microscope objective lens

Abstract

An optical system for a nonlinear microscope is disclosed. The optical system comprises, in order from the object side, a first lens group, one or more optical elements, and a second lens group. The first lens group is configured to focus illumination light of a first wavelength onto a sample and to collect light of at least a second wavelength from the sample. The first lens group has a transmission coefficient of at least 0.5 for light of the first wavelength and at least 0.3 for light of the at least second wavelength. The one or more optical elements are configured to split the light of the at least second wavelength from the light of the first wavelength. The second lens group is configured to correct aberrations of the first lens group for the first wavelength. The optical system is substantially object-space telecentric for light of the first wavelength.

Description

[Technical Field]

[0001] The present disclosure relates to microscope objectives, particularly, but not exclusively, to microscope objectives for non-linear microscopy systems. [Background technology]

[0002] Traditionally, optical pathology transmission microscopy relies on creating thin sections from specimens to obtain depth information. This is a time-consuming procedure and therefore not well suited for intraoperative use. Furthermore, creating thin sections typically requires fixation and staining of the specimen, which can affect specimen properties. Therefore, there is a need for a more rapid pathology method that does not require slicing and / or extensive and time-consuming preparation of the specimen.

[0003] Confocal microscopy can partially overcome the need to create thin slices by spatially filtering the light emitted from the sample. In this method, the detected light is limited to the light emitted from the focal point of the illumination source. By moving the focal point through the specimen, depth information can be obtained. Signals can be created through autofluorescence and / or staining with fluorescent dyes.

[0004] Nonlinear microscopy, such as second harmonic generation (SHG), third harmonic generation (THG), and multi-photon fluorescence microscopy, can provide contrast and multi-modal information without any sample preparation. Furthermore, nonlinear microscopy can improve image quality by generating signals only within the focal point of the illumination spot, with a very steep drop-off away from the focal point. Typically, a high-intensity infrared pulsed laser source is used to generate the illumination spot, while signals from the sample are detected in the visible and ultraviolet regions. When measuring thick samples (e.g., not sliced or even excised), epidetection is commonly used. That is, the lens system used to focus light into the sample is also used to collect signals from the sample. Therefore, microscope objectives with a very wide spectral transmission window, typically from the near infrared to the near ultraviolet, must be used. For example, a laser light source with a wavelength of 1060 nm requires high transmittance from about 350 to about 1100 nm.

[0005] However, currently available microscope objectives are typically optimized for either infrared or ultraviolet light and have very low or even no transmission for light outside the optimized range. In particular, currently available microscope objectives designed for confocal microscopy are optimized to image the full spectral window used and require highly complex apochromatic corrections to allow spatial filtering of the detected light. This typically results in the use of glasses, such as Schwer Flint, that have limited or no ultraviolet transmission.

[0006] Furthermore, to generate a high-resolution signal, the focal spot of the illumination light needs to be very narrow. This typically requires a microscope objective with a high numerical aperture (NA) and excellent focusing properties over the entire field of view, which in pathology systems needs to be very large, e.g., (0.5 mm). 2 Furthermore, a flat focal field is highly desirable. To the extent that microscope objectives with sufficiently wide transmission bands exist, they compromise one of several other parameters.

[0007] For example, US Patent Application Publication No. US2016 / 0116724A1 discloses a microscope objective lens with a high numerical aperture and a wide field of view, but does not disclose transmittance outside the visible range.

[0008] Japanese Patent Publication No. 2008 / 197127A discloses an objective lens for a multiphoton-excitation laser scanning microscope. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a need in the art for a microscope objective or similar optical system that has high transmittance from at least the near infrared to the near ultraviolet, combined with a high numerical aperture and a wide field of view.

[0010] It is an object of embodiments of the present disclosure to provide a microscope objective for a nonlinear microscope that avoids or at least reduces the drawbacks of the prior art. [Means for solving the problem]

[0011] In a first aspect, the present disclosure relates to an optical system for a nonlinear microscope, the optical system comprising, in order from an object side, a first lens group, one or more optical elements, and a second lens group. The first lens group is configured to focus illumination light of a first wavelength onto a sample and to collect light of at least a second wavelength from the sample. The first wavelength may be selected from 700 nm to 1200 nm, preferably 1000 nm to 1200 nm or 750 nm to 850 nm, more preferably 1040 nm to 1080 nm, or more preferably about 800 nm. Preferably, the first lens group has a transmission coefficient for light of the first wavelength of at least 0.5, more preferably at least 0.6, and for light of the at least second wavelength of at least 0.3, preferably at least 0.5, more preferably at least 0.6. The one or more optical elements are configured to separate the light of the at least second wavelength from the light of the first wavelength. The second lens group is configured to correct aberrations of the first lens group for the first wavelength. The optical system may be configured to be substantially object space telecentric for light at the first wavelength, preferably with a deviation from telecentricity of less than 1 mrad.

[0012] The optical system may be referred to as a microscope objective. As used herein, the term "microscope objective" refers to an optical system that includes one or more lenses for focusing light onto and / or collecting light from a sample and that is designed as a complete unit.

[0013] When the microscope objective is substantially object-space telecentric with respect to the light at the first wavelength (i.e., excitation light), the focus geometry is substantially constant across the field of view, and the angle and numerical aperture are substantially constant. As a result, the resolution is substantially constant across the field of view, as are the contrast and signal level.

[0014] In typical embodiments, the light of the at least second wavelength includes light of a second wavelength different from the first wavelength, and light of a third wavelength different from the first wavelength and the second wavelength.

[0015] The first lens group can have one or more aberrations, such as chromatic aberration or distortion, and the second lens group is configured to correct the one or more aberrations of the first lens group for the light at the first wavelength, e.g., the second lens group can have chromatic aberration or distortion for the first wavelength that is complementary to the chromatic aberration or distortion of the first lens group.

[0016] By dividing the microscope objective lens into a first lens group and a second lens group and dividing the light of the detection wavelength (i.e., the second wavelength and the third wavelength) between the first lens group and the second lens group, only the first lens group needs to have a high transmission coefficient over the entire spectral window, while the second lens group used to correct the illumination light may comprise glass with low transmission in the detection range. For example, the first lens group may comprise only lenses made of fluorite and / or (synthetic) quartz and / or crown glass, which typically have sufficient (near) UV transmittance, while the second lens group may comprise corrective lenses, which typically comprise dense or heavy flint glasses, which have low (near) UV transmittance or no (near) UV transmittance.

[0017] Nonlinear microscopy typically uses pulsed laser sources with very short pulse durations, such as so-called femtosecond lasers with pulse durations of less than 100 fs. Short pulses necessarily have relatively large spectral widths; for example, a femtosecond laser source with a central wavelength of 1050 nm may have a spectral width (FWHM) of about 20 to about 150 nm. The shorter the pulse, the wider the spectral width. As used herein, wavelength refers to the central wavelength unless otherwise specified. As used herein, parameters specified for a first wavelength, a second wavelength, or a third wavelength should be understood to apply to the entire wavelength spectrum of the pulse around the respective wavelength.

[0018] As used herein, a lens group comprises at least one lens. The lens group may comprise one or more lens subgroups. The at least one lens may comprise a simple lens and / or a compound lens, e.g., a cemented lens.

[0019] In one embodiment, the light of the first wavelength is infrared light, and the light of the at least second wavelength comprises visible light and / or ultraviolet light.In another embodiment, the light of the first wavelength is ultraviolet light, and the light of the at least second wavelength comprises visible light and / or infrared light.In such an embodiment, different wavelengths may have different requirements.For example, the transmission coefficient for visible light may be at least 0.7, and the transmission coefficient for ultraviolet light may be at least 0.5.

[0020] In one embodiment, the optical system has a numerical aperture of at least 0.9, preferably at least 1.1, and more preferably at least 1.2. Other embodiments may use even higher numerical apertures (NA), such as NA≧1.3 or NA≧1.4. The resulting numerical aperture also depends on the immersion fluid used. In some embodiments, a non-toxic (biocompatible) immersion fluid may be used, such as glycerol or a water / glycerol mixture. This may limit the achievable numerical aperture.

[0021] To obtain high optical resolution in a raster scanning microscope, the focusing of the laser light must be very precise. Therefore, a high numerical aperture over a wide field of view significantly improves the quality of the resulting image. In nonlinear microscopy, this argument is repeated many times (see below), further enhancing the advantages of high-NA objectives. When the numerical aperture is greater than 1, an immersion objective may be required, which is used in combination with an appropriately selected immersion medium, typically an immersion oil with a refractive index close to that of the first lens.

[0022] The lateral (i.e., in-plane) resolution of an optical system is inversely proportional to its NA, where the lateral resolution is given by the Abbe diffraction limit: dxy = λ / (2NA). The axial (i.e. depth) resolution is inversely proportional to the square of the NA: d z =2λ / NA 2 When very thin samples are used, as in classical pathology, the axial resolution is of little concern. However, in the case of microscope systems using thick samples (e.g., fresh biopsies or even living tissue), the axial resolution is determined by the size of the focal spot. This is particularly relevant for nonlinear microscopes, such as multi-photon microscopy. It is worth noting that in the case of harmonic signals, the squared / cubed scaling of the second / third harmonic signals results in a resolution improvement of a factor √2 or √3 for the second / third harmonic signals.

[0023] Furthermore, tight focus is not only important for resolution, but also for the strength / quality of the generated signal, which depends nonlinearly on the quality of the focusing. Therefore, both resolution and signal quality are affected by the focusing properties of the objective lens.

[0024] In the case of third harmonic signals, the generated signal has a peak intensity (W / m 2 ) because the area of the focal spot is inversely proportional to the square of the NA. For a small object in a relatively large focal area, the magnitude of the signal generated at the focal spot will be NA 6 In the case of a second harmonic signal, the signal is proportional to the square of the peak intensity, and therefore NA 4 is proportional to.

[0025] However, a high numerical aperture requires a wide aperture and a relatively large diameter lens, which results in increased aberrations. By correcting aberrations only for light at the first wavelength, a high numerical aperture can be combined with a substantially aberration-free optical system.

[0026] In one embodiment, the optical system is configured to be substantially object-space telecentric, optionally substantially bi-telecentric, with respect to light at the first wavelength, and the deviation from telecentricity may be less than 1 mrad.

[0027] The high dependence of signal intensity on the (effective) NA causes a strong drop-off in signal away from the center of the image, resulting in dark corners. This appears as vignetting, but is typically caused by imperfections / aberrations in the focusing of the laser light away from the center of the objective lens. This means that the usable field of view is limited by aberrations (rather than being diffraction limited). By making the optical system telecentric, the focusing properties (geometry) remain constant over a wide field of view. This improves image quality, especially at the periphery of the field of view. The apparent shape and position of features on the object do not change when the object is moved to a different position within the field of view.

[0028] An additional advantage of a telecentric system is that it provides constant magnification even at different distances between the focal plane and the objective, i.e., at different depths. This makes it easy to combine multiple 2D images into a 3D volume, for example. Furthermore, by eliminating magnification errors, a telecentric objective minimizes unintended and uncontrollable variations in magnification caused by the objective moving relative to the sample during focusing and translation. This significantly improves the accuracy of optical measurements.

[0029] Furthermore, because objects that are not in best focus are symmetrically blurred, features on a sample can be accurately measured even when the features are out of focus. This keeps the centroid position constant and allows for accurate location of features and edges without distortion. This also makes it easier to combine multiple images. This is particularly relevant in the case of raster scanning, where it is important to be able to accurately recombine multiple images from multiple scans together.

[0030] Further advantages include equal sightlines to all points within the field of view and equal collected light throughout the field of view. As a result, all features are visualized despite height variations within the sample. In addition, the light signal collected by the objective lens is collected identically within the entire field of view, thereby ensuring a homogeneous stimulation and response of the sample and allowing comparison of signal strength between different points / regions scanned.

[0031] In one embodiment, the light of the at least second wavelength includes light having a wavelength equal to half the first wavelength and / or light having a wavelength equal to one-third of the first wavelength, such as when the optical system is used for nonlinear microscopy including second and third harmonic signals.

[0032] Additionally or alternatively, the light at the at least second wavelength may also include one or more other wavelengths, typically one or more other wavelengths in the visible and / or (near) infrared spectrum, which may for example be associated with multi-photon fluorescence signals.

[0033] In one embodiment, the one or more optical elements comprise a first dichroic mirror, preferably a first long-pass dichroic mirror, for reflecting light in at least a first spectral portion of the one or more second wavelengths, and optionally a second dichroic mirror, preferably a second long-pass dichroic mirror, for reflecting light in a second spectral portion of the one or more second wavelengths, different from the first spectral portion.

[0034] In one embodiment, the first dichroic mirror is configured to compensate for optical distortion, eg, shift and / or dispersion, of the light at the first wavelength caused by the second dichroic mirror.

[0035] In one embodiment, the first wavelength is 700 nm to 1200 nm, preferably 1000 to 1200 nm, more preferably 1040 to 1080 nm, e.g., about 1050 nm or about 1060 nm. Laser sources in this wavelength range are relatively economical. Furthermore, light absorption by water has a minimum near 1050 nm and increases substantially at longer wavelengths. Because biological samples typically contain a significant amount of water, minimizing energy absorption by water reduces tissue heating. At shorter wavelengths, the third harmonic signal shifts toward the ultraviolet side of the spectrum, where sample absorption again becomes high and the signal becomes weaker. As described above, in such an embodiment, the light at the first wavelength can have a spectral width of greater than 35 nm, e.g., about 50 nm or greater.

[0036] In other embodiments, the laser can have a wavelength of 700-900 nm, preferably 750-850 nm, and more preferably about 800 nm, where light absorption by water is even lower than at 1050 nm, both the illumination light and the second harmonic signal (i.e., the light at the second wavelength) are in or at least close to the visible spectrum, and there is generally good availability of suitable optical components (including sensors).

[0037] When the first wavelength has a center wavelength of 700 to 1200 nm, the second wavelength is 350 to 600 nm, and the third wavelength is 233 to 400 nm. Thus, in these embodiments, the first wavelength is in the infrared range, while the third wavelength is in the ultraviolet range. The second wavelength can be in either the visible range or the ultraviolet range.

[0038] As used herein, ultraviolet light is defined as light having a wavelength between 10 nm and 400 nm, visible light having a wavelength between 400 nm and 700 nm, while infrared light is defined as light having a wavelength between 700 nm and 1 mm. When an optical element is described as having a parameter value, such as a transmission coefficient, in the infrared or ultraviolet range, the relevant subrange is intended, typically 700 to 1200 nm for infrared or 233 to 400 nm for ultraviolet. For example, when one embodiment is discussed having an illumination source with a central wavelength of 1050 nm, the relevant ultraviolet subrange can be about 340 nm to about 360 nm.

[0039] In one embodiment, the first lens group and the second lens group are configured such that, for the light at the first wavelength, the curvature of the focal field is less than 4 μm, preferably less than 2 μm, more preferably less than 1 μm, within a radial distance of at least 250 μm, preferably at least 400 μm, from a central axis of the optical system.

[0040] In one embodiment, the optical system has a resolution of at least (0.25 mm) 2 , preferably at least (0.5 mm) 2 For example, the field of view can be configured as a disk with a radius of 0.4 mm or as a square with sides of 0.5 mm.

[0041] In one embodiment, the optical system further comprises a correction element for the light of the second wavelength and / or the third wavelength. For example, the correction element may comprise a focusing element, e.g., a focusing lens, for focusing the light of the at least second wavelength onto one or more detectors. Alternatively or additionally, the correction element may be configured to correct chromatic aberration of the light of at least the second wavelength, e.g., caused by the first lens group.

[0042] The present disclosure also relates to a scanning nonlinear microscope system comprising the optical system described above, further comprising a laser light source, a 2D or 3D scanner system for moving a focal spot of the light at the first wavelength on or through the sample, a first detector for detecting the light at the second wavelength, and optionally a second detector for detecting the light at the third wavelength.

[0043] The invention will be further explained with reference to the accompanying drawings, which show, in a simplified form, embodiments according to the invention, it being understood that the invention is in no way limited to these particular embodiments.

[0044] Aspects of the present invention will be explained in more detail by reference to exemplary embodiments shown in the drawings. [Brief explanation of the drawings]

[0045] [Figure 1] FIG. 1 illustrates a schematic diagram of a microscope system according to one embodiment. [Figure 2A] FIG. 2A illustrates a schematic diagram of a microscope objective according to one embodiment. [Figure 2B] FIG. 2B illustrates a schematic diagram of a microscope objective according to one embodiment. [Figure 3] FIG. 3 illustrates schematically an example of a lens assembly of an optical system according to one embodiment. [Figure 4A] FIG. 4A illustrates various optical properties of the embodiment illustrated in FIG. [Figure 4B] FIG. 4B illustrates various optical properties of the embodiment illustrated in FIG. [Figure 4C] FIG. 4C illustrates various optical properties of the embodiment illustrated in FIG. [Figure 4D] FIG. 4D illustrates various optical properties of the embodiment illustrated in FIG.

[0046] The same reference numbers refer to identical or at least similar elements. DETAILED DESCRIPTION OF THE INVENTION

[0047] Embodiments of the present disclosure describe methods and systems for obtaining high-speed images of unprepared samples, such as fresh biopsies or biological tissue, using non-linear optics for contrast imaging. The non-linear optics may include, for example, second harmonic, third harmonic, autofluorescence, and multi-photon interactions. Thus, the embodiments enable high-resolution (pathological) images to be obtained during surgery and / or endoscopy, allowing surgical and / or endoscopic procedures to be adjusted based on pathological information. While the examples provided herein are primarily directed to pathological applications, other applications, such as industrial quality control, are not excluded.

[0048] It is an object of embodiments of the present disclosure to provide a microscope objective lens that is substantially planar, substantially telecentric, has a high NA over the entire field of view, has a diffraction-limited focus of the fundamental wavelength over the entire field of view, and has a wide field of view.

[0049] FIG. 1 schematically illustrates a microscope system according to one embodiment. The microscope system 100 includes a light source 102, which includes a laser. In the illustrated example, the laser is a pulsed laser generating a pulse train, such as a femtosecond laser, e.g., a mode-locked Ti:sapphire laser or a Yb fiber laser. Selecting a suitable laser light source may involve several trade-offs. Generally, the higher the peak intensity of each pulse, the stronger the nonlinear optical signal. For example, the second harmonic is proportional to the square of the peak intensity, and the third harmonic is proportional to the cube of the peak intensity. However, if the peak intensity is too high, it may cause so-called nonlinear damage to the sample 114. On the other hand, if the peak intensity is too low, a longer illumination time may be required to obtain sufficient image quality, which may be achieved by using longer pulses (hence, higher energy per pulse) and / or a larger number of pulses per pixel. This can result in thermal damage due to overheating of the tissue. The energy at which tissue damage occurs can depend, among other things, on the type of tissue, pulse intensity, pulse duration, wavelength, numerical aperture, and focal spot size. For imaging of a typical example of a biological sample, a system with a numerical aperture of 1 or more and a laser source with a wavelength of about 1060 nm can be used, with a pulse duration of less than 100 fs and an energy per pulse of about 1 nJ.

[0050] Similarly, the wavelength of the laser light source can be selected based on the intended use of the microscope system. Biological samples (including pathological samples) typically have a high water content. Therefore, to increase the maximum penetration depth and reduce energy absorption by the sample, it can be beneficial to select an illumination wavelength with low water absorption, such as about 800 nm or 1000-1200 nm, preferably about 1060 nm. The absorption spectrum of water has a minimum around 800 nm and a local minimum around 1060 nm.

[0051] The second harmonic signal has a wavelength that is half the illumination wavelength, and the third harmonic signal has a wavelength that is one-third the illumination wavelength. Thus, for an illumination wavelength of 800 nm, the second harmonic signal has a wavelength of 400 nm, and the third harmonic signal has a wavelength of 267 nm. The latter are in the (mid) ultraviolet part of the electromagnetic spectrum and have high absorption by the sample, resulting in a weak signal.

[0052] Therefore, in a microscope system not configured to measure third harmonic signals, a laser light source having a wavelength of approximately 0.80 μm may be used. In a microscope system configured to measure third harmonic signals, the laser light source may generate light having a wavelength of approximately 1.06 μm. In that case, the second harmonic has a wavelength of approximately 0.53 μm, and the third harmonic has a wavelength of approximately 0.35 μm. These wavelengths have relatively low absorption by typical biological samples.

[0053] Femtosecond pulses necessarily have significant spectral widths. For example, a femtosecond laser source with a central wavelength of 1050 nm or 1060 nm can have a spectral width of approximately 35 nm or more, and in some cases even 50 nm or more, depending on the characteristics of the laser source. This results in a spectral width of approximately 17.5 nm or more for the second harmonic signal (25 nm or more, respectively) and approximately 11.7 nm or more for the third harmonic signal (16.7 nm or more, respectively).

[0054] When the microscope system is used for fluorescence imaging, the imaging wavelength is typically longer than the excitation wavelength, in which case the laser source can be selected to optimize the fluorescence signal.

[0055] Optionally, a control optics system 103 downstream of the laser source can modify the pulse train generated by the laser source 102. The control optics system can, for example, modify the pulse intensity and / or selectively transmit pulses. In this way, bursts of pulses can be generated. The number of pulses per burst can vary based on, among other things, the sample, imaging depth, repetition rate, and energy per pulse. For example, a pulse picker such as that described in co-pending Dutch application NL2029545 can be used.

[0056] The illustrated system further comprises a two-dimensional raster scanner 104. The raster scanner may comprise a galvoscanner comprising a pair of galvomirrors. Other embodiments may use different raster scanners, for example based on MEMS mirrors or based on resonant mirrors. The control optical system 103 may be used to effectively switch off the illumination during the backsweep of the 2D raster scanner 104.

[0057] The system further includes an objective 105 for focusing the pulses onto a sample 114 and for collecting light that has interacted with the sample. The objective includes a main objective 108 and a correction objective 106 spatially separated from the main objective. The objective further includes one or more optical elements 107 disposed in the light beam between the main objective and the correction objective. The one or more optical elements are configured to split the light of the at least second wavelength from the light of the first wavelength.

[0058] In the illustrated example, the one or more optical elements 107 include one or more beam splitters 110 and 112. In the illustrated example, the first beam splitter is configured to deflect the third harmonic signal, and the second beam splitter is configured to deflect the second harmonic signal. The first dichroic mirror may be a first long-pass dichroic mirror, and the second dichroic mirror may be a second long-pass dichroic mirror. When two or more beam splitters are used, the two or more beam splitters can be configured to compensate for each other's distortions. For example, the first dichroic mirror can be configured to compensate for distortions, such as shifts and / or dispersion, of the light of the first wavelength caused by the second dichroic mirror. Optionally, the objective lens includes correcting optics 122 and 124. The objective lens is described in more detail below with reference to FIG. 2. An exemplary lens assembly is described below with reference to FIG.

[0059] The system further includes one or more detectors 116-120 for detecting light that interacts with the sample, such as light reflected and / or refracted by the sample 114 and / or light transmitted through the sample. For example, the system may include a first detector 116 for detecting a transmitted signal from the sample on the opposite side from the objective lens.

[0060] The system may include a second detector 118 for detecting light at a second wavelength, such as a second harmonic signal or a first fluorescence signal. The light at the second wavelength is split to the second detector using a beam splitter 110, such as a first dichroic mirror. Optionally, a correction optical system 122 may be located upstream of the second detector. However, if the focal spot is small enough so that all received signals can be assumed to be generated at the focal spot, a correction optical system may not be necessary.

[0061] The system may include a third detector 120 for detecting light at a third wavelength, e.g., a third harmonic signal or a second fluorescence signal. The light at the third wavelength is split to the third detector using a beam splitter 112, e.g., a second dichroic mirror. Optionally, a correction optical system 124 may be located upstream of the third detector.

[0062] In some embodiments, a single beam splitter may split light at both the second and third wavelengths, which may then be split into different signals.

[0063] The system may include one or more further detectors (not shown) for detecting other signals, for example a detector for detecting autofluorescence light.

[0064] One or more of the detectors may comprise a photomultiplier tube. In other embodiments, other types of detectors, for example, based on charge-coupled devices (CCDs), may be used. Photomultiplier tubes typically have very high sensitivity and can measure single photons. They are therefore suitable for detecting weak nonlinear signals, such as second and third harmonics, even at relatively low, and therefore non-damaging, peak light intensities. When photomultiplier tubes are used, the control optical system 103 is preferably configured to generate pulse bursts containing multiple pulses, which improves the linearity of the detector response. A linear response is typically preferred in many applications, such as quantitative analysis of microscopy images, particularly AI-based analysis.

[0065] The system further includes a controller 126, e.g., a field programmable gate array (FPGA). Preferably, a single controller controls the raster scanner, the data acquisition system, and, optionally, the control optical system. In this manner, real-time control and optimal synchronization are achieved. The controller may be connectable to a host computer via a data connection 130. System parameters, such as scan parameters (e.g., number of pulses per burst and energy per pulse), can be loaded into the controller from the host computer, and acquired data can be streamed from the controller to the host computer.

[0066] 2A is a schematic diagram of a microscope objective according to one embodiment, which includes, from the object side, a first lens group 108 (which may also be referred to as a main objective), one or more optical elements 107, and a second lens group 106 (which may also be referred to as a correction objective). Light beams between the first lens group and the second lens group may be essentially parallel.

[0067] The first lens group 108 is configured to focus illumination light of a first wavelength onto the sample and collect light of at least a second wavelength from the sample. Typically, there is a substantial spectral distance between the light of the first wavelength and at least a portion of the light of the at least second wavelength. For example, the light of the first wavelength λ can be infrared light, and the light of at least the second wavelength includes visible light λ / 2 and / or ultraviolet light λ / 3. Alternatively, the light of the first wavelength can be ultraviolet light, and the light of the at least second wavelength can be visible light and / or infrared light. This can be the case, for example, in fluorescence imaging.

[0068] For example, the microscope objective can be used for nonlinear microscopy or multi-photon imaging based on second and / or third harmonic signals, in which case the at least second wavelength includes light having a wavelength equal to half the first wavelength and light having a wavelength equal to one-third the first wavelength.

[0069] The first lens group 108 is substantially transparent to light of the first wavelength and the at least second wavelength. A high transmission coefficient for the at least second wavelength, i.e., the signal, is generally particularly important because a somewhat lower transmission coefficient for the first wavelength can typically be compensated for by increasing the intensity of the light source (or adjusting the control optical system). However, many glasses have relatively low transmission coefficients for ultraviolet light. When the at least second wavelength includes ultraviolet light, the first lens group preferably includes only glasses that are transparent to at least the relevant portion of the ultraviolet spectrum. For example, the first lens group may have a transmission spectrum 202 whose transmission coefficient exceeds a predetermined threshold across at least the relevant portion of the spectrum, in this case in the range of λ / 3 to λ, e.g., from about 340 nm to about 1100 nm.

[0070] In some embodiments, the first wavelength is between 700 nm and 1200 nm, e.g., between 1000 and 1200 nm, more particularly between 1040 and 1080 nm, e.g., about 1050 nm or about 1060 nm. In other embodiments, the laser can have a wavelength between 700 and 900 nm, more particularly between 750 and 850 nm, e.g., about 800 nm. When the first wavelength is between 700 and 1200 nm, the second harmonic signal has a wavelength between 350 and 600 nm, and the third wavelength signal has a wavelength between 233 and 400 nm. Thus, in these embodiments, the first wavelength is in the infrared range, while one or more second wavelengths may include light in the visible and / or ultraviolet ranges.

[0071] The limitations on glass selection imposed by transparency requirements, and possibly other requirements, can result in wavelength-dependent focus errors 204, such as chromatic or other optical aberrations.

[0072] The one or more optical elements 107 are configured to split the light of the at least second wavelength from the light of the first wavelength. In the illustrated embodiment, the one or more optical elements comprise a single beamsplitter 210, in this case a first dichroic cube beamsplitter, configured to deflect the (collected) light of the at least second wavelength while transmitting the light of the first wavelength.

[0073] In the illustrated example, a second beam splitter 212, in this case a second dichroic cube beam splitter, is provided downstream of first beam splitter 210 for separating the light at the second wavelength from the light at the third wavelength. In some embodiments, the second beam splitter may be included in the optical system, while in other embodiments, it may be a separate element or may be omitted, depending on the use case. Optional optical systems 122 and 124 are described below.

[0074] Other embodiments may use different types of beam splitters for the first beam splitter and / or the second beam splitter, for example, a plate beam splitter or a prism.

[0075] The second lens group 106 is configured to (pre-)correct a focus error 204 of the light at the first wavelength. For example, the second lens group can have a focus error 208, e.g., a second chromatic aberration, that is complementary to the focus error, e.g., a first chromatic aberration, of the first lens group. The second lens group can be optimized to reduce the focus error at the first wavelength, without needing to reduce the focus error at other wavelengths, particularly one or more second wavelengths. This is shown by the dotted line portions of graphs 204 and 206, which schematically represent the individual focus errors of the first and second lens groups, and does not require compensation. Only the solid line portions in a relatively small spectral window around the first wavelength are optimized.

[0076] By splitting the microscope objective into a first lens group 108 and a second lens group 106 and using one or more optical elements 107 to split light at the detection wavelengths (i.e., one or more second wavelengths) between the first and second lens groups, only the first lens group needs to have a high transmission coefficient 202 across the entire spectral window, while the second lens group, used to (pre-)correct the illumination light, can comprise glass 206 with low transmission in the detection range. For example, the first lens group may comprise only lenses made from fluorite and / or (synthetic) quartz, while the second lens group may comprise correction lenses comprising dense or heavy flint glass (which is typically not transparent to ultraviolet light).

[0077] The objective lens, and therefore the first lens group 108, preferably has a high numerical aperture, for example, at least 1, at least 1.1, or even at least 1.2, to enable a small focal spot. A high numerical aperture results in a smaller focal spot size and therefore higher spatial resolution. Furthermore, an increase in numerical aperture results in an increase in peak light intensity and therefore an increase in signal strength. Depending on the immersion liquid used, even higher numerical apertures can be obtained.

[0078] The first and second lens groups may be configured to provide a substantially flat field, i.e., minimize field curvature for the light at the first wavelength. For example, the objective lens may have a focal field curvature of less than 4 μm, less than 2 μm, or even less than 1 μm within a radial distance of at least 250 μm, or at least 400 μm, from a central axis of the optical system (as determined for the light at the first wavelength). A flat focal plane provides a sharp cross-section, thereby enhancing image sharpness and providing accurate depth information.

[0079] The first and second lens groups may be configured so that the field of view is essentially free of geometric distortion. A field of view free of geometric distortion results in a sharp and dimensionally accurate image. Therefore, regular image patches can be obtained from scanning (with minimal, if any, calibration), and the multiple patches can be further processed with high precision into a larger image by stitching together multiple undistorted image patches.

[0080] The first and second lens groups may be configured such that the objective is substantially object-space telecentric, or even substantially bi-telecentric. This allows high resolution and good signal quality to be maintained across the entire field of view. In particular, a substantially uniform image intensity across the entire field of view can be achieved for all relevant wavelengths. This includes a uniform illumination numerical aperture for all positions within the field of view for the first wavelength and a substantially uniform imaging (collection) numerical aperture for the one or more second wavelengths. Furthermore, the objective may have substantially no lateral chromatic aberration, even with chromatic focus plane shifts of 50 μm. As a result, chromatic focal plane shifts, such as those due to manufacturing tolerances, do not induce lateral chromatic shifts, because the lateral chromatic shift is less than 50 nm for deviations from telecentricity of less than 1 mrad, even with a chromatic focal plane shift of 50 μm. Obviously, the longitudinal chromatic aberration, ie the chromatic focal plane shift, is typically much smaller than 50 μm.

[0081] A further advantage of telecentric objectives is the high reproducibility of the images obtained by scanning. Even if thermal deviations and other effects induce focal plane shifts, a nearly constant magnification can be achieved. Deviations from telecentricity on the sample side of less than 1 mrad do not significantly change the magnification: a focal plane shift of 25 μm results in a magnification change of only 0.0005% (less than 25 nm over the full field of view). This also makes it easy to stitch together multiple scans to form a 2D mosaic image or a 3D image cube. Especially in the latter case, combining images obtained at different depths is very easy, since the shape or size of the object does not change even at different focal depths.

[0082] To this end, the microscope objective may include an aperture stop 210. The aperture stop may be located, for example, in the first lens group. Alternatively, the aperture stop may be located (as shown here) between the second lens group 106 and one or more optical elements 107. In this way, the collected light of the one or more second wavelengths is not attenuated by the aperture stop, maximizing signal strength.

[0083] In some embodiments, such as that illustrated in FIG. 2 , the optical system further includes correction optics 122 and 124 configured to correct the light of one or more second wavelengths, e.g., by correcting focus errors across a relevant portion of the light spectrum. In this manner, an objective lens can be used to obtain high-quality images (i.e., spatially resolved measurements) based on the light of the one or more second wavelengths. However, in other embodiments, only the light intensity of the one or more second wavelengths need be detected; in such embodiments, these additional correction optics may be omitted. Because the objective lens enables a very narrow focus of the illumination light, any light received by a detector measuring the light of the at least second wavelengths may be associated with the focal spot, and spatial resolution of this light may not be required.

[0084] 2B schematically illustrates a microscope objective lens in a different configuration according to one embodiment. In this example, the objective lens is essentially folded. This can shorten the length of the space in which the microscope objective lens must be installed. The combined length of the optical elements of the second lens group 106 can be significant, for example, on the order of decimeters.

[0085] The microscope objective in this example further uses dichroic plate-beamsplitters 220 and 222. However, in other embodiments, different types of beamsplitters may be used for the first beamsplitter and / or the second beamsplitter, such as a cube beamsplitter or a prism.

[0086] FIG. 3 schematically illustrates an example of a lens assembly of an optical system according to one embodiment. The left side of the figure is the object side, and the right side of the figure is the image side. In the illustrated example, the first lens group 108 includes six lenses L1-L6 and one aperture stop S1. Lenses L1, L2, and L4 are simple lenses, while lenses L3, L5, and L6 are compound lenses. The second lens group 106 includes four lenses L7-L10, of which lens L9 is a simple lens, while lenses L7, L8, and L10 are compound lenses. Between the first and second lens groups, there is space for one or more dichroic beam splitters (not shown). The one or more dichroic beam splitters may include cubic beam splitters and / or dichroic plates. If a dichroic plate is used, the dichroic plate may be at a 45° angle with the optical axis of the optical system. The dichroic plate may also be positioned under another angle, such as the Brewster angle. In the region between the first lens group and the second lens group, the light beam is essentially collimated.

[0087] Table 1 shows exemplary dimensions and materials for the optical system shown in FIG. 3 . The results shown in FIGS. 4A-4D were obtained using these exemplary values. These values were obtained by automated optimization in Zemax based on the following constraints and considerations: The first wavelength was selected as 1050±50 nm. The first lens group was optimized to maximize light collection for wavelengths of 350-750 nm (inclusive) while providing diffraction-limited imaging for wavelengths of 1000-1100 nm (first wavelength). The second lens group was optimized to minimize distortion of the entire lens assembly at the first wavelength, providing a diffraction-limited optical system with a substantially flat field and minimal geometric distortion. Additionally, the lens assembly was optimized to provide a substantially telecentric object side.

[0088] Clearly, those skilled in the art can determine many other suitable configurations based on these or similar requirements. For example, in this case, the first wavelength was selected as 1050±50 nm. If a different first wavelength is used, the optimization may result in a different lens assembly. Furthermore, in this example, the lens dimensions were limited to spherical lenses, but other embodiments may use aspherical lenses. However, this may result in increased costs and possibly reduced manufacturing precision. Generally, there are trade-offs between various optimization goals; for example, different goals may trade off slightly higher field curvature for higher light concentration at least at the second wavelength.

[0089] For example, a lens assembly according to one embodiment may be optimized at the first wavelength, e.g., 1000-1100 nm, to achieve a high-numerical-aperture diffraction-limited design using the following constraints: - the field curvature is flat, e.g., less than 1 μm within the field of view; -Distortion-free image, e.g., less than 0.025%; -Telecentric on the sample side, i.e. deviation from telecentricity less than 1 mrad; - a total transmittance of greater than 50% for light at a first wavelength and one or more second wavelengths (e.g., 335-1100 nm) using a suitably selected coating (e.g., a broadband MgF2 coating) on the lenses of said first lens group; - in a full optical system, a total transmittance of light at the first wavelength (e.g., 1000-1100 nm) greater than 50% using suitably selected coatings (e.g., near-infrared optimized coatings) on the lenses of the second lens group; a parallel beam between the first lens group and the second lens group; and The distance between the first lens group and the second lens group is sufficient (for example, 100 mm or more) to accommodate an optical component, such as a beam splitter.

[0090] Alternatively, one or more of these constraints can be part of the optimization function. In other embodiments, fewer, different, and / or additional constraints can be used. For example, constraints may be limited to the first lens group and the second lens group rather than the entire optical system.

[0091] In addition, the following manufacturability constraints may be imposed: - a sufficiently large clear semi-diameter margin, e.g. 1.5 mm; - Limited diameter of certain lenses; - Limited curvature of achromatic lenses (achromats); -etc.

[0092] Again, one or more of these constraints can be part of the optimization function.

[0093] Additionally, broadband anti-reflective coatings (e.g., MgF2 @ 400 nm) were used for the first lens group, while coatings highly efficient for light having wavelengths between 1000 and 1100 nm (e.g., MgF2 / ZrO2 / CeF2) were used for the second lens group. Broadband anti-reflective coatings are particularly advantageous when the light collected from a sample may have a relatively broad wavelength range, such as fluorescent light with multiple potential fluorescent markers or (possibly multiphoton) autofluorescence signals. Generally, a low overall transmittance of the light at the first wavelength can be tolerated because it can be compensated for, at least to some extent, by increasing the power of the light source.

[0094] However, depending on the envisaged use case, different coatings may be used, especially for the first lens group: for example, if the optical system is used for nonlinear microscopy, a double or triple waveband coating may be used that has good anti-reflective properties at the first (dominant) wavelength and at least the second wavelength, e.g., the second and / or third harmonic.

[0095] The illustrated lens assembly is optimized for use with glycerol as the immersion medium, in which case it has a numerical aperture of approximately 1.2. The advantage of using glycerol is that it is non-toxic and available in a sterile state, making it safe for use on living specimens, and it is generally easier to handle than toxic immersion media. This is particularly relevant for bedside microscopy applications. However, glycerol has a relatively low refractive index (approximately 1.46 for pure glycerol and lower for mixtures with water). Higher numerical apertures can be obtained if an immersion medium with a higher refractive index is used.

[0096] The illustrated lens assembly has a magnification of approximately 11.5x.

[0097] [Table 1]

[0098] 4A-4D illustrate various optical characteristics of the embodiment illustrated in FIG. 3. In particular, FIG. 4A illustrates the total transmittance, including both internal transmission losses and reflection losses, of the first lens group for wavelengths in the range of 370-1100 nm. For wavelengths in the range of 370-770 nm, the total transmittance is greater than 0.7, and for wavelengths in the range of 770-1100 nm, the total transmittance is greater than 0.65. This graph was obtained using a broadband MgF2 coating optimized at 400 nm. As previously mentioned in the description of FIG. 3, the total light transmittance can be improved, particularly for one or more wavelengths of interest, by using a different anti-reflection coating, perhaps at the expense of reducing transmittance at other wavelengths.

[0099] Figure 4B illustrates the total transmittance, including both internal transmission and reflection losses, of the entire full lens assembly (including the first and second lens groups) for wavelengths in the range 1000-1100 nm, where the transmission loss is dominated by the (reflection) loss of the first lens group.

[0100] Table 2 lists the internal transmittance at 10 mm of the glasses used in the lens assembly shown in FIG. 3 and listed in Table 1. Values are obtained from Schott. The first lens group uses only glasses with high to very high transmittance in the UV range, while the second lens group includes several glasses with much lower internal transmittance, and even one with essentially zero internal transmittance. It should be noted that the total transmittance will be lower due to reflective losses at the surfaces. These can be reduced using anti-reflective coatings.

[0101] Combining the data in Tables 1 and 2, the internal transmittance for the first lens group is 0.760, 0.986, and 0.988 for light with wavelengths of 350 nm, 546 nm, and 1060 nm, respectively; the internal transmittance for the second lens group is 0.000, 0.977, and 0.986 for light with wavelengths of 350 nm, 546 nm, and 1060 nm, respectively; and the internal transmittance for the full lens assembly is 0.000, 0.963, and 0.974 for light with wavelengths of 350 nm, 546 nm, and 1060 nm, respectively. This clearly shows that at least ultraviolet light needs to be deflected between the first and second lens groups.

[0102] [Table 2]

[0103] Figure 4C illustrates the field curvature and distortion of the lens assembly shown in Figure 3. The graph on the left shows the field curvature for light having a wavelength of 1000-1100 nm (from left to right) in 25 nm increments. Both the tangential (solid line) and sagittal (dotted line) components are shown. The horizontal axis shows the curvature in mm, and the vertical axis shows the radial distance from the optical axis in mm in the sample plane. It can be seen that the curvature is less than about 1 μm out to a radius of approximately 0.5 mm.

[0104] The graph on the right side of Figure 4C shows the corresponding distortion relative to the image center for light with wavelengths of 1000-1100 nm (from right to left) in 25 nm increments. The horizontal axis shows distortion in %, and the vertical axis shows radial distance from the optical axis in mm within the sample plane. It can be seen that distortion is less than about 0.02% up to a radius of approximately 0.5 mm.

[0105] Figure 4D illustrates spot diagrams on the sample for light with wavelengths between 1000 and 1100 nm. The circle represents an Airy disk with a radius of 0.527 μm. The graph on the left illustrates the spot image at the image center (i.e., on the optical axis), while the graph on the right illustrates the spot image at a radial distance of 0.4 mm from the image center. The root mean square (RMS) of the spot radius increases from 0.142 μm to 0.246 μm at a distance of 0.4 mm. This indicates that the lens assembly is diffraction-limited for light with wavelengths in the range 1000 to 1100 nm, at least for radial distances of 0.4 mm.

[0106] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that the words "comprises" and / or "comprising," when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0107] The corresponding structure, material, acts, and equivalents of any means or step-function-added element in the following claims are intended to encompass any structure, material, or acts for performing that function in combination with other claimed elements as specifically claimed. The description of the present invention has been presented for purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the invention. The embodiments were chosen and described to best explain the principles and practical application of the invention and to enable those skilled in the art to understand the invention in its various embodiments, with various modifications suited to the particular uses contemplated.

Claims

1. An optical system for a nonlinear microscope, in which, starting from the object side, A first lens group configured to focus illumination light of a first wavelength onto a sample and to collect light of at least one second wavelength, wherein the first wavelength is 700 nm to 1200 nm, and the first lens group has a transmittance coefficient of at least 0.5 for light of the first wavelength and at least 0.3 for light of the at least one second wavelength. One or more optical elements configured to split light of a first wavelength from light of at least one second wavelength, and A second lens group configured to correct the aberration of the first lens group with respect to the light of the first wavelength. It is equipped with, Here, the optical system is configured to be substantially telecentric in object space with respect to light of the first wavelength, and the deviation from telecentricity is less than 1 mrad. The aforementioned optical system.

2. The optical system according to claim 1, wherein the light of the first wavelength is infrared light, and the light of at least one second wavelength includes visible light and / or ultraviolet light.

3. The optical system according to claim 1 or 2, wherein the optical system has an numerical aperture of at least 0.

9.

4. The optical system according to claim 1 or 2, wherein the light of at least one second wavelength includes light having a wavelength equal to half the first wavelength and / or light having a wavelength equal to one-third the first wavelength.

5. The optical system according to claim 1 or 2, wherein the one or more optical elements include a first dichroic mirror for reflecting light of at least a first spectral portion of the at least one second wavelength.

6. The optical system according to claim 5, further comprising a second dichroic mirror for reflecting light of a second spectral portion of the at least one second wavelength that is different from the first spectral portion, and the first dichroic mirror is configured to compensate for distortion, e.g., shift and / or dispersion, of the light of the first wavelength caused by the second dichroic mirror.

7. The optical system according to claim 1 or 2, wherein the first lens group and the second lens group are configured such that, with respect to light of the first wavelength, the curvature of the focal field is less than 4 μm within a radial distance of at least 250 μm from the central axis of the optical system.

8. The optical system has at least (0.25 mm) 2 The optical system according to claim 1 or 2, configured to have a field of view.

9. The optical system according to claim 1 or 2, further comprising a correcting element for the light of at least one second wavelength.

10. A scanning nonlinear microscope system, An optical system, in which order from the object side, A first lens group configured to focus illumination light of a first wavelength onto a sample and to collect light of at least one second wavelength, wherein the first wavelength is 700 nm to 1200 nm, and the first lens group has a transmittance coefficient of at least 0.5 for light of the first wavelength and at least 0.3 for light of at least the second wavelength. One or more optical elements configured to split light of a first wavelength from light of at least one second wavelength, and A second lens group configured to correct the aberration of the first lens group with respect to the light of the first wavelength. It is equipped with, Here, the optical system is configured to be substantially telecentric in object space with respect to light of the first wavelength, and the deviation from telecentricity is less than 1 mrad. The optical system and, A laser light source and A 2D or 3D scanner system that moves the focal spot of the light of the first wavelength on or through the sample, At least one detector for detecting the light of at least one second wavelength and It is equipped with The aforementioned scanning nonlinear microscope system.

11. The scanning nonlinear microscope system according to claim 10, wherein the first wavelength is 1000 to 1200 nm.

12. The scanning nonlinear microscope system according to claim 10 or 11, wherein the light of at least one second wavelength includes at least one of light having a wavelength equal to half the first wavelength and light having a wavelength equal to one-third the first wavelength.

13. The scanning nonlinear microscope system according to claim 10 or 11, wherein the optical system has an numerical aperture of at least 0.

9.

14. The scanning nonlinear microscope system according to claim 10 or 11, wherein the first lens group and the second lens group are configured such that, with respect to light of the first wavelength, the curvature of the focal field is less than 4 μm within a radial distance of at least 250 μm from the central axis of the optical system.

15. The scanning nonlinear microscope system according to claim 10 or 11, wherein the optical system is configured to have a field of view of at least (0.25 mm) 2.

16. The optical system according to claim 1 or 2, wherein the first wavelength is 1000 to 1200 nm.

17. The optical system according to claim 1 or 2, wherein the first lens group has a transmittance coefficient of at least 0.5 for the light of at least one second wavelength.

18. The optical system according to claim 1 or 2, wherein the optical system has an numerical aperture of at least 1.

1.

19. The optical system according to claim 1 or 2, wherein the first lens group and the second lens group are configured such that, with respect to light of the first wavelength, the curvature of the focal field is less than 2 μm within a radial distance of at least 400 μm from the central axis of the optical system.

20. The optical system according to claim 1 or 2, wherein the optical system is configured to have a field of view of at least (0.5 mm) 2.

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