Dynamic remote refocusing microscope

Abstract

A remote refocusing microscope system is disclosed, comprising a first objective lens having a first numerical aperture NA1 and a sample refractive index n1 having a first immersion medium having a first refractive index n1. s a first microscope arranged to receive light from a sample in a medium having a second numerical aperture NA2 and a second refractive index n2; and an optical compensator disposed between the first and second microscopes, wherein (n s / n2) ratio. RR and an optical compensator comprising at least one lens having a linearly adjustable position to provide a refocusing function. The system is modular, and the first objective lens can be changed to allow operation in one of three modes. Methods for configuring the remote refocusing microscope in one of the three modes are also disclosed.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Patent Application No. 63 / 418,256, filed October 21, 2022, the entire contents of which are incorporated herein by reference in their entirety. Summary of the Invention [Means for solving the problem]

[0002] According to one aspect, a remote refocusing system is provided that is configured to image a sample. The system has a refractive index (RI) of the sample n s The system may include a first microscope arranged to receive light from a sample in a medium having a first numerical aperture NA1. The first microscope may include a first objective lens having a first numerical aperture NA1 and a first immersion medium having a first refractive index n1. The system may include a second microscope including a second objective lens having a second numerical aperture NA2 and a second refractive index n2. The second objective lens may be positioned and arranged to receive light passing through the first microscope. The combination of the first microscope and second microscope may have a magnification M RR The system may be configured to generate an intermediate image of the sample at M. The system may further include an optical compensator disposed between the first microscope and the second microscope. The optical compensator may RR (n s The optical system may include at least one lens having a linearly adjustable position such that the optical system is continuously adjusted to approximately equal a ratio of (n / n2).

[0003] In some embodiments, the sample refractive index n s may be greater than the first refractive index n1. In some embodiments, the sample refractive index n s may be less than the first refractive index n1. In some embodiments, the sample refractive index n s may be substantially the same as the first refractive index n1.

[0004] In some embodiments, MRR is the refractive index (n s / n2).

[0005] In some embodiments, n s can be in the range of 1.00 to 2.00, e.g., 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00. In certain embodiments, n s is in the range of 1.33 to 1.51.

[0006] In some embodiments, n1 can be in the range of 1.00 to 2.00, e.g., 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00. In certain embodiments, n1 is in the range of 1.00 to 1.51.

[0007] In some embodiments, the first objective lens is configured to focus the image at a focal length of the system (n s / n2) ratio is approximately equal to M RR The objective lens may be constructed and arranged to be switched between different objective lenses of the same focal length but with different refractive indices n1 so as to maintain

[0008] In some embodiments, the first objective lens has an optical compensator (n s / n2) ratio is approximately equal to M RR The objective lens may be constructed and arranged to be switchable between different objective lenses having different focal lengths and different refractive indices n1 so as to maintain

[0009] In some embodiments, the collection half angle of the second objective lens is greater than or approximately equal to the collection half angle of the first objective lens.

[0010] According to one aspect, a modular microscopy system for imaging a sample is provided. The system comprises: sThe system may include a sample stage constructed and arranged to hold a sample immersed in a sample medium having a first numerical aperture NA1. The system may include a first microscope having a first objective lens having a first numerical aperture NA1 and a first immersion medium having a first refractive index n1. The system may further include a second microscope having a second objective lens having a second numerical aperture NA2 and a second refractive index n2, the combination of the first and second microscopes providing a magnification M RR The system may further include an optical compensator disposed between the first microscope and the second microscope, and configured to generate an intermediate image of the sample at (n s / n2) ratio. RR The modular microscopy system includes at least one lens having a linearly adjustable position to provide a sample refractive index n s and a first refractive index n1, and a sample refractive index n s and the first refractive index n1 is determined by the three modes, namely: Sample refractive index n s a first mode in which n is equal to a first refractive index n1; Sample refractive index n s a second mode in which n exceeds the first refractive index n1; Sample refractive index n s a third mode in which n is less than the first refractive index n1; The oscilloscope is selected to allow operation in the oscilloscope.

[0011] In some embodiments, n s can be in the range of 1.00 to 2.00, e.g., 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00. In certain embodiments, n s is in the range of 1.33 to 1.51.

[0012] In some embodiments, n1 can be in the range of 1.00 to 2.00, e.g., 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00. In certain embodiments, n1 is in the range of 1.00 to 1.51.

[0013] According to one aspect, a method of configuring a remote refocusing system including a first microscope and a second microscope for imaging a sample is provided. The method may include selecting a first objective for the first microscope based on a selected compromise between first, second, and third operating modes for the remote refocusing system and the refractive index of the sample. The first, second, and third operating modes are based on: 1) the sample refractive index n s a first operating mode using a non-immersion objective for the first microscope when n is greater than the first refractive index of the first objective; and 2) a first operating mode using a non-immersion objective for the first microscope when n is greater than the first refractive index of the first objective. s is substantially the same as the first refractive index n1 of the first objective lens; and 3) a second operating mode including an extended focal range of the first objective lens when the sample refractive index n s and a third operating mode for maximizing the numerical aperture (NA) of the first objective lens when n is less than a first refractive index n of the first objective lens. The method may include selecting a second objective lens having a second refractive index n for the second microscope, wherein the first microscope and the second microscope have a magnification M RR The method may further include selecting an optical system combination for an optical compensator disposed between the first objective and the second objective to collect substantially all of the emitted light from the first objective. The optical compensator may be configured to generate an intermediate image of the sample at (n s / n2) ratio. RR The position may be linearly adjustable to provide

[0014] In some embodiments, selecting the first objective lens may include selecting an air immersion objective lens. In some embodiments, selecting the first objective lens may include selecting a water immersion objective lens. In some embodiments, selecting the first objective lens may include selecting an oil immersion objective lens. For example, the oil immersion objective lens may include a silicone oil immersion objective lens or a glycerol immersion objective lens. [Brief explanation of the drawings]

[0015] Various aspects of at least one embodiment are discussed below with reference to the accompanying drawings, which are not intended to be drawn to scale. The drawings are included to provide illustration and a further understanding of the various aspects and embodiments, and are incorporated into and constitute a part of this specification, but are not intended as a definition of the limits of the invention. In the drawings, each identical or nearly identical component illustrated in the various drawings is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing.

[0016] [Figure 1A] 1A-1C illustrate remote refocusing microscopy systems including an optical compensator according to different embodiments. FIG. 1A illustrates a system for imaging a sample, where the sample refractive index ns is substantially equal to a first refractive index n1 . FIG. 1B illustrates a system for imaging a sample, where the sample refractive index ns is greater than the first refractive index n1 . FIG. 1C illustrates a system for imaging a sample, where the sample refractive index ns is less than the first refractive index n1 . [Figure 1B] 1A-1C illustrate remote refocusing microscopy systems including an optical compensator according to different embodiments. FIG. 1A illustrates a system for imaging a sample, where the sample refractive index ns is substantially equal to a first refractive index n1 . FIG. 1B illustrates a system for imaging a sample, where the sample refractive index ns is greater than the first refractive index n1 . FIG. 1C illustrates a system for imaging a sample, where the sample refractive index ns is less than the first refractive index n1 . [Figure 1C] 1A-1C illustrate remote refocusing microscopy systems including an optical compensator according to different embodiments. FIG. 1A illustrates a system for imaging a sample, where the sample refractive index ns is substantially equal to a first refractive index n1 . FIG. 1B illustrates a system for imaging a sample, where the sample refractive index ns is greater than the first refractive index n1 . FIG. 1C illustrates a system for imaging a sample, where the sample refractive index ns is less than the first refractive index n1 .

[0017] [Figure 2A] Figures 2A-2B illustrate the focal range of a standard single-objective wide-field microscope using air, water, and oil immersion objectives. Figure 2A illustrates the focal range for the objective with the highest numerical aperture (NA). Figure 2B illustrates the focal range for the objective with the largest number of pixels. [Figure 2B] Figures 2A-2B illustrate the focal range of a standard single-objective wide-field microscope using air, water, and oil immersion objectives. Figure 2A illustrates the focal range for the objective with the highest numerical aperture (NA). Figure 2B illustrates the focal range for the objective with the largest number of pixels.

[0018] [Figure 3A] Figures 3A-3B illustrate the focal range of a remote refocusing microscope using an air-immersion objective, a water-immersion objective, and an oil-immersion objective as the first objective and remote optics optimized for samples with refractive indices between 1.33 and 1.51. Figure 3A illustrates the focal range for the objective with the highest numerical aperture (NA). Figure 3B illustrates the focal range for the objective with the largest number of pixels. [Figure 3B]Figures 3A-3B illustrate the focal range of a remote refocusing microscope using an air-immersion objective, a water-immersion objective, and an oil-immersion objective as the first objective and remote optics optimized for samples with refractive indices between 1.33 and 1.51. Figure 3A illustrates the focal range for the objective with the highest numerical aperture (NA). Figure 3B illustrates the focal range for the objective with the largest number of pixels.

[0019] [Figure 4A] 4A-4B illustrate a zoom lens system according to one or more non-limiting embodiments. [Figure 4B] 4A-4B illustrate a zoom lens system according to one or more non-limiting embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Aspects and embodiments are directed to methods and apparatus for improved microscopic imaging of samples with mismatched RI.

[0021] Choosing the objective lens is an important consideration when using a visible-light microscope. For example, the numerical aperture (NA) and field of view (FOV) of the objective lens will determine the maximum available resolution and object size in the image. When a 3D semi-transparent sample is imaged with a high NA, the focal plane should be adjusted to access the available volumetric information. For example, a series of 2D images can be obtained at different depths by moving the microscope objective lens toward the object, a method known as "standard focusing." With standard focusing, the maximum imaging depth of a typical wide-field microscope is therefore limited by the objective lens's working distance (WD), i.e., the amount of advancement of the objective lens.

[0022] Positive working distance for standard focusing involves the "final element" in objective design, where shape and refractive index are considerations. The shape is fixed by the imaging plane and the final solid surface of the objective, typically a glass lens. However, the RI of this space can vary; i.e., immersion media and / or coverslips alter the RI of the space. In standard focusing experiments to image deeper into a sample, a "slab" of the intended immersion medium, e.g., air, water, or oil, is effectively replaced with a slab of sample. If the RI of the sample matches that of the objective, this results in null operation, and the objective will image as designed. However, if there is a refractive index mismatch, for example, a sample slab with an unintended RI will produce spherical aberration at a finite NA, blurring the image at sufficient depth.

[0023] Therefore, for aplanatic standard focusing, the refractive index n1 of the first objective lens should be set equal to the sample refractive index.

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[0024] The need to match the refractive index of the objective lens with that of the sample for 3D imaging presents challenges. The RI of biological samples varies significantly, creating numerous objective lens options. Air-immersion objectives are the most convenient and excellent for high-speed tiling, but have the lowest numerical aperture (NA) and low penetration depth due to the large RI difference. Water-immersion objectives generally provide a good RI match for biological samples, such as aqueous suspensions of cells, but require periodic rehydration to reduce evaporation; water has the lowest numerical aperture of any liquid immersion objective. Oil-immersion objectives offer the highest numerical aperture and are not affected by coverslip thickness, but generally have lower depth capabilities due to RI mismatch. Silicone oil-immersion objectives offer a compromise between NA and RI match, but challenges remain for specific applications. Therefore, there is a need for improved microscopy that eliminates the challenges of sample selection and RI mismatch.

[0025] Remote refocusing (RR) optics are used in high-speed 3D imaging, such as in single-objective light-sheet (SOLS) microscopes. One feature of the RR optical setup is that either the primary objective or the sample is focused. Without moving For example, in contrast to standard focusing experiments, the ability to adjust the focal plane by moving one of the downstream objective lenses. In remote refocusing experiments, slabs of different refractive index between the sample and the downstream remote space can be swapped. As a non-limiting example, an air-immersion optical setup can be used in the downstream remote space with an aqueous sample. In this configuration, spherical aberration can be avoided by setting the magnification and preserving the angle between the sample and the downstream remote space.

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[0026] In the formula, M RR is the remote refocusing (RR) magnification, and n2 is the RI of the immersion of the downstream remote space, which is equal to the RI of the second objective lens n2. Here, improved imaging can be achieved by optimizing remote refocusing for the refractive index of the sample rather than the immersion of the first objective lens.

[0027] In a typical RR system, the sample and first objective RI are equal, i.e., n1=n sRR range, thus enabling the maximum range for both standard focusing and remote refocusing experiments. If the RI of the sample and the RI of the first objective differ, the standard focusing range can be reduced, but the remote refocusing range can remain. Alternatively, if the RI of the sample and the RI of the first objective match, but the remote refocusing optics are optimized for different RIs, the RR range will be reduced and the standard focusing range will remain. As disclosed herein, it is therefore counterintuitive to deviate from typical system designs in which the first objective and remote refocusing are optimized for the same sample RI. For example, the systems and methods disclosed herein with mismatched RIs can provide improved microscopy imaging by enabling objective lens selection to increase the range of time-lapse imaging, high-speed tiling, or increased NA for imaging live biological samples.

[0028] According to one aspect, a remote refocusing system is provided that is configured to image a sample. The system includes a sample refractive index n s The system includes a first microscope arranged to receive light from a sample in a medium having a first numerical aperture NA1. The first microscope includes a first objective lens having a first numerical aperture NA1 and a first immersion medium having a first refractive index n1. The system includes a second microscope having a second objective lens having a second numerical aperture NA2 and a second refractive index n2. The second objective lens is positioned and arranged to receive light passing through the first microscope. The combination of the first microscope and second microscope has a magnification M RR The system further includes an optical compensator disposed between the first microscope and the second microscope. The optical compensator is configured to generate an intermediate image of the sample at M RR (n s The optical system may include at least one lens having a linearly adjustable position such that the optical system is continuously adjusted to be approximately equal to the ratio of (n / n2).

[0029] Refractive index n s1A-1C, an embodiment of a remote refocusing system configured to image a sample dispersed in a medium of n is illustrated. Referring to FIG. 1A-1C, remote refocusing systems 100a, 100b, and 100c are configured to image a sample dispersed in a medium of n. s 1A-1C, the first microscope 102 includes first objective lenses 102a, 102b, and 102c having a first numerical aperture NA1 and a first immersion medium having a first refractive index n1. As illustrated in FIGS. 1A-1C, the three first objective lenses 102a, 102b, and 102c of the first microscope 102 provide a choice of first refractive index n1, providing a modular microscopy system capable of operation in three modes. The first microscope 102 also includes an optional folding mirror 102d for directing light, and a first tube lens 102e positioned to collect light from the optional folding mirror 102d and direct it into other optical components of the remote refocusing systems 100a, 100b, and 100c. s / n2) ratio is approximately equal to M RR Alternatively, or in addition, the first objective lenses 102a, 102b, and 102c are constructed and arranged to be switched between different objective lenses having the same focal length but different refractive indices n1 so as to maintain the refractive index (n s / n2) ratio is approximately equal to M RR The first objective lenses 102a, 102b, and 102c are constructed and arranged to be switchable between different objective lenses having different focal lengths and different refractive indices n1 so as to maintain the refractive index n1. The available choices for the first objective lenses 102a, 102b, and 102c and focal lengths are n less than n1. s This maximizes the NA for samples with λ / 2 and allows for imaging that increases the imaging depth for no-immersion imaging beyond the typical depth for standard focusing or wide-field microscopy setups.

[0030] The remote refocusing systems 100a, 100b, and 100c further include a second microscope 104 including a second objective lens 104a having a second numerical aperture NA2 and a second refractive index n2. The second objective lens 104a is positioned and arranged to receive light, e.g., substantially all light, passing through the first microscope 102, e.g., from the first objective lenses 102a, 102b, and 102c, the optional folding mirror 102d, and the first tube lens 102e. The collection half angle of the second objective lens 104a is greater than or approximately equal to the collection half angles of the first objective lenses 102a, 102b, and 102c. The second microscope 104 further includes a second tube lens 104b positioned between the first tube lens 102e and the second objective lens 104a. The image plane of the first microscope 102 is between the first tube lens 102e and the second tube lens 104b. When arranged together, the combination of the first microscope 102 and the second microscope 104 provides a magnification M RR 10 to generate an intermediate image 107 of the sample.

[0031] 1A-1C, systems 100a, 100b, and 100c include an optical compensator 106 disposed between first microscope 102 and second microscope 104. As shown, optical compensator 106 includes at least one of a first tube lens 102e and a second tube lens 104b, where one or both of first tube lens 102e and second tube lens 104b have linearly adjustable positions (n s / n2) ratio. RR Other configurations for the optical compensator, such as a zoom image relay system in which the optical compensator is disposed between the tube lenses of the first and second microscopes, are within the spirit of the disclosure, and the specific design of the optical compensator is in no way limited to the specific embodiments disclosed herein. At least the linearly adjustable lens of the optical compensator may be n s is usually M of the first objective lens n1 RR with the expectation that there will be no substantial deviation from a particular ns M against RR This is an advancement over existing remote refocusing microscopy systems, which have generally used static or fixed optical configurations to set the refractive index of the optical compensator 106. The use of at least one lens with a linearly adjustable position allows for tuning of the refractive index of the optical compensator 106 to a biologically relevant range, e.g., a refractive index range of about 1.33 to 1.51, such that the systems disclosed herein are suitable for imaging a wide range of live biological samples.

[0032] 1A-1C, systems 100a, 100b, and 100c include additional components for providing a display of an image of sample 101 to an end user or operator. As shown, systems 100a, 100b, and 100c can include an optional third microscope 108 having suitable optics for directing substantially all of the light from second microscope 104 to camera 109. Camera 109 can be any suitable camera used for microscopy and can be connected to any suitable display 110 to show a representation of imaged sample 101. The present disclosure is in no way limited by the choices for optional third microscope 108, camera 109, and display 110.

[0033] As disclosed herein, the system illustrated in FIGS. 1A-1C is configured to measure the refractive index of a fixed sample, n s and the remote region, i.e., the second microscope 104 and the optical compensator 106, provide for a choice of refractive indexes of the first objective lenses 102a, 102b and 102c that allow different modes of operation for n s The first mode of operation is illustrated in FIG. 1A, where the sample refractive index n s is equal to, or matches, the first refractive index n1, e.g., sample 101 is an aqueous sample and first objective lens 102a is a water immersion objective. In this configuration, n1 and n sMatching the sample refractive index n provides a microscopy system that increases the maximum range of the complete system for both the standard focusing objective, i.e., the first microscope 102 with the first objective 102a, and the remote focusing objective, i.e., the second microscope 104 with the second objective 104 and optical compensator 106. The second mode of operation is illustrated in FIG. 1B, in which the sample refractive index n s where n is above the first refractive index n1, e.g., a mismatched RI experiment in which sample 101 is an aqueous sample and first objective 102b is an air-immersion objective, i.e., no immersion. In this configuration, first objective 102b has a limited focal range in aqueous samples, and second microscope 104, having a remote-focusing objective, i.e., second objective 104 and optical compensator 106, is configured to image any sample with a refractive index between 1.33 and 1.51, which can be imaged by the optical compensator. This configuration extends the range of typical no-immersion microscopy for uses where it is beneficial not to use an immersion objective. A third mode of operation is illustrated in Figure 1C, in which the sample refractive index n s is less than the first refractive index n1, e.g., a mismatched RI experiment in which sample 101 is an aqueous sample and first objective lens 102c is an oil-immersion objective. In this configuration, first objective lens 102c provides a limited focal range for aqueous samples, while second microscope 104, having a remote-focusing objective, i.e., second objective lens 104 and optical compensator 106, is configured to image any sample with a refractive index between 1.33 and 1.51, which can be imaged by the optical compensator. This configuration, due to the large NA value of the oil-immersion first objective lens, increases the light collection of the microscopy system for improved imaging of samples that benefit from increased light collection, such as live biological samples.

[0034] In some embodiments, n sis within the range of 1.00 to 2.00, for example, 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00, for example, about 1.00, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.10, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.20, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.40, about 1.41, about 1.42, about 1.43, about 1.44 , about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.50, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.60, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.70, about 1.71, about 1.72, about 1 0.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.80, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.90, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, or about 2.00. In certain embodiments, n s is in the range of 1.33 to 1.51, e.g., the range for live biological samples.

[0035] In some embodiments, n1 is within the range of 1.00 to 2.00, e.g., 1.00 to about 1.40, about 1.25 to about 1.60, about 1.50 to about 1.80, or about 1.75 to about 2.00, e.g., about 1.00, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.10, about 1.11, about 1.12, about 1.13, or about 1. 14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.20, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.40, about 1.41, about 1.42, about 1.43 3, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, about 1.50, about 1.51, about 1.52, about 1.53, about 1.54, about 1.55, about 1.56, about 1.57, about 1.58, about 1.59, about 1.60, about 1.61, about 1.62, about 1.63, about 1.64, about 1.65, about 1.66, about 1.67, about 1.68, about 1.69, about 1.70, about 1.71, about 1.72 , about 1.73, about 1.74, about 1.75, about 1.76, about 1.77, about 1.78, about 1.79, about 1.80, about 1.81, about 1.82, about 1.83, about 1.84, about 1.85, about 1.86, about 1.87, about 1.88, about 1.89, about 1.90, about 1.91, about 1.92, about 1.93, about 1.94, about 1.95, about 1.96, about 1.97, about 1.98, about 1.99, or about 2.00. In certain embodiments, n1 is within the range of 1.00 to 1.51. For example, the range for n1 of 1.00 to 1.51 applies to the first objective lens for air immersion objectives, water immersion objectives, and oil immersion objectives. Other objective lenses for the first objective lens are also contemplated by this disclosure, and this disclosure is in no way limited by the selection of an objective lens and its associated refractive index for the first objective lens.

[0036] According to one aspect, a method of configuring a remote refocusing system including a first microscope and a second microscope for imaging a sample is provided. The method may include selecting a first objective for the first microscope based on a selected compromise between first, second, and third operating modes for the remote refocusing system and the refractive index of the sample. The first, second, and third operating modes are based on: 1) the sample refractive index n s a first operating mode using a non-immersion objective for the first microscope when n is greater than the first refractive index of the first objective; and 2) a first operating mode using a non-immersion objective for the first microscope when n is greater than the first refractive index of the first objective. s is substantially the same as the first refractive index n1 of the first objective lens, the focal range of the first objective lens is extended; and 3) the sample refractive index n s and a third operating mode maximizing the numerical aperture of the first objective lens when n is less than a first refractive index n of the first objective lens. The method may include selecting a second objective lens having a second refractive index n for the second microscope, wherein the first microscope and the second microscope have a magnification M RR The method may further include selecting an optical system combination for an optical compensator disposed between the first objective and the second objective to collect substantially all of the emitted light from the first objective. The optical compensator may be configured to generate an intermediate image of the sample having a wavelength of 1000 nm (n s / n2) ratio. RR The position may be linearly adjustable to provide

[0037] In some embodiments, selecting the first objective lens may include selecting an air immersion objective lens. In some embodiments, selecting the first objective lens may include selecting a water immersion objective lens. In some embodiments, selecting the first objective lens may include selecting an oil immersion objective lens. For example, the oil immersion objective lens may include a silicone oil immersion objective lens or a glycerol immersion objective lens. [Example]

[0038] The function and advantages of these and other embodiments may be better understood from the following examples, which are intended to be illustrative in nature and are not to be construed as limiting the scope of the invention in any way.

[0039] (Example 1) Standard wide-field imaging In this example, optical components compatible with a single-objective light-sheet (SOLS) microscope design were used to evaluate several configurations, allowing for control of the numerical aperture, focal length, and immersion medium for a given setup. Objectives used in the test setups included a 40x0.95 air-immersion objective, a 60x1.27 water-immersion objective, a 100x1.35 silicone oil-immersion objective, and a 100x1.45 oil-immersion objective. Using these settings, standard focusing and remote refocusing ranges were determined to be 1.33≦n s Sample refractive index n over the biological range of ≤ 1.51 s was calculated as a function of

[0040] 2A-2B illustrate the accessible imaging range of different objectives in a typical wide-field microscope, with FIG. 2A showing the range for the objective with the largest NA and FIG. 2B showing the range for the objective with the largest number of pixels, i.e., Nyquist pixels. In FIG. 2A-2B, the diffraction-limited depth of focus for standard focusing is plotted as a function of sample RI and calculated as follows:

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[0041] Example 2: Remote refocusing imaging To evaluate the deeper imaging potential of a remote refocusing microscope as disclosed herein, this example models a series of commercially available objectives used in the remote refocusing setup disclosed herein compared to a standard widefield microscope, i.e., a microscope without remote optics, as described in Example 1 and illustrated in Figures 2A-2B.

[0042] 3A-3B illustrate the accessible imaging ranges of different objectives in a combined microscopy system including a standard focusing, i.e., first microscope as described herein, and a remote refocusing microscope, i.e., a second microscope and optical compensator as described herein, e.g., as illustrated in FIGS. 1A-1C. FIG. 3A shows the range for the objective with the largest NA, and FIG. 3B shows the range for the objective with the largest pixel count, i.e., Nyquist pixels. In the combined standard focusing, i.e., first microscope as described herein, and remote refocusing, i.e., a second microscope and optical compensator as described herein, the first objectives used in the test setup included a 40x0.95 air-immersion objective, a 60x1.27 water-immersion objective, a 100x1.35 silicone oil-immersion objective, and a 100x1.45 oil-immersion objective. The inclusion of a second microscope and an optical compensator with an adjustable tube lens allows for the measurement of the sample RI n s This allowed for the optimization, i.e., tuning, of the remote refocusing optics, i.e., the tube lenses of one or both of the first or second microscopes, continuously over a range of . As illustrated in Figures 3A-3B, each of the tested configurations benefited from the additional imaging range provided by the optical compensator, and the maximum focusing range in the sample was the sum of the standard range and the remote range, i.e.,

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[0043] (Example 3) Zoom lens system 4A-4B illustrate a constant track length zoom lens design for incorporation into the second tube lens of a remote refocusing microscope as disclosed herein. Referring to the microscope configuration illustrated in FIGS. 1A-1C, the location of second tube lens 104b was selected as a convenient mounting point for the zoom lens, allowing first objective lenses 102a, 102b, 102c and first tube lens 102e to remain in their conventional, or stock, configuration. The constant track length, or double telecentric, design of the zoom lens maintained the axial position of both the front and back focal planes of the zoom lens, allowing the focal length to be adjusted over the full range, or 132.5-150 mm, without disrupting the microscope's optical train. In this example, the zoom lens was chromatically corrected across the visible spectrum, i.e., 450-700 nm, and was designed to be paired with a second microscope objective, i.e., objective 104a in Figures 1A-1C, having a back focal plane diameter of up to 9.5 mm, e.g., a Nikon 40x0.95 air-immersion objective suitable for diffraction-limited performance over the 13.5 mm diameter field typical of sCMOS imaging cameras. The zoom lens, with a focal length of 132.5-150 mm, and the second objective, with a focal length of 5 mm, allowed for continuously adjustable magnification of the second microscope in the range of 26.5-30x. Using the first microscope at 40x magnification in combination with the second microscope described above provided a remote refocusing magnification (M), which was continuously adjustable in the range of 1.33-1.51. RR ) were generated. Figure 4A illustrates a 1.51 magnification (f=132.5 mm) constant track length zoom lens configuration, and Figure 4B illustrates a 1.33 magnification (f=150 mm) constant track length zoom lens configuration.

[0044] While several aspects of at least one embodiment have been described above, it should be understood that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure and are intended to be within the scope of the present invention. Accordingly, it should be understood that the method and apparatus embodiments discussed herein are not limited in application to the details of construction and the arrangements of components set forth in the foregoing description or illustrated in the accompanying drawings. The methods and apparatuses are capable of implementation in other embodiments and of being practiced or carried out in various ways. Examples of specific implementations are provided herein for illustrative purposes only and are not intended to be limiting. Additionally, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. References to "or" may be construed as inclusive, such that any term described using "or" may refer to either a single one, a plurality, and all of the described term. Any references to front and back, left and right, top and bottom, upper and lower, and vertical and horizontal are intended for convenience of description and are not intended to limit the existing systems and methods or their components to any one location or spatial orientation. Accordingly, the foregoing description and drawings are by way of example only, and the scope of the invention should be determined from proper construction of the appended claims and their equivalents.

Claims

1. 1. A remote refocusing system configured to image a sample, the system comprising: Sample refractive index n ｓ a first microscope arranged to receive light from the sample in a medium having a first numerical aperture NA １ a first objective lens having a first refractive index n １ a first immersion medium having Second Numerical Aperture NA ２ and the second refractive index n ２ a second microscope including a second objective lens having a magnification M ＲＲ a second microscope configured to generate an intermediate image of the sample at an optical compensator disposed between the first microscope and the second microscope, ｓ / n ２ ) ratio. ＲＲ an optical compensator comprising at least one lens having a linearly adjustable position to provide A system comprising:

2. The sample refractive index n ｓ is the first refractive index n １ The system of claim 1 .

3. The sample refractive index n ｓ is the first refractive index n １ The system of claim 1 , wherein the

4. The sample refractive index n ｓ is the first refractive index n １ The system of claim 1 , wherein the system is substantially identical to

5. M ＲＲ is the ratio of the refractive index of the sample to the refractive index of the second microscope (n ｓ / n ２ 2. The system of claim 1, wherein the ratio is approximately equal to:

6. n ｓ The system of claim 1, wherein is in the range of 1.00 to 2.

00.

7. n ｓ The system of claim 6, wherein is in the range of 1.33 to 1.

51.

8. n １ The system of claim 1, wherein is in the range of 1.00 to 2.

00.

9. n １ The system of claim 8, wherein is in the range of 1.00 to 1.

51.

10. The first objective lens is configured such that the system (n ｓ / n ２ ) is approximately equal to the ratio of M ＲＲ The same focal length but different refractive index n １ 10. The system of claim 1, constructed and arranged to be switched between different objective lenses having:

11. The first objective lens is configured such that the optical compensator (n ｓ / n ２ ) is approximately equal to the ratio of M ＲＲ different focal lengths and different refractive indices n １ 10. The system of claim 1, constructed and arranged to be switched between different objective lenses having:

12. The system of claim 1 , wherein a collection half angle of the second objective lens is greater than or approximately equal to a collection half angle of the first objective lens.

13. 1. A modular microscopy system for imaging a sample, the system comprising: Sample refractive index n ｓ a sample stage constructed and arranged to hold a sample immersed in a sample medium having a First Numerical Aperture NA １ a first objective lens having a first refractive index n １ a first microscope having a first immersion medium having Second Numerical Aperture NA ２ and the second refractive index n ２ a second microscope having a second objective lens having a magnification M ＲＲ a second microscope configured to generate an intermediate image of the sample at Located between the first microscope and the second microscope, (n ｓ / n ２ ) ratio. ＲＲ an optical compensator comprising at least one lens having a linearly adjustable position to provide Equipped with The modular microscopy system operates in three modes: The sample refractive index n ｓ is the first refractive index n １ a first mode equal to The sample refractive index n ｓ is the first refractive index n １ a second mode exceeding The sample refractive index n ｓ is the first refractive index n １ a third mode, which is less than The sample refractive index n ｓ and the first refractive index n １ The system will be selected.

14. n ｓ The system of claim 13, wherein is in the range of 1.00 to 2.

00.

15. n ｓ The system of claim 14, wherein is in the range of 1.33 to 1.

51.

16. n １ The system of claim 13, wherein is in the range of 1.00 to 2.

00.

17. n １ The system of claim 16, wherein is in the range of 1.00 to 1.

51.

18. 1. A method of configuring a remote refocusing system for imaging a sample, the system including a first microscope and a second microscope, the method comprising: a first mode of operation, a second mode of operation, and a third mode of operation for the remote refocusing system; and a refractive index n of the sample. ｓ and selecting a first objective lens for the first microscope based on a selected compromise between: The sample refractive index n ｓ is the first refractive index n of the first objective lens １ a first mode of operation using a non-immersion objective for said first microscope when The sample refractive index n ｓ is the first refractive index n of the first objective lens １ a second operating mode in which the focal range of the first objective lens is expanded when The sample refractive index n ｓ is the first refractive index n of the first objective lens １ a third operating mode that maximizes the numerical aperture (NA) of the first objective lens when and a second refractive index n for the second microscope; ２ and selecting a second objective lens having a magnification M ＲＲ and selecting an optical system combination for an optical compensator disposed between the first objective lens and the second objective lens to collect substantially all of the emitted light from the first objective lens, wherein the optical compensator is configured to: ｓ / n ２ ) ratio. ＲＲ and having a linearly adjustable position to provide A method comprising:

19. 20. The method of claim 18, wherein selecting the first objective lens comprises selecting an air immersion objective lens.

20. 20. The method of claim 18, wherein selecting the first objective lens comprises selecting a water immersion objective lens.

21. 20. The method of claim 18, wherein selecting the first objective lens comprises selecting a silicone oil immersion objective lens.

22. 20. The method of claim 18, wherein selecting the first objective lens comprises selecting a glycerol immersion objective lens.

23. 20. The method of claim 18, wherein selecting the first objective lens comprises selecting an oil immersion objective lens.

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