Hyperspectral microscopy systems & methods

Abstract

A hyperspectral widefield microscope and a kit for converting a microscope into a hyperspectral widefield microscope are disclosed. The kit comprises a hyperspectral transmission illumination source and hyperspectral camera having a linear spectral filter, a translation device arranged to move the linear spectral filter such that the relative position of the light captured by the microscope and the spectral filter can be adjusted, and an image capture device to capture spectrally filtered light. The kit may also include a z-translation adapter configured to move a sample holder of the microscope towards and away from an objective lens of the microscope. The kit may also include a hyperspectral lateral lighting system suitable for dark-field microscopy.

Description

HYPERSPECTRAL MICROSCOPY SYSTEMS & METHODSCROSS-REFERENCE TO PREVIOUS APPLICATION

[0001] This application claims priority from United States provisional patent application 63 / 778,728 filed on March 27, 2025, which is incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure generally relates to the field of microscopy. In particular, various embodiments are described herein that relate to hyperspectral microscopy systems and methods.INTRODUCTION

[0003] The following paragraphs are provided by way of background to the present disclosure. They are not, however, an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art.

[0004] Advances in digital imaging and optical technologies, supported by new powerful microelectronics and computational tools, have enabled the development and introduction to the market of advanced types of instruments and tools that overcome the limitations of existing methods and protocols.

[0005] An example of such advancements is multi-hyperspectral imaging, which provides a more informative dataset and a more precise and accurate characterization of an object under study, leading to better decision-making and more accurate results in various scientific, industrial, and medical fields.

[0006] Hyperspectral white and dark-field microscopy and material characterization is a rapidly evolving field with growing applications across various industries, from healthcare to agriculture and environmental monitoring. While the technology has made a number of improvements in terms of efficiency and data analysis, significant challenges remain relating to cost, miniaturization and accessibility.

[0007] There is therefore a clear need for hyperspectral microscopy systems and methods that address the challenges and / or shortcomings described above.SUMMARY

[0008] Various embodiments of hyperspectral microscopy systems and methods using tunable linear spectral filters are provided according to the teachings herein.

[0009] According to an aspect of the present disclosure, there is disclosed a hyperspectral widefield microscope including a linear spectral filter and an objective lens to direct captured light onto the linear spectral filter. The hyperspectral widefield microscope further comprises a translation device arranged to move the linear spectral filter such that the relative position of the captured light and the spectral filter can be adjusted. The hyperspectral widefield microscope further comprises an image capture device to capture spectrally filtered light.

[0010] In some examples, the hyperspectral widefield microscope further comprises a hyperspectral transmission illumination source.

[0011] In some examples, the hyperspectral transmission illumination source is a Light Emitting Diode (LED) illumination source.

[0012] In some examples, the hyperspectral transmission illumination source includes wavelengths from 400nm to 950nm.

[0013] In some examples, the hyperspectral widefield microscope further comprises a z-translation adapter configured to move a sample holder of the microscope towards and away from the objective lens.

[0014] In some examples, the hyperspectral widefield microscope further comprises a hyperspectral lateral lighting system suitable for dark-field microscopy.

[0015] In some examples, the hyperspectral lateral lighting system comprises a plurality of colour LEDs.

[0016] In some examples, the plurality of colour LEDs produce light in the wavelengths between 400nm and 950nm.

[0017] In some examples, the hyperspectral widefield microscope further comprises a computer control system configured to control one or more of: the intensity of the hyperspectral transmission illumination source, the position of the objective lens by controlling the z-translation adapter; and the position of the linear spectral filter by controlling the translation device.

[0018] In some examples, the computer control system is configured to synchronize the position of the objective lens and the position of the linear spectral filter to mitigate chromatic aberrations.

[0019] In some examples, the computer control system is configured to synchronize the intensity of the hyperspectral transmission illumination source and the position of the linear spectral filter to at least partially compensate for spectral variations in the intensity of the hyperspectral transmission illumination source.

[0020] In some examples, the computer control system is further configured to control each of the plurality of colour LEDs individually to mitigate phototoxicity.

[0021] In some examples, the computer control system is further configured to control the image capture device to capture a series of images and move the position of the objective lens by controlling the z-translation adapter between each image in the series. The resulting series of images is suitable to be converted into a 3D image of a sample.

[0022] According to another aspect of the present disclosure, there is disclosed a kit for converting a microscope into a hyperspectral widefield microscope. The kit comprises a hyperspectral camera and a hyperspectral transmission illumination source.

[0023] In some examples, the hyperspectral camera comprises a linear spectral filter and a translation device arranged to move the linear spectral filter such that the relative position of the light captured by the microscope and the spectral filter can be adjusted. The kit also comprises an image capture device to capture spectrally filtered light.

[0024] In some examples, the kit further comprises a z-translation adapter configured to move a sample holder of the microscope towards and away from an objective lens of the microscope.

[0025] In some examples, the kit further comprises a hyperspectral lateral lighting system suitable for dark-field microscopy.

[0026] In some examples, the hyperspectral lateral lighting system comprises a plurality of colour LEDs.

[0027] In some examples, the plurality of colour LEDs produce light in the wavelengths between 400nm and 950nm.

[0028] In some examples, the kit further comprises a computer control system configured to control one or more of: the intensity of the hyperspectral transmission illumination source, the position of the objective lens by controlling the z-translation adapter, and the position of the linear spectral filter by controlling the translation device.

[0029] In some examples, the computer control system is configured to synchronize the position of the objective lens and the position of the linear spectral filter to mitigate chromatic aberrations.

[0030] In some examples, the computer control system is configured to synchronize the intensity of the hyperspectral transmission illumination source and the position of the linear spectral filter to at least partially compensate for spectral variations in the intensity of the hyperspectral transmission illumination source.

[0031] In some examples, the computer control system is further configured to control each of the plurality of colour LEDs individually to mitigate phototoxicity.

[0032] In some examples, the computer control system is further configured to: control the image capture device to capture a series of images and move the position of the objective lens by controlling the z-translation adapter between each image in the series. The resulting series of images is suitable to be converted into a 3D image of a sample.

[0033] Other features and advantages of the present disclosure will become apparent from the following detailed description taken together with the accompanying drawings. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.DRAWINGS

[0034] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which showat least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein. In the drawings:

[0035] FIG. 1 shows a hyperspectral microscopy system in accordance with various embodiments of the present disclosure;

[0036] FIG. 2 shows a simplified schematic diagram of a hyperspectral microscopy system in accordance with various embodiments of the present disclosure;

[0037] FIGs. 3A and 3B show simplified schematic diagrams of a hyperspectral camera in accordance with various embodiments of the present disclosure;

[0038] FIG. 4 shows a graph of a spectral characteristic of a hyperspectral transmission light source;

[0039] FIG. 5 shows a simplified schematic diagram of a lateral lighting system in accordance with various embodiments of the present disclosure; and

[0040] FIGs. 6A and 6B include flowcharts showing simplified representations of various methods of operation of a hyperspectral microscopy system in accordance with various embodiments of the present disclosure.

[0041] Further aspects and features of the example embodiments described herein will appearfrom the following description taken togetherwith the accompanying drawings.DESCRIPTION OF VARIOUS EMBODIMENTS

[0042] Various embodiments in accordance with the teachings herein will be described below to provide an example of at least one embodiment of the claimed subject matter. No embodiment described herein limits any claimed subject matter. The claimed subject matter is not limited to devices, systems, or methods having all of the features of any one of the devices, systems, or methods described below or to features common to multiple or all of the devices, systems, or methods described herein. It is possible that there may be a device, system, or method described herein that is not an embodiment of any claimed subject matter. Any subject matter that is described herein that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors, or owners do not intend to abandon, disclaim, or dedicate to the public any such subject matter by its disclosure in this document.

[0043] It will be appreciated that for simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the embodiments described herein. Also, the description is not to be considered as limiting the scope of the embodiments described herein.

[0044] It should also be noted that the terms “coupled” or “coupling” as used herein can have several different meanings depending on the context in which these terms are used. For example, the terms coupled or coupling can have a mechanical or electrical connotation. For example, as used herein, the terms coupled or coupling can indicate that two elements or devices can be directly connected to one another or connected to one another through one or more intermediate elements or devices via an electrical signal, electrical connection, ora mechanical element depending on the particular context.

[0045] It should also be noted that, as used herein, the wording “and / or” is intended to represent an inclusive-or. That is, “X and / or Y” is intended to mean X or Y or both, for example. As a further example, “X, Y, and / or Z” is intended to mean X or Y or Z or any combination thereof.

[0046] It should be noted that terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree may also be construed as including a deviation of the modified term, such as by 1%, 2%, 5%, or 10%, for example, if this deviation does not negate the meaning of the term it modifies.

[0047] Further, although method steps may be described (in the disclosure and / or in the claims) in a sequential order, such methods may be configured to work in alternate orders. In other words, any sequence or order of steps that may be described does not necessarily indicate a requirement that the steps be performed in that order. The steps of methods described herein may be performed in any order that is practical. Further, some steps may be performed simultaneously.

[0048] The example embodiments of the devices, systems, or methods described in accordance with the teachings herein may be implemented as a combination of hardware and software. For example, the embodiments described herein may be implemented, at least in part, by using one or more computer programs, executing on one or more programmable devices comprising at least one processing element and at least one storage element (i.e., at least one volatile memory element and at least one nonvolatile memory element). The hardware may comprise input devices including one or more of a touch screen, a keyboard, a mouse, buttons, keys, sliders, and the like, as well as one or more of a display, a printer, and the like depending on the implementation of the hardware.

[0049] It should also be noted that there may be some elements that are used to implement at least part of the embodiments described herein that may be implemented via software that is written in a high-level programming language. The program code may be written in Rust, C++, C#, JavaScript, Python, or any other suitable programming language and may comprise modules or classes, as is known to those skilled in the art. Alternatively, or in addition thereto, some of these elements implemented via software may be written in assembly language, machine language, orfirmware as needed. In either case, the language may be a compiled or interpreted language.

[0050] At least some of the programs associated with the devices, systems, and methods of the embodiments described herein may be capable of being distributed in a computer program product comprising a computer readable medium that bears computer usable instructions, such as program code, for one or more processing units. The medium may be provided in various forms, including non-transitory forms such as, but not limited to, one or more diskettes, compact disks, tapes, chips, and magnetic and electronic storage. In alternative embodiments, the medium may be transitory in nature such as, but not limited to, wire-line transmissions, satellite transmissions, internet transmissions (e.g., downloads), media, digital and analog signals, and the like. The computer useable instructions may also be in various formats, including compiled and non-compiled code.

[0051] As used herein, the term “multi-spectral imaging” means any imaging technique that captures images across several discrete wavelength bands.

[0052] As used herein, the term “hyperspectral imaging” means any imaging technique that captures a wider range of electromagnetic spectrum bands than multi-spectral imaging. Unlike traditional imaging systems that capture images in only a few bands, hyperspectral imaging records data across many (often hundreds) of contiguous spectral bands. This results in detailed spectral information for each pixel in an image.

[0053] As used herein, the term “wide field” or “wide field microscopy” means any optical system capable of capturing an image of the entire object (e.g., specimen sample) in a single exposure.

[0054] As used herein, the term “dark-field” or “dark-field microscopy” means a technique used in light microscopy that enhances the contrast in unstained samples by illuminating a sample with light that is angled in such a way that it does not enter the objective lens directly. Instead, only light that has been scattered by the sample enters the objective lens, thereby creating a bright image against a dark background.

[0055] As used herein, the term “3D microscopy” means a set of techniques used to create three-dimensional (3D) images or models of samples by collecting data at multiple focal planes and / or angles.

[0056] As used herein, the term “3D hyperspectral microscopy” means an imaging technique that combines three-dimensional (3D) imaging with hyperspectral analysis to obtain detailed spatial and spectral information about a sample.

[0057] In accordance with the teachings herein, there are provided various embodiments of a new optical approach and microscopy system for optical imaging. This novel system is advantageous when used, for example, in the spectroscopic identification of individual plasmonic nanoparticles (NPs) in a homogeneous medium, in fixed or live cell preparations as well as on the surface of thick bio-tissues.

[0058] The system includes hyperspectral optical illumination and detection by using a linear spectral filter. In some embodiments, the system also includes a reflected light microscopy (RLM) dark-field mode with a high numerical aperture imaging objective. The presently disclosed system removes many of the inherent limitations of conventional dark-field microscopy solutions.

[0059] Hyperspectral imaging systems can be broadly categorized based on how they capture spectral data. Two of the most common methods are wide-field hyperspectral imaging and pushbroom hyperspectral imaging. These methods differ mainly in their design, data acquisition strategy, and the way an object is scanned.

[0060] Pushbroom hyperspectral imaging is a type of remote sensing technology that captures images by continuously scanning a scene with a linear array of detectors, typically using a spectrometer to capture data across multiple spectral bands.

[0061] Wide-field hyperspectral imaging is a technique that captures images over a broad range of wavelengths, providing both spatial and spectral information. It combines the ability to capture large areas (i.e. , wide-field) with the detailed spectral information from multiple wavelengths (i.e., hyperspectral), which is beyond what the human eye can perceive.

[0062] Most industrial equipment on the market is based on the pushbroom method, which provides high spectral resolution and is optimal for satellite imaging, remote sensing, aerial mapping, and industrial inspections (e.g., quality control on production lines). Pushbroom hyperspectral imaging systems however acquire spectral data by scanning a scene line by line, rather than capturing an entire object at once. This is why wide-field hyperspectral imaging is advantageous for applications in microscopy.

[0063] There is a growing need for affordable wide-field hyperspectral technology that leverages recent advances in spectral sensor design and optical microscopy integration. Another aspect of existing multi-hyperspectral imaging systems is that they are usually limited to conventional transmission or reflection microscopy.

[0064] Dark-field microscopy is a microscopy technique that enhances the contrast in unstained, transparent specimens, making it easier to observe structures that would otherwise be difficult to see under bright field illumination. Unlike traditional light microscopy, which uses direct light to illuminate the sample, dark-field microscopy uses specialized optics to direct light at an angle, which causes scattered light to enter an objective lens, creating a high-contrast image against a dark background.

[0065] The present disclosure provides systems that increase the efficiency and flexibility of dark-field hyperspectral microscopy, by providing new, more efficient and optimal dark-field illumination methods that allow for rapid and accurate spectral discrimination while maintaining spatial, preferably 3D resolution of objects and compatible with conventional transmission microscope modes.

[0066] FIG. 1 shows a hyperspectral microscopy system in accordance with various embodiments of the present disclosure and FIG. 2 shows a simplified schematicdiagram of a hyperspectral microscopy system 100 in accordance with various embodiments of the present disclosure.

[0067] The hyperspectral microscopy system 100 comprises a hyperspectral camera 101. The hyperspectral camera 101 is configured to operate in either a tunable hyperspectral mode or a monochrome imaging mode, as described in more detail elsewhere herein. The hyperspectral microscopy system also comprises a high magnification numerical aperture (NA) objective 102. In some embodiments, the NA objective 102 is coupled to a z-translation adapter 103 configured to adjust the focal position of the NA objective 102 during hyperspectral scanning, as described in more detail elsewhere herein. The hyperspectral microscopy system 100 comprises a stage 105 upon which can be placed a specimen 104 for observation. The hyperspectral microscopy system 100 also comprises a transmission light source 106 configured to illuminate the specimen from the opposite side of the NA objective 102. In some embodiments, the hyperspectral microscopy system 100 comprises a lateral illumination module 107 comprising an RGB colour light source 108 and a hyperspectral light source 109.

[0068] The hyperspectral microscopy system 100 also includes a computer control system 201. The computer control system 201 may include a processor 202, memory 203 including one or more data storage devices. Processor 202 may comprise one or more processors for performing processing operations that implement functionality of the various methods described herein with reference to FIGs. 6A and 6B, for example. Processor 202 may be a general-purpose processor executing program code stored in memory 203 to which it has access via internal data-communication bus 206. Alternatively, a processor of the processors 202 may be a specific-purpose processor comprising one or more preprogrammed hardware or firmware elements (e.g., application-specific integrated circuits (ASICs), electrically erasable programmable readonly memories (EEPROMs), etc.) or other related elements.

[0069] Memory 203 comprises one or more storage devices for storing program code executed by processor 202 and data used during operation of the hyperspectral microscopy system 100. Memory 203 may be a semiconductor medium (including, e.g., a solid-state memory), a magnetic storage medium, an optical storage medium, and / orany other suitable type of memory. Storage devices of memory 203 may be read-only memory (ROM) and / or random-access memory (RAM), for example.

[0070] In some embodiments, two or more elements of processor 202 may be implemented by devices that are physically distinct from one another and may be coupled to one another via data-communication bus 206. As will be appreciated by the skilled reader, the hardware components of the computer control system 201 may be implemented in any suitable way in order to implement the methods disclosed herein.

[0071] In some embodiments, the computer control system 201 can include a communication module 205 configured to communicate with, and control operation of, hyperspectral camera 101, z-translation adapter 103, RGB colour light source 108, hyperspectral light source 109, and transmission light source 106 in accordance with the methods disclosed herein.

[0072] The computer control system 201 may also be configured to interface and be controlled by external computer devices (not shown) which may be configured to implement a User Interface (Ul) for allowing users to monitor, control and / or receive imaging information from hyperspectral microscopy system 100.

[0073] In some embodiments, transmission light source 106 may be a hyperspectral light source. Non-limiting examples of hyperspectral light sources include white Light Emitting Diodes (LEDs). In some embodiments, the white LEDs may be used in conjunction with known linear drivers and thermostabilization means provided by, for example, Meerstetter Engineering™. The thermostabilization may use one or more thermoelectric coolers (TEC) for temperature regulation. TECs, also known as Peltier devices, are solid-state devices that use the Peltier effect to transfer heat from one side of the device to another. In preferred embodiments, the thermostabilization means is adapted to the specific number and type of LED.

[0074] As will be appreciated by the skilled reader, the temperature of the transmission light source 106 (e.g., an LED source) must be stable to avoid changes in spectral properties. Modulation of the transmission light source 106 by computer control system 201 may be used to synchronize the illumination time with the exposure time of hyperspectral camera 101 to minimize the effect of phototoxicity. Modulation of the hyperspectral camera 101 may also be used by computer control system 201 to synchronize a short pulse of illumination with the highest possible LED intensity (current)(e.g., 3 to 10 times higher than continuous illumination) with the exposure time of hyperspectral camera 101 to maximize the intensity of the transmitted light and make the exposure shorter and scans faster.

[0075] Computer control system 201 may be operable to control the intensity, modulation and temperature of the transmission light source 106. As can be seen from FIG. 4, most hyperspectral light sources have characteristics that vary over a spectrum. FIG. 4 shows the radiant intensity of a hyperspectral LED as a function of the wavelength of interest. In preferred embodiments, computer control system 201 may be used to increase or decrease the intensity of the transmission light source 106 in order to keep the transmission light 113 incident on the NA objective 102 at a relatively stable intensity. In other embodiments, computer control system 201 may be used to generate a light having a flat spectral profile of intensity on a sample and to then vary hyperspectral camera 101 exposure time to maintain an optimal dynamic range according to the spectral sensitivity of hyperspectral camera 101. In yet other embodiments, the computer control system 201 may be used to simply generate a light using the original spectral profile of intensity of the LED and then to vary the hyperspectral camera 101 exposure time to maintain an optimal dynamic range. In yet other embodiments, the computer control system 201 may be used to generate a light using the spectral profile of intensity of the LED adapted to the spectral sensitivity of the hyperspectral camera 101 , and the hyperspectral camera 101 may then use the same exposure time for all spectral scans.

[0076] FIGs. 3A and 3B show a simplified schematic diagram of a hyperspectral camera 101 in accordance with various embodiments of the present disclosure. Hyperspectral camera 101 comprises a lens 301 for focusing light onto an image sensor 302. The hyperspectral camera also comprises a linear spectral bandpass filter 304 coupled to a translation device 303 operable to move spectral filter 304 into and across the area situated between the lens 301 and the image sensor 302 so as to tune the camera to capture only a part of the spectrum of incoming light. As will be appreciated by the skilled reader, the width of spectral bandpass filter 304 can be adapted to the size of the image sensor 302. In some embodiments a 12mmx60mm filter may be used. In other embodiments, a 25mmx60mm filter may be used.

[0077] In some embodiments, the image sensor may be a (Complementary Metal-Oxide-Semiconductor) CMOS image sensor to capture images or video. CMOS sensorsare a type of semiconductor technology commonly used in modern cameras, including smartphones, digital cameras, and webcams. For example, the hyperspectral camera 101 may use an IMX676 CMOS sensor commercialized by the Sony Corporation®.

[0078] In some embodiments, the linear spectral bandpass filter 304 may be a linear variable interference filter. In some embodiments, the spectral range of the spectral bandpass filter 304 may be one of 400nm to 800nm, 380nm to 1100nm, and 1100nm to 1700nm. In some embodiments, the spectral bandpass filter 304 used may be one commercialized by Materion Balzers Optics™.

[0079] In some embodiments, the translation device 303 may be a high-resolution servo motor. The translation device 303 may be functionally coupled to and operable to move the spectral bandpass filter 304 between the lens 301 and the image sensor 302, as shown in FIG. 3B, so as to have hyperspectral camera 101 operate in a tunable hyperspectral mode or, to move the spectral bandpass filter 304 out of the area between the lens 301 and the image sensor 302, as shown in FIG. 3A, so as to have the hyperspectral camera 101 operate in a monochrome mode.

[0080] As shown in FIG. 1, hyperspectral microscopy system 100 provides a system to compensate for wavelength-dependent displacement of the image plane (i.e., optical chromatic aberration). Optical chromatic aberration is a type of distortion in optical systems caused by the different wavelengths of light being refracted by different amounts. This occurs because lenses have varying refractive indices for different colors of light, and shorter wavelengths (e.g., blue light) are bent more strongly than longerwavelengths (e.g., red light). This results in a situation where the lens cannot focus all the colors onto the same point, leading to a visible blur or color fringing along the edges of objects, especially at the borders of the image or in high-contrast areas. In order to mitigate this effect, hyperspectral microscopy system 100 provides z-translation adapter 103, which is functionally coupled to stage 105 and operable by computer control system 201 to move stage 105 up and down along the z-axis (as shown in FIGs. 1, 2 and 5). In some embodiments, z-translation adapter 103 may include a portable stepper motor adapter to fine-tune the focal position of the NA objective 102. The focal position of the NA objective 102 may be fine-tuned during hyperspectral scans in order to provide spectral compensation, as described in more detail elsewhere herein.

[0081] FIG. 6A shows a flowchart showing a simplified representation of a method 600 of operating hyperspectral microscopy system 100 to reduce chromatic aberration. First, at step 601, a user and / or computer control system 201 may select a desired spectral target, namely a target wavelength, or target wavelength band, to which hyperspectral camera 101 is to be tuned. Then, at step 602, under control of the computer control system 201 , translation device 303 moves the linear filter such that the target wavelength, or target wavelength band, is positioned between the lens 301 and the image sensor 302. Concurrently, or subsequently, at step 603, z-translation adapter 103, under control of the computer control system 201 , may fine-tune the focal position (along the z-axis) of NA objective 102 to a predetermined position associated with the target wavelength, or target wavelength band to mitigate chromatic aberration. As will be appreciated, this can be achieved by any suitable means including, but not limited to, using a lookup table providing correspondence between target wavelengths, or target wavelength bands, and advantageous NA objective 102 positions.

[0082] Concurrently, or subsequently, at step 604, the intensity of transmission light source 106 may be adjusted by computer control system 201 in order to compensate for variations in radiant intensity associated with the target wavelength, or target wavelength band (as shown in FIG. 4). As will be appreciated, this can also be achieved by any suitable means including, but not limited to, using a lookup table providing correspondence between target wavelengths, or target wavelength bands, and normalized values of radiant intensity for a particular transmission light source 106.

[0083] In some embodiments, z-translation adapter 103 may also provide 3D hyperspectral scanning by moving the focal point of the NA objective 102 through specimen 104 along the z-axis. In some embodiments, z-translation adapter 103 may also be used to provide a 3D image scan by moving the focal point of the NA objective 102 through specimen 104 along the z-axis while the hyperspectral camera 101 is in monochromatic mode.

[0084] FIG. 6B shows a flowchart showing a simplified representation of a method 610 of operating hyperspectral microscopy system 100 to produce 3D images. First, at step 611 , a user and / or computer control system 201 may select an initial capture plane (in the z-axis, as shown in FIGs. 1 , 2 and 5. Then, at step 612, z-translation adapter 103, under control of the computer control system 201 , moves the focal plane of NA objective102 to the selected capture plane in the z-axis. Then, at step 613, under control of the computer control system 201, hyperspectral camera 101 captures image information at the selected capture plane. At step 614, the computer control system 201 then sets a new selected capture plane position either above or below the previous capture plane along the z-axis. Z-translation adapter 103 may enable reliable scanning with steps from 100nm, providing sufficient axial (z-axis) resolution for a microscope objective with a numerical aperture of up to 1.3.

[0085] Once the new capture plane position is determined by computer control system 201, the method may return to step 612. By repeating the aforementioned steps, it is possible for computer control system 201 to build a 3D image (volume) by superimposing multiple 2D images captured at various depths (i.e. , along the z-axis). 3D image reconstruction may be performed using proprietary software or known software such as Amira™ or Dragonfly™.

[0086] FIG. 5 shows a simplified schematic diagram of a lateral lighting system 500 in accordance with various embodiments of the present disclosure. The lateral lighting system 500 may comprise one or more lateral red-green-blue (RGB) colour light sources 108, and one hyperspectral light source 109. Lateral lighting system 500 provides darkfield imaging capabilities which enhance the contrast of transparent or minimally contrasting specimens, enabling the observation of fine structural details without the need for staining.

[0087] The lateral lighting system 500 provides illumination directed onto the specimen at an oblique angle, causing the light to scatter due to the specimen’s inherent properties, such as irregularities or particle structures. The scattered light 114 (generally along the z-axis) is captured by NA objective 102 of hyperspectral camera 101, while direct light from the illumination source is excluded by means of the total internal reflection (TIR) phenomenon. This selective capture of scattered light results in a visual representation where the specimen appears bright against a dark background, thereby providing improved contrast for detailed observation.

[0088] The lateral lighting system 500 described herein offers several advantages over conventional microscopy techniques. Notably, it enables the visualization of transparent or unstained samples, which would otherwise be difficult to distinguish under standard bright field illumination. This method is particularly beneficial for applications inmicrobiology, nanotechnology, and cell biology, where high-contrast images of small structures, microorganisms, nanoparticles, and cellular components are essential for accurate analysis and observation.

[0089] In some embodiments, the lateral lighting system 500 may comprise a plurality of RGB LEDs. In an advantageous example, the lateral lighting system 500 comprises 48 individual LEDs. The intensity and modulation of each individual LED in the lateral lighting system 500 may be controlled by computer control system 201 to provide an improved level of image synchronization, visualization contrast, and spatial illumination positioning. For example, in some embodiments, while a specimen is being moved along the x-axis, computer control system 201 may control illumination of the LEDs (i.e. , turn them on or off) in order to ensure that a certain portion / section of the specimen remains illuminated, thereby providing optimal spatial control.

[0090] A significant advantage of the dark-field imaging capabilities provided by the lateral lighting system 500 is that they do not require oil immersion objectives, and they are therefore compatible with clinical practice to prevent sample contamination.

[0091] As will be appreciated by the skilled reader, one of the advantages of the hyperspectral microscopy system 100 of the present disclosure is that hyperspectral camera 101, z-translation adapter 103 and lateral lighting system 500 may be integrated into a kit and retrofitted to a standard microscope (as shown in FIG. 1), thereby providing simple integration on any upright or inverted microscope while preserving original functionality. In some embodiments, each element of such kit may communicate with and be monitored and controlled by computer control system 201. In some examples, computer control system 201 may be implemented as part of a software package operating on a laptop, desktop, or in a mobile communication device, such as a smartphone, tablet, head-mounted display, or other communication device which is carried or worn by the user. Elements of the kit may communicate amongst each other and with computer control system 201 by way of known data communication means, such as wired (e.g. IEEE 802.3) or wireless (e.g., Wi-Fi, WiMAX, or Bluetooth®) technologies, or combinations thereof.

[0092] The systems and methods of the present disclosure therefore provide hyperspectral capabilities at reduced cost and complexity when compared to previous systems and methods. Moreover, compared to existing commercial systems, theproposed systems and methods disclosed herein can be implemented as a modular platform (i.e., integration of individual adapters into the imaging setup) or as a complete multi-hyperspectral microscopy system. Depending on the application, the systems and methods of the present disclosure can be used as a fully automated digital research platform or a portable automated analytical system. Accordingly, the systems and methods of the present disclosure offer a modular integration into existing microscopy imaging setups while preserving all existing microscopy modes and initial optical characteristics.

[0093] As discussed in more detail elsewhere herein, the systems and methods described herein capture information in multiple spectral ranges and therefore allow the identification and differentiation of materials based on their unique spectral features. This is particularly useful in industries such as agriculture, geology, food quality control and especially for improving diagnostic accuracy in medicine. In such applications multi-hyperspectral capabilities can improve the detection of diseases or abnormalities by providing more detailed images and identifying tissue characteristics by detecting biomarkers that are not visible with traditional imaging.

[0094] The systems and methods of the present disclosure also provide an easy switch between conventional monochrome camera for reference images and conventional transmission and multispectral dark-field RGB microscope modes. Moreover, the systems and methods of the present disclosure are compatible with many other modalities such as fluorescence microscopy, standard dark-field or bright field imaging. Other advantages include relatively fast manual or automatic spectral tuning and acquisition for microscopy observation in a required spectral range.

[0095] The embodiments have been described above with reference to flow, sequence, and block diagrams of methods, apparatuses, systems, and computer program products. In this regard, the depicted flow, sequence, and block diagrams illustrate the architecture, functionality, and operation of implementations of various embodiments. For instance, each block of the flow and block diagrams and operation in the sequence diagrams may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified action(s). In some alternative embodiments, the action(s) noted in that block or operation may occur out of the order noted in those figures.

[0096] For example, two blocks or operations shown in succession may, in some embodiments, be executed substantially concurrently, or the blocks or operations may sometimes be executed in the reverse order, depending upon the functionality involved. Some specific examples of the foregoing have been noted above but those noted examples are not necessarily the only examples. Each block of the flow and block diagrams and operation of the sequence diagrams, and combinations of those blocks and operations, may be implemented by special purpose hardware-based systems that perform the specified functions or acts, or combinations of special purpose hardware and computer instructions.

[0097] While the applicant’s teachings described herein are in conjunction with various embodiments for illustrative purposes, it is not intended that the applicant’s teachings be limited to such embodiments as the embodiments described herein are intended to be examples. On the contrary, the applicant’s teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments described herein, the general scope of which is defined in the appended claims.

Claims

CLAIMS1. A hyperspectral widefield microscope including:a linear spectral filter;an objective lens to direct captured light onto the linear spectral filter;a translation device arranged to move the linear spectral filter such that the relative position of the captured light and the spectral filter can be adjusted; andan image capture device to capture spectrally filtered light.

2. The hyperspectral widefield microscope of claim 1 , further comprising:a hyperspectral transmission illumination source.

3. The hyperspectral widefield microscope of claim 2, wherein the hyperspectral transmission illumination source is a Light Emitting Diode (LED) illumination source.

4. The hyperspectral widefield microscope of claim 3, wherein the hyperspectral transmission illumination source includes wavelengths from 400nm to 950nm.

5. The hyperspectral widefield microscope of any one of claims 2 to 4, further comprising a z-translation adapter configured to move a sample holder of the microscope towards and away from the objective lens.

6. The hyperspectral widefield microscope of any one of claims 1 to 5, further comprising:a hyperspectral lateral lighting system suitable for dark-field microscopy.

7. The hyperspectral widefield microscope of claim 6, wherein the hyperspectral lateral lighting system comprises a plurality of colour LEDs.

8. The hyperspectral widefield microscope of claim 7, wherein the plurality of colour LEDs produce light in the wavelength range of between 400nm and 950nm.

9. The hyperspectral widefield microscope of any one of claims 5 to 8, further comprising a computer control system configured to control one or more of:the intensity of the hyperspectral transmission illumination source;the position of the objective lens by controlling the z-translation adapter; and the position of the linear spectral filter by controlling the translation device.

10. The hyperspectral widefield microscope of claim 9, wherein the computer control system is configured to synchronize the position of the objective lens and the position of the linear spectral filter to mitigate chromatic aberrations.

11. The hyperspectral widefield microscope of claim 9 or 10, wherein the computer control system is configured to synchronize the intensity of the hyperspectral transmission illumination source and the position of the linear spectral filter to at least partially compensate for spectral variations in the intensity of the hyperspectral transmission illumination source.

12. The hyperspectral widefield microscope of any one of claims 9 to 11, when dependent on claim 7, wherein the computer control system is further configured to control each of the plurality of colour LEDs individually to mitigate phototoxicity.

13. The hyperspectral widefield microscope of any one of claims 9 to 12, wherein the computer control system is further configured to:control the image capture device to capture a series of images; andmove the position of the objective lens by controlling the z-translation adapter between each image in the series,wherein the resulting series of images is suitable to be converted into a 3D image of a sample.

14. A kit for converting a microscope into a hyperspectral widefield microscope, the kit comprising:a hyperspectral camera; anda hyperspectral transmission illumination source.

15. The kit of claim 14, wherein the hyperspectral camera comprises:a linear spectral filter;a translation device arranged to move the linear spectral filter such that the relative position of the light captured by the microscope and the spectral filter can be adjusted; andan image capture device to capture spectrally filtered light.

16. The kit of claim 15, further comprising:a z-translation adapter configured to move a sample holder of the microscope towards and away from an objective lens of the microscope.

17. The kit of any one of claims 14 to 16, further comprising:a hyperspectral lateral lighting system suitable for dark-field microscopy.

18. The kit of claim 17, wherein the hyperspectral lateral lighting system comprises a plurality of colour LEDs.

19. The kit of claim 18, wherein the plurality of colour LEDs produce light in the wavelength range of between 400nm and 950nm.

20. The kit of any one of claims 16 to 19, further comprising a computer control system configured to control one or more of:the intensity of the hyperspectral transmission illumination source;the position of the objective lens by controlling the z-translation adapter; and the position of the linear spectral filter by controlling the translation device.

21. The kit of claim 20, wherein the computer control system is configured to synchronize the position of the objective lens and the position of the linear spectral filter to mitigate chromatic aberrations.

22. The kit of claim 20 or 21, wherein the computer control system is configured to synchronize the intensity of the hyperspectral transmission illumination source and the position of the linear spectral filter to at least partially compensate for spectral variations in the intensity of the hyperspectral transmission illumination source.

23. The kit of any one of claims 20 to 22, when dependent on claim 18, wherein the computer control system is further configured to control each of the plurality of colour LEDs individually to mitigate phototoxicity.

24. The kit of any one of claims 20 to 23, wherein the computer control system is further configured to:control the image capture device to capture a series of images; andmove the position of the objective lens by controlling the z-translation adapter between each image in the series,wherein the resulting series of images is suitable to be converted into a 3D image of a sample.

Images