control system in communication with the sample interrogation system and the light collection system to regulate electrical and optical properties of the sample interrogation system and the light collection system.

18. a detection system that receives the collected light from the collection system; 20. The optical microscope apparatus of claim 17, wherein the control system is in communication with the detection system to form an image of a sample from the light collected from the light collection system by interrogating the sample with the sample interrogation system.

Images