Infrared imaging system and related methods

Abstract

An infrared imaging system and method for imaging a sample having a fluorescent marker is provided. The system includes a light source configured to illuminate a sample contact surface. The light source includes first and second illumination modules, each configured to project corresponding first and second infrared illumination beams toward the sample holder, where the infrared illumination beams interact at the imaging plane to define an illumination area having a rectangular and homogenous power profile. The system also includes a control unit operably connected to the motor assembly and the opto-mechanical mechanism. The control unit is configured to superimpose the sample plane and the imaging plane at one of a plurality of positions within the enclosure. The system includes a detector configured to receive light emitted by the fluorescent marker of the sample upon illumination of the sample at the imaging plane when the sample plane is superimposed with the imaging plane.

Description

Technical Field

[0001] The technical field generally relates to imaging systems and related methods, and more particularly to infrared imaging systems and related methods.

Background Art

[0002] Many preclinical imaging systems are available on the market. Non-limiting examples include the IVIS® Spectrum in vivo imaging system from Perkin Elmer, Lago from Spectral Instruments Imaging, Pearl Trilogy® from LiCor, iBox® from UVP, and Newton from Viber. These commercially available solutions typically rely on silicon-based detectors and as a result are essentially limited in their applications. This is because they can only image from the visible portion of the electromagnetic spectrum to the first near-infrared window (NIR-I) portion of the electromagnetic spectrum (i.e., from about 400 nm to about 1000 nm).

[0003] Light absorption and scattering in biological tissues are significantly weaker in the second near-infrared window (NIR-II, i.e., from about 1000 nm to about 1700 nm) of the electromagnetic spectrum compared to the visible or NIR-I portion of the electromagnetic spectrum, which means that small animals are more transparent in the NIR-II imaging window.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, there is a need for technologies, methods, systems, and devices that solve or at least mitigate at least some of the problems shown above.

Means for Solving the Problems

[0005] According to one embodiment, an infrared imaging system for imaging a sample having a fluorescent marker is provided. The infrared imaging system includes an enclosure, a sample holder, a light source, a motor assembly, an optics-mechanical mechanism, a control unit, and a detector. The sample holder is mounted in the enclosure. The sample holder has a sample contact surface and a sample plane. The light source is configured to illuminate the sample contact surface and includes a first illumination module and a second illumination module, each illumination module configured to project corresponding first and second infrared illumination beams toward the sample holder. The first and second infrared illumination beams interact in the imaging plane to define an illumination area having a rectangular and homogeneous power profile. The motor assembly is configured to move the sample holder to multiple positions within the enclosure. The optics-mechanical mechanism is configured to orient the first and second infrared illumination beams to move the illumination area within the enclosure. The control unit is operably connected to the motor assembly and the optical-mechanical mechanism, and is configured to superimpose the sample plane and the imaging plane at one of several positions within the enclosure. The detector is configured to receive light emitted by the sample's fluorescent markers due to the illumination of the sample on the imaging plane when the sample plane is superimposed with the imaging plane.

[0006] In some embodiments, the enclosure defines an internal volume and further includes a door or drawer for accessing the contents of the internal volume.

[0007] In some examples, the sample plane is offset perpendicularly from the sample contact surface.

[0008] In some embodiments, the sample plane is offset perpendicularly from the sample contact surface by a value corresponding to the thickness of the sample or a fraction thereof.

[0009] In some embodiments, the sample plane coincides with the sample contact surface.

[0010] In some embodiments, the sample contact surface is formed from a black powder-coated steel sheet.

[0011] In some embodiments, the infrared imaging system further includes one or more anesthetic ports, the one or more of which are configured to inject anesthetic gas into and from an enclosure.

[0012] In some embodiments, the infrared imaging system further includes a heating element in thermal contact with the sample holder.

[0013] In some embodiments, the infrared imaging system further includes a barrier attached to the sample holder, the barrier protruding upward from the sample plane.

[0014] In some embodiments, each of the first and second lighting modules includes one or more laser diodes.

[0015] In some embodiments, the first and second infrared illumination beams have wavelengths of approximately 750 nm, approximately 808 nm, or approximately 980 nm.

[0016] In some embodiments, the illumination area was approximately 1 mW / mm². 2 ~about 3mW / mm 2 It has a lighting power density that falls within a wide range.

[0017] In some embodiments, each of the first and second lighting modules includes a Köhler integrator.

[0018] In some embodiments, the first and second illumination modules are arranged symmetrically on either side of the detector.

[0019] In some embodiments, the first and second illumination modules are calibrated based on calibration data that maps a plurality of orientations of the first and second illumination modules to corresponding plurality of illumination power densities of the first and second infrared illumination beams and to corresponding plurality of positions of a sample holder within the enclosure.

[0020] In some embodiments, the detector includes an InGaAs camera.

[0021] In some embodiments, the detector includes a sensor, a first optical circuit configured to collect and collimate light emitted by a fluorescent marker, and a second optical circuit configured to form an image of the sample on the sensor.

[0022] In some embodiments, the infrared imaging system further includes a motorized focusing mechanism connected to the detector, the motorized focusing mechanism being configured to vary the distance between the first optical circuit and the second optical circuit.

[0023] In some embodiments, the infrared imaging system further includes a filter wheel positioned between the first optical circuit and the second optical circuit, the filter wheel including a plurality of filters.

[0024] According to another aspect, a method for imaging a sample having a fluorescent marker is provided. The method includes providing a sample on a sample holder having a sample contact surface and a sample plane, generating first and second infrared illumination beams interacting in an imaging plane to define an illumination area having a rectangular and uniform power profile towards the sample by first and second illumination modules, moving the power profile sample holder to a plurality of positions within an enclosure, orienting the first and second infrared illumination beams to move the illumination area within the enclosure, overlaying the sample plane and the imaging plane at any of a plurality of positions within the enclosure, and collecting light emitted by the fluorescent marker of the sample by illumination of the sample by the illumination beam light in the imaging plane when the sample plane is overlaid with the imaging plane.

[0025] In some embodiments, the method further includes shifting the sample plane vertically from the sample contact surface.

[0026] In some embodiments, the sample plane is shifted vertically from the sample contact surface by a value corresponding to the thickness of the sample or a fraction thereof.

[0027] In some embodiments, the sample plane coincides with the sample contact surface.

[0028] In some embodiments, the method further includes heating the sample holder.

[0029] In some embodiments, the first and second infrared illumination beams have a wavelength of about 750 nm, about 808 nm or about 980 nm.

[0030] In some embodiments, the method further includes adjusting each of the first and second infrared illumination beams by a Keller integrator.

[0031] In some embodiments, the method further includes calibrating the first and second illumination modules based on calibration data, which maps multiple orientations of the first and second illumination modules to corresponding multiple illumination power densities of the first and second infrared beams and to corresponding multiple positions of the sample holder within the enclosure.

[0032] In some embodiments, the method is The light emitted by the fluorescent marker is collected and aligned by the first optical circuit. The method further includes forming an image of the sample on the sensor using a second optical circuit.

[0033] Other features and advantages of the methods and systems described herein will be better understood by referring to the accompanying drawings and reading the preferred embodiments. While certain features described in the above summary and the following detailed description may be described in relation to specific embodiments or aspects, it should be noted that, unless otherwise specified, these specific features can be combined with each other. [Brief explanation of the drawing]

[0034] [Figure 1] This figure shows one embodiment of an infrared imaging system for imaging a sample having a fluorescent marker. [Figure 2] This figure shows one embodiment of a lighting module. [Figure 3] This is a diagram of a detector according to one embodiment. [Figure 4] This figure shows an example in which the sample holder is initially in a first position (the left side of the image) and then moved to a subsequent or second position (the right side of the image). [Figure 5] This is a block diagram showing the operational connections between some of the components included in an infrared imaging system. [Modes for carrying out the invention]

[0035] In this description, the same features in the drawings are given the same reference numerals. To avoid cluttering the drawings, some elements may be omitted if they have already been shown in previous drawings. It should also be understood that the elements in the drawings are not necessarily shown to scale, as the emphasis is on clearly illustrating the elements and structure of this embodiment. Furthermore, positional descriptors indicating the position and / or orientation of one element relative to another are used herein for ease of explanation and clarity. Unless otherwise specified, these positional descriptors should be interpreted in relation to the drawings and not considered limiting. More specifically, it should be understood that such spatially relative terms are intended to encompass different orientations in the use or operation of this embodiment, in addition to the orientations illustrated in the drawings.

[0036] Unless otherwise specified, the terms “connected” and “joined,” as well as their derivatives and variations, refer herein to any direct or indirect structural or functional connection or joining between two or more elements. For example, the connection or joining between elements may be mechanical, optical, electrical, thermal, logical, or any combination thereof.

[0037] The terms “a,” “an,” and “one” are defined herein as meaning “at least one,” and therefore, unless otherwise specified, these terms do not exclude multiple items.

[0038] Terms such as “substantially,” “generally,” and “about” that modify the values, conditions, or characteristics of the exemplary embodiments should be understood to mean that the values, conditions, or characteristics are defined within tolerances that are permissible for the proper operation of this exemplary embodiment for its intended use, or within the permissible range of experimental error. In particular, the term “about” refers generally to a range of numbers that a person skilled in the art would consider to be equal to (e.g., having the same or similar function or result) the stated value. In some examples, the term “about” means a variation of ±10 percent of the stated value. Note that all numerical values ​​used herein are assumed to be modified by the term “about” unless otherwise specified.

[0039] Similarly, the terms “superimposed,” “to superimpose,” “superimposed,” and “superimposed” are intended herein to refer to the condition that two elements are in the same position or within a given tolerance of each other in terms of spatial alignment. That is, these terms mean not only to superimpose two elements “exactly” or “identically,” but also to superimpose two elements “substantially,” “almost,” or “subjectively,” and to provide a higher or best superimposition among multiple superimposition possibilities.

[0040] In this explanation, the expression "based on" is intended to mean "based on at least partially," that is, it can mean "based only on" or "based partially on," and therefore should not be interpreted in a limited sense. More specifically, the expression "based on" can also be understood as meaning "depending on," "representing," "indicating," "related to," or similar expressions.

[0041] In this description, the terms “light” and “optical,” as well as their variations and derivatives, are used to refer to radiation in any appropriate region of the electromagnetic spectrum. Therefore, the terms “light” and “optical” are not limited to visible light, but may also include, and may not be limited to, the infrared and ultraviolet regions. For example, in some implementations, the technology can be used with electromagnetic signals having wavelengths in the range of approximately 400 nm to approximately 1700 nm, for example, 1000 nm to 1700 nm. However, this range is provided for illustrative purposes only, and some implementations of the technology may operate outside this range. Furthermore, those skilled in the art will acknowledge that the definitions of the ultraviolet, visible, infrared, and near-infrared regions in terms of spectral ranges, and the dividing lines between them, may vary depending on the art or the definitions considered, and are not intended to limit the scope of applications of the technology.

[0042] In this explanation, the term "illumination beam spectrum," its synonyms, or derivatives are used broadly to refer to the spectral power distribution of an illumination beam. An illumination spectrum can represent the distribution of radiated power per unit area and per unit wavelength or frequency across the spectral domain of the electromagnetic spectrum.

[0043] This description generally relates to infrared imaging systems, related methods, and techniques for preclinical imaging purposes. In the context of this description, infrared imaging systems may sometimes be referred to as “IR VIVO devices.” It should be noted that the term “preclinical imaging” is understood herein to mean techniques that enable the visualization and examination of living animals (e.g., small animals such as mice and rats). Preclinical imaging techniques may be particularly useful for research purposes (e.g., drug development).

[0044] The infrared imaging systems described in more detail below are fluorescence-based imaging instruments. Such instruments generally include a light source that uses excitation light to excite fluorescent probes in a sample, which can generally be small animals from the perspective of preclinical imaging systems. The instrument also includes a detector configured to detect the fluorescence signals produced by these probes. Other optical components, not limited to those mentioned above, may be placed between the sample and the detector, such as imaging lenses, spectral filters, dichroism elements (for example, to separate the output signal into two spectral bands which may be detected by two different cameras), and other optical components.

[0045] Here, with reference to the drawings, different embodiments of infrared imaging systems and methods are provided.

[0046] Referring to Figure 1, an infrared imaging system 20 for imaging sample 22 is shown. It should be noted that sample 22 may include one or more animals. Therefore, the expression “sample” is not limited to, and is not intended to be limited to, a single animal. Fluorescent markers (not shown) are provided for sample 22. For example, but not limited to, fluorescent markers may be injected into the small animal being imaged. Note that the expressions “fluorescent marker” and “fluorescent probe” are used interchangeably throughout this description. Non-limited examples of fluorescent markers include quantum dots (e.g., PbS,Ag2S), organic molecules such as indocyanine green (ICG) and IR800 dye molecules, single-walled carbon nanotubes, and rare earth element nanoparticles.

[0047] Enclosure and sample holder The infrared imaging system 20 includes an enclosure 24 (sometimes called a “chamber”). The enclosure 24 includes walls defining an internal volume 26, within which at least some of the other components of the infrared imaging system 20 can be mounted. The enclosure 24 generally includes a door or drawer (not shown in Figure 1) for accessing the internal volume 26 (i.e., its contents) when required, for example, to set up a preclinical trial or to prepare a sample 22. In some embodiments, the entire enclosure 24 may be light-proof. In some embodiments, the door or drawer may be light-proof. Note that the enclosure 24 may be equipped with components such as, for example, an anesthetic gas manifold, gas tubing, and a heat plate, as will be described in more detail below.

[0048] The infrared imaging system 20 also includes a sample holder 28. The sample holder 28 is positioned within an enclosure 24 and has a sample contact surface 30. The sample contact surface 30 has a sample plane 32. Note that in some implementations, the sample plane 32 may be offset perpendicularly from the sample contact surface 30 by a value corresponding to the thickness (or height) of the animal being imaged, or a fraction of the thickness (or height) of the animal being imaged. Alternatively, the sample plane 32 may coincide with or substantially coincide with the sample contact surface 30. The sample 22 can be positioned on the sample contact surface 30 such that the sample plane 32 intersects the sample 22 or at least a portion thereof. As will be described in more detail below, the sample holder 28 can be adjusted by translation along three dimensions, e.g., the x-axis, y-axis, and z-axis. Thus, the position of the sample plane 32 can be changed or adjusted.

[0049] In some embodiments, the sample contact surface 30 is formed from black powder-coated steel sheet. In other embodiments, the sample contact surface 30 can be formed from aluminum. Alternatively, the sample contact surface 30 can be formed from any type of material (e.g., anodized aluminum, heated glass, etc.) that has suitable properties for preclinical trials or related medical applications, as long as it has low reflectivity and fluorescence in infrared light and is relatively easy to clean with ethanol solution or bleach. It should be noted that the material forming the sample contact surface 30 can be selected based on various chemical properties (e.g., composition) and / or physical properties (e.g., optical and magnetic properties).

[0050] The infrared imaging system 20 generally includes components found in typical preclinical devices. For example, the infrared imaging system 20 may generally include one or more anesthetic ports. In some embodiments, the infrared imaging system 20 includes three anesthetic ports, meaning that three samples 22 can be placed on the sample contact surface 30. It should be noted that the number of anesthetic ports may differ from three, and the fact that the infrared imaging system 20 may include three anesthetic ports serves only as an illustrative purpose and should therefore not be considered limiting. For example, the infrared imaging system 20 may include one, two, three, four, five, or more anesthetic ports. Similarly, the number of animals forming the sample 22 may also differ from three. For example, without limitation, the sample 22 may include one, two, three, four, five, or more animals. In some embodiments, the number of anesthetic ports may be the same as the number of animals forming the sample 22. In other embodiments, the number of anesthetic ports may be proportional to, or at least related to, the number of animals forming the sample 22. For example, the ratio of the number of anesthesia ports to the number of animals forming the sample 22 may be 1:1, 1:2, 1:3, or any other ratio that allows the anesthesia ports to achieve their function. In this regard, it should be noted that the anesthesia ports allow for the injection and collection of anesthetic gas to bring anesthetic gas into the enclosure 24 / and to release anesthetic gas from the enclosure 24. The anesthetic gas is generally useful for keeping the sample 22 immobile during imaging of the sample 22.

[0051] The infrared imaging system 20 may also include a heating plate or similar device. It should be noted that a heating plate can maintain the entire internal volume 26 or only a portion thereof (e.g., the sample contact surface 30) at any desired temperature. Heating plates are generally useful for maintaining the sample 22 at any desired temperature. In fact, in general, small animals or mammals tend to have a decrease in mean body temperature under anesthesia. Therefore, a heating plate can mitigate this effect. The infrared imaging system 20 may also include a barrier or fence attached to or on the sample holder 28. This feature can be particularly useful in the rare opportunity for the sample 22 (e.g., a small animal) to awaken while under anesthesia, or in the event of failure in the anesthesia process.

[0052] The dimensions and geometric configuration of the sample holder 28 can be varied. However, the dimensions of the sample holder 28 are preferably such that the sample holder 28 substantially matches the field of view of the imaging system 20, as will be described in more detail below. In one example of implementation, the field of view has the following dimensions: approximately 15.6 cm × approximately 12.5 cm.

[0053] In some embodiments, the sample holder 28 may include a lower platform and an upper platform. The lower platform may extend across the entire floor (i.e., width and depth) of the enclosure 24. The upper platform may be smaller and may have the following dimensions: 300 mm × 250 mm. The upper platform may be mounted on the lower platform. In some embodiments, the upper platform may be mechanically connected to the lower platform by a two-dimensional translation stage. In the context of this description, the two-dimensional stage is configured to translate its upper surface along the X and Y axes and to move the sample 22 laterally along these two axes and relative to the camera's field of view.

[0054] light source The infrared imaging system 20 includes a light source 34 configured to illuminate the sample contact surface 30. The light source 34 includes a first illumination module 36 and a second illumination module 38. The first and second illumination modules 36 and 38 are configured to project corresponding first and second infrared illumination beams 40 and 42 toward the sample holder 30, respectively. The first and second illumination modules 36 and 38 can each generate relatively high-power infrared illumination beams. It should be noted that the first and second illumination modules 36 and 38 may each include one or more laser diodes, each related to corresponding optical properties (e.g., intensity and / or spectral profile). Relatively high power is sufficient to produce a sufficiently strong fluorescence signal to be detected (approximately 0.05 to approximately 3 mW / mm²). 2 It is useful for generating fluorescent markers. It should be noted that fluorescent markers in the NIR-II portion of the electromagnetic spectrum typically have relatively lower efficiency than fluorescent markers that can be used in the visible portion of the electromagnetic spectrum. It should also be noted that detectors configured to operate in the NIR-II portion of the electromagnetic spectrum are generally less sensitive than detectors configured to operate in the visible portion of the electromagnetic spectrum. It should be noted that the wavelengths of the infrared illumination beams 40,42 emitted by the first and second illumination modules 36,38 can be selected or varied. The selection may be manual (e.g., by the user) or automatic (e.g., each module 36,38 can sequentially and / or automatically select the infrared illumination wavelength). This feature may be useful for exciting fluorescent markers of different properties. It should be noted that illumination with different wavelengths is generally performed sequentially rather than simultaneously. Non-limiting examples of wavelengths that may be used are 750 nm, 808 nm, 860 nm and 980 nm.

[0055] The first and second infrared illumination beams 40, 42 interact in an imaging plane 44 to define an illumination region 46. The imaging plane 44 extends along the X and Y axes. The illumination region 46 has a rectangular and homogeneous power profile (sometimes called the "illumination profile" or "power density profile") and also extends along the X and Y axes. The power or illumination density in the illumination region represents the cumulative power of the first and second infrared illumination beams 40, 42. In some embodiments, the power or illumination density is approximately 1 mW / mm² for a total power of approximately 20 W. 2 These values ​​are relatively close to, but lower than, the limits of power or illumination density for living tissue (i.e., about 3 mW / mm²). 2 ).

[0056] Those skilled in the art should note that illumination in existing visible preclinical imagers is generally provided by halogen white lamps. These lamps are typically optically coupled with filters (e.g., filter wheels) for excitation filtering. Existing visible preclinical imagers can also use LED and / or laser sources. In these cases, the illumination density is lower than that which can be obtained using the first and second illumination modules 36,38 included in the techniques described herein. In fact, the illumination densities achievable by the techniques presented herein are an order of magnitude greater than those of existing techniques that rely on halogen and LED devices.

[0057] The light generated by each laser diode contained in the first and second illumination modules 36,38 is optically structured by the optical components contained in the first and second illumination modules 36,38. This allows for the definition of an illumination area 46 having a substantially homogeneous rectangle on the sample 22. It should be noted that the illumination area 46 generally has the same dimensions as the field of view of the infrared imaging system 20. Those skilled in the art will understand that homogeneous illumination is important to ensure that all parts of the sample 22 receive the same illumination density. For example, if three mice are to be imaged and placed in the field of view, all mice must be illuminated with the same power density so that the imaging results can be compared with one another. It is also advantageous to limit the illumination area 46 to the field of view of the infrared imaging system 20 to avoid wasting laser power. In fact, if too much laser power is lost, a more powerful laser will be required, which increases the cost of the system and also increases the complexity of thermal management of the first and second illumination modules 36,38. Furthermore, it should be noted that projecting portions of the first and second infrared illumination beams 40, 42 contributes to adding undesirable stray light to the infrared imaging system 20, which may be detected by the detector, thereby reducing the overall sensitivity of the infrared imaging system 20.

[0058] Referring here to Figure 2, the first and second illumination modules 36, 38 structure the light emitted by the laser diode using a Köhler integrator design. Although only the first illumination module 36 is shown in Figure 2, it will be readily apparent that the description also applies to the second illumination module 38. The Köhler integrator design is known in the art and involves the use of a collimation lens 48, a first fly-eye lens 50, a second fly-eye lens 52, a projection lens 54, and a fold mirror 55. These elements define an optical path between them, which extends along the optical axis 56. In some embodiments, the projection lens 54 may be replaced by an optical assembly (not shown). The optical assembly may include multiple optical elements. Such optical elements include, but are not limited to, lenses, mirrors, filters, and other suitable reflective, refractive, and / or diffractive optical components.

[0059] In the illustrated embodiment, the first and second illumination modules 36,38 are positioned to project light (i.e., the first and second infrared illumination beams 40,42) from above the sample 22. Referring again to Figure 1, it can be seen that the first and second illumination modules 36,38 are positioned on both sides of the detector 58. In some embodiments, the first and second illumination modules 36,38 are positioned symmetrically on both sides of the detector 58. However, it will be understood that the first and second illumination modules 36,38 may be positioned in other locations relative to the detector and / or asymmetrically with respect to the detector, without departing from the scope of protection.

[0060] The first and second illumination modules 36, 38, and consequently the first and second infrared illumination beams 40, 42, are generally not parallel to the Z-axis, but form a slight angle with respect to the Z-axis. If the sample 22 contains multiple animals (i.e., two or more animals), it should be noted that the angle between the first and second illumination beams and the Z-axis should be kept as small as possible to prevent the animals from casting shadows on each other.

[0061] As will be described in more detail below, the first and second illumination modules 36, 38 can be rotated, which allows the projection of the first and second infrared illumination beams 40, 42 onto the center or near the center of the field of view of the infrared imaging system 20, for example, when the sample holder 28 moves upward and / or downward. The rotation of the first and second illumination modules 36, 38 makes it possible to maintain homogeneous illumination in the illumination area 46.

[0062] Motor assembly A block diagram showing the operational connections between some of the components included in the infrared imaging system 20 is shown in Figure 5. The infrared imaging system 20 includes a motor assembly 60 configured to move the sample holder to multiple positions within the enclosure 24. The motor assembly 60 may include one or more motors. The motors can be of any type of design.

[0063] In some embodiments, the sample holder 28 can be moved or translated along two axes (e.g., the X-axis and the Y-axis) by a motor assembly 60. The motor assembly 60 can be configured to translate the sample holder 28 sequentially or simultaneously along the X-axis and Y-axis. For example, the sample holder 28 can be translated sequentially in a direction parallel to the X-axis, then in a direction parallel to the Y-axis, or in the reverse order. Alternatively, the sample holder 28 can be configured to be adjustable simultaneously along the X-axis and Y-axis. Note that the motor assembly 60 can monitor or record the displacement of the sample holder 28 along each axis. The monitored or recorded information is included in the calibration data.

[0064] In some embodiments, the motor assembly 60 may include two motors, each configured to move the sample holder 28 along its respective direction (e.g., the X-axis or the Y-axis).

[0065] The sample holder 28 can also be moved or translated along the Z-axis. Displacement of the sample holder 28 is generally performed after the sample holder 28 has been aligned or positioned along the X and Y axes within the enclosure 24. Movement of the sample holder 28 along this direction can be provided by one or more motors.

[0066] It will be observed that the motor assembly 60 can move the sample holder 28, allowing a transition from "wide view mode" to "close view mode." Switching between these two view modes may be useful when the sample 22 contains multiple animals. For example, the motor assembly 60 may, but is not limited to, allow a transition from a first field of view containing all the animals in the sample 22 to a second field of view containing only one animal or a portion of the animals that make up the sample 22. In some embodiments, the field of view may be adjusted to simultaneously image a portion of each animal that makes up the sample 22, which may be useful in relation to comparative characterization of specific parts of animals.

[0067] In some embodiments, the motor assembly 60 is manual. In these embodiments, the translation of the sample holder 28 involves two steps. In the first step, a spring-loaded, normally-on brake that prevents the upper platform from sliding is released. The brake can be released instantaneously by pressing a button, or permanently by pressing the same button and locking it. Once the brake is released, the upper platform can be pushed relatively smoothly in the "lateral direction" (e.g., along the X-axis) and "forward and backward direction" (e.g., along the Y-axis). In other embodiments, the motor assembly 60 is automatic.

[0068] Optical mechanical mechanisms Referring to Figure 5, the infrared imaging system 20 includes an optico-mechanical mechanism 62 configured to orient first and second infrared illumination beams 40, 42 to move an illumination area 46 within an enclosure 24. Orienting the first and second infrared illumination beams 40, 42 generally involves changing the spatial configuration of the first and second illumination modules 36, 38 (by rotation, translation, or a combination thereof). More specifically, the adjustment of the orientation of the first and second infrared illumination beams 40, 42 by the optico-mechanical mechanism 62 in combination with a motor assembly 60 can be used to control the size of the illumination area 46.

[0069] In some embodiments, referring here to Figure 2, oriented the first and second illumination modules 36, 38 includes rotating them about the optical axis 56. By rotating the illumination modules 36, 38 from an initial position to a subsequent position, the first and second infrared illumination beams 40, 42 interact from the first imaging plane (relating to the initial position of modules 36, 38) to the subsequent imaging plane (relating to the subsequent position of modules 36, 38), defining a subsequent illumination region that also has a substantially rectangular and substantially homogeneous power profile, as will be described in more detail with reference to Figure 4. Note that the first imaging plane and the subsequent plane are not generally in the same position along the Z-axis. For example, the subsequent imaging plane is generally higher or lower than the first imaging plane. Thus, oriented the first and second illumination modules 36, 38 changes the position of the imaging plane 44 within the enclosure 24 (e.g., along the Z-axis). In other embodiments, only the fold mirror 55 can be rotated. It should be noted that, in addition to their orientation, the power of the first and second infrared illumination beams 40, 42 can also be changed (i.e., modified or altered). Changing or adjusting the orientation of the first and second infrared illumination beams 40, 42 to move the illumination area 46 within the enclosure is generally referred to as "illumination modulation."

[0070] It should be noted that the first and second illumination modules 36,38 are generally calibrated. The calibration data includes, but is not limited to, a mapping between multiple orientations of the first and second illumination modules 36,38 and the corresponding optical properties of the first and second infrared illumination beams 40,42. Therefore, if the position of the sample holder 28 in the enclosure 24 is known, this information is included in the calibration data, or the orientation of the first and second illumination modules 36,38 to be achieved can be determined by calculations based on the calibration data, e.g., interpolation, extrapolation, and other techniques. The interpolation may be linear, polynomial (Lagrange, Newton, etc.), spline, etc. In one example, the calibration step is performed at any position in the enclosure 24, i.e., even if the distance between the sample holder 28 and the detector 58 changes, approximately 1 mW / mm 2 ~about 3mW / mm 2 This is useful for maintaining a relatively constant power density within the enclosure 24. More specifically, by changing the orientation of the first and second infrared illumination beams 40,42, a rectangular and homogeneous power profile can be maintained at least partially, almost or substantially, at any position within the enclosure 24. In some embodiments, the calibration data may further include information about the illumination power that needs to be provided or generated to maintain a constant or desired power density. It should be noted that the power density generally increases as the sample holder 28 rises, i.e., with respect to the distance between the sample holder 28 and the first and second illumination modules 36,38. As the sample holder 28 rises, the rectangular and homogeneous illumination becomes smaller. In some embodiments, the power density of the infrared illumination beams 40,42 may be controlled to maintain a relatively constant power density in the illuminated area. In some embodiments, the power density of the illumination modules 36,38 may be constant, which allows the power illumination density of the illuminated area to increase as the platform rises.

[0071] control unit As shown in Figure 5, the infrared imaging system 20 includes a control unit 64. The control unit 64 is operably connected to a motor assembly 60 and an optical-mechanical mechanism 62. The control unit 64 is configured to superimpose the sample plane 32 and the imaging plane 44 at one of several positions within the enclosure 24. When the sample plane 32 and the imaging plane 44 are superimposed, the two planes are substantially in the same position along the Z-axis. It should be noted that if the sample plane 32 is vertically offset from the sample contact surface 30, for example, if it is required to accommodate the thickness (or a fraction thereof) of the sample 22, the control unit 64 may be configured to position the sample plane 32 and the imaging plane 44 according to this vertical offset. In some embodiments, the value of the vertical offset may be automatically determined by the infrared imaging system 20. In other embodiments, the value of the vertical offset may be provided by the user, for example, manually. In yet another embodiment, the value of the vertical offset may be obtained from a database. Such a database may, for example, associate the average thickness of the animal forming the sample 22 with the corresponding position in the sample holder 28 within the enclosure 24.

[0072] For example, the control unit 64 may be embodied by a programmable computer comprising at least one processor, a data storage system (including volatile and non-volatile memory and / or storage elements), at least one input device, and at least one output device. In some embodiments, the programmable computer can run a computer program that enables control of the motor assembly 60 and the optical-mechanical mechanism 62. The control unit 64 is configured to continuously track, monitor, or record the position of the sample holder 28 in the enclosure 24. More specifically, the control unit 64 receives the position of the sample holder 28 in the enclosure 24 as input and outputs signals to be transmitted to the first and second illumination modules 36, 38 based on calibration data.

[0073] Referring to Figure 4, an example is shown in which the sample holder 28 starts in an initial or first position (left side of the figure) and is then moved to a subsequent or second position (right side of the figure).

[0074] In the initial position, the sample holder 28 is positioned such that the distance between the detector 58 and the sample holder 28 is approximately 400 mm (in the Z-axis). Therefore, the sample plane 32 is approximately 400 mm away from the detector 58. In the initial position, the first and second illumination modules 36 and 38 are oriented such that the illumination area 46 covers the sample 22. The imaging plane 44 and the sample plane 32 are superimposed.

[0075] In the subsequent position, the sample holder 28 is positioned such that the distance between the detector 58 and the sample holder 28 is approximately 200 mm (in the Z-axis), meaning that the sample holder 28 has been brought closer to the detector 58. Therefore, the sample plane 32' is approximately 200 mm away from the detector 58. In the subsequent position, the orientation of the first and second illumination modules 36, 38 has been changed from the initial position. Note that in the subsequent position shown in the non-limiting embodiment of Figure 4, the first and second illumination modules 36, 38 are oriented so that the illumination area 46' covers the sample 22. The imaging plane 44' and the sample plane 32' are superimposed.

[0076] The shape and dimensions of the illumination area 46 may change in accordance with changes in the orientation of the first and second illumination modules 36, 38 and / or changes in the position of the sample holder 28 in the enclosure 24.

[0077] detector As described above, the infrared imaging system 20 includes a detector 58 (see, for example, Figures 1 and 3). The detector 58 is configured to receive light emitted by fluorescent markers in the sample 22 by illuminating the sample 22 with first and second infrared illumination beams 40, 42 in the imaging plane 46 when the sample plane 32 is superimposed with the imaging plane 44. In the shown embodiment, the detector 58 includes an InGaAs camera. Such a camera allows for relatively precise localization of fluorescence emanating from within the sample 22, generating valuable information for biologists.

[0078] As shown in more detail in Figure 3, the detector 58 includes two optical circuits 66, 68 separated by a filter wheel 70. The detector 58 also includes a sensor 72.

[0079] The first optical circuit 66 collects light emitted from the sample 22 and makes it nearly parallel. The resulting light then passes through a double filter wheel 70. The filter wheel 70 generally includes multiple filters. In some embodiments, the filter wheel 70 includes a bandpass filter and an edgepass filter. In some embodiments, the first optical circuit 66 may include one or more detection lenses and / or any other optical elements. For example, in one embodiment, the optical circuit 66 may include two lenses. The second optical circuit 68 forms an image of the sample 22 on the sensor 72. In some embodiments, the second optical circuit 68 may include one or more lenses and / or any other optical elements. In some embodiments, the detector 58 is provided with an electric focusing mechanism 74. The electric focusing mechanism 74 is positioned between the second optical circuit 68 and the sensor 72 and can change the distance between them to adjust the focus.

[0080] The filters contained in the filter wheel 70 are typically dielectric interference filters, for which the wavelength of transmission depends on the angle. In some embodiments, the optical design of the first optical circuit 66 is such that light passes through the filter wheel 70 as close to perpendicular incidence (i.e., parallel to the Z-axis) as possible. Light coming from different field points in the sample 22 collides with the filter at different angles. Therefore, optimizing the optical design of the first optical circuit 66 to allow light to pass through the filter in a near-perpendicular incidence manner ensures that the detected wavelength is close at all points in the field of view.

[0081] The positioning of the filter wheel 70 between the two optical circuits 66 and 68 also prevents stray light from colliding with the filter at an angle away from the vertical, which results in unwanted light not being blocked by the filter and causing undesirable artifacts in the image formed on the sensor 72.

[0082] Varying the working distance (i.e., the distance between the sample 22 and the sensor 72) and adjusting the focus of the infrared imaging system 20 (e.g., the motorized focus), in combination with the operation of the motor assembly 60 and the optical mechanical mechanism 62, makes it possible to image an area with dimensions of approximately 156 mm × approximately 125 mm (i.e., illumination area 46) to an area with dimensions of approximately 50 mm × approximately 40 mm (i.e., illumination area 46), which means that the field of view of the detector 58 can be controlled. In the first configuration, up to three mice (or other similar samples 22) can be imaged. In the second configuration, approximately one-third of the body of one mouse (or similar sample 22) can be imaged in a 50 mm × 40 mm area (at a Z distance of 200 mm).

[0083] By combining illumination modulation, XY translation, and FOV adjustment, the infrared imaging system can capture images of sample 22 anywhere in a 156mm × 125mm × 50mm three-dimensional space with a spatial sampling rate of 80μm per pixel when the field of view is approximately 50mm × 40mm.

[0084] method According to the examples, a method for imaging a sample having a fluorescent marker is also provided.

[0085] The method may include the step of providing a sample to a sample holder, the sample holder having a sample contact surface and a sample plane.

[0086] The method may include the step of generating first and second infrared illumination beams toward a sample using first and second illumination modules, wherein the first and second infrared illumination beams interact in the imaging plane to define an illumination region having a rectangular and homogeneous power profile.

[0087] The method may include the step of moving the sample holder to multiple positions within the enclosure.

[0088] The method may include the step of aligning first and second infrared illumination beams to move the illumination area within the enclosure.

[0089] The method may include the step of superimposing the sample plane and the imaging plane at one of several locations within the enclosure.

[0090] The method may include the step of collecting or receiving light emitted by a fluorescent marker on a sample by illuminating the sample with an illumination beam on the imaging plane when the sample plane is superimposed on the imaging plane.

[0091] In some embodiments, the method further includes shifting the sample plane perpendicular to the sample contact surface.

[0092] In some embodiments, the sample plane is offset perpendicularly from the sample contact surface by a value corresponding to the thickness of the sample or a fraction thereof.

[0093] In some embodiments, the sample plane coincides with the sample contact surface.

[0094] In some embodiments, the method further includes heating the sample holder.

[0095] In some embodiments, the first and second infrared illumination beams have wavelengths of approximately 750 nm, approximately 808 nm, or approximately 980 nm.

[0096] In some embodiments, the method further includes adjusting the first and second infrared illumination beams respectively using a Köhler integrator.

[0097] In some embodiments, the method further includes calibrating the first and second illumination modules based on calibration data, which maps multiple orientations of the first and second illumination modules to corresponding multiple illumination power densities of the first and second infrared beams and to corresponding multiple positions of the sample holder within the enclosure.

[0098] In some embodiments, the method further includes collecting and aligning light emitted by a fluorescent marker using a first optical circuit, and forming an image of the sample on a sensor using a second optical circuit.

[0099] Several alternative embodiments and examples are described and explained herein. The embodiments described above are intended to be illustrative only. Those skilled in the art will recognize the characteristics of each embodiment, as well as possible combinations and variations of the components. Those skilled in the art will further recognize that any of the embodiments can be provided in any combination with other embodiments disclosed herein. Therefore, these embodiments and examples should be considered illustrative and not limiting in all respects. Thus, although certain embodiments are described and explained, many modifications can be conceived without significantly departing from the scope set forth in the appended claims.

Claims

1. An infrared imaging system for imaging a sample having a fluorescent marker, wherein the infrared imaging system is Enclosure and, A sample holder attached to the enclosure, the sample holder having a sample contact surface and a sample plane, A light source configured to illuminate the sample contact surface, the light source comprising a first illumination module and a second illumination module, each illumination module configured to project corresponding first and second infrared illumination beams toward the sample holder, the first and second infrared illumination beams interacting in the imaging plane to define an illumination area having a rectangular, homogeneous power profile, the first and second illumination modules being calibrated based on calibration data, the calibration data mapping multiple orientations of the first and second illumination modules to corresponding multiple illumination power densities of the first and second infrared illumination beams and to corresponding multiple positions of the sample holder within the enclosure, A motor assembly configured to move the sample holder to multiple positions within the enclosure, An optical mechanical mechanism configured to direct the first and second infrared illumination beams in order to move the illumination area within the enclosure, A control unit operably connected to the motor assembly and the optical mechanical mechanism, the control unit configured to superimpose the sample plane and the imaging plane at any of the plurality of positions within the enclosure, An infrared imaging system comprising: a detector configured to receive light emitted by the fluorescent marker of the sample upon illumination of the sample on the imaging plane when the sample plane is superimposed on the imaging plane, wherein the calibration data ensures that the density of the rectangular homogeneous power profile is maintained even if the distance between the sample holder and the detector changes.

2. The infrared imaging system according to claim 1, wherein the enclosure defines an internal volume, and the enclosure further includes a door or drawer for accessing the contents of the internal volume.

3. The infrared imaging system according to claim 1 or 2, wherein the sample plane is offset perpendicularly from the sample contact surface.

4. The infrared imaging system according to claim 3, wherein the sample plane is offset perpendicularly from the sample contact surface by a value corresponding to the thickness of the sample or a fraction thereof.

5. The infrared imaging system according to claim 1 or 2, wherein the sample plane coincides with the sample contact surface.

6. The infrared imaging system according to any one of claims 1 to 5, wherein the sample contact surface is formed from a black powder-coated steel plate.

7. An infrared imaging system according to any one of claims 1 to 6, further comprising one or more anesthetic ports, the one or more of which are configured for injecting anesthetic gas into the enclosure and collecting the anesthetic gas from the enclosure.

8. The infrared imaging system according to any one of claims 1 to 7, further comprising a heating element in thermal contact with the sample holder.

9. The infrared imaging system according to any one of claims 1 to 8, further comprising a barrier attached to the sample holder, wherein the barrier protrudes upward from the plane of the sample.

10. The infrared imaging system according to any one of claims 1 to 9, wherein each of the first illumination module and the second illumination module includes one or more laser diodes.

11. The infrared imaging system according to any one of claims 1 to 10, wherein the first and second infrared illumination beams have wavelengths of about 750 nm, about 808 nm, or about 980 nm.

12. The infrared imaging system according to any one of claims 1 to 11, wherein the illumination area has an illumination power density that is in the range of about 1 mW / mm² to about 3 mW / mm².

13. The infrared imaging system according to any one of claims 1 to 12, wherein each of the first and second illumination modules includes a Köhler integrator.

14. The infrared imaging system according to any one of claims 1 to 13, wherein the first and second illumination modules are arranged symmetrically on both sides of the detector.

15. The infrared imaging system according to any one of claims 1 to 14, wherein the detector includes an InGaAs camera.

16. The aforementioned detector, Sensors and, A first optical circuit configured to collect and parallelize the light emitted by the fluorescent marker, An infrared imaging system according to any one of claims 1 to 15, comprising a second optical circuit configured to form an image of the sample on the sensor.

17. The infrared imaging system according to claim 16, further comprising an electric focusing mechanism connected to the detector, wherein the electric focusing mechanism is configured to change the distance between the first optical circuit and the second optical circuit.

18. The infrared imaging system according to claim 16 or 17, further comprising a filter wheel positioned between the first optical circuit and the second optical circuit, wherein the filter wheel comprises a plurality of filters.

19. A method for imaging a sample having a fluorescent marker, To provide the sample on a sample holder of an infrared imaging system having a sample contact surface and a sample plane, First and second infrared illumination beams are generated toward the sample by first and second illumination modules, interacting in the imaging plane to define an illumination region having a rectangular and homogeneous power profile. Calibrating the first and second illumination modules based on calibration data, wherein the calibration data maps a plurality of orientations of the first and second illumination modules to a plurality of corresponding illumination power densities of the first and second infrared beams and a plurality of corresponding positions of the sample holder within the enclosure of the infrared imaging system. Moving the sample holder to multiple positions within the enclosure of the infrared imaging system, The first and second infrared illumination beams are oriented to move the illumination area within the enclosure of the infrared imaging system, The sample plane and the imaging plane are superimposed at any of the plurality of positions within the enclosure of the infrared imaging system. When the sample plane is superimposed on the imaging plane, the light emitted by the fluorescent marker of the sample by the illumination beam on the imaging plane is collected by a detector, and the calibration data is collected in such a way that the density of the rectangular, homogeneous power profile is maintained even if the distance between the sample holder and the detector changes. Methods that include...

20. The method according to claim 19, further comprising shifting the sample plane perpendicular to the sample contact surface.

21. The method according to claim 20, wherein the sample plane is offset perpendicularly from the sample contact surface by a value corresponding to the thickness of the sample or a fraction thereof.

22. The method according to claim 19, wherein the sample plane coincides with the sample contact surface.

23. The method according to any one of claims 19 to 22, further comprising heating the sample holder.

24. The method according to any one of claims 19 to 23, wherein the first and second infrared illumination beams have wavelengths of about 750 nm, about 808 nm, or about 980 nm.

25. The method according to any one of claims 19 to 24, further comprising adjusting each of the first and second infrared illumination beams with a Köhler integrator.

26. The light emitted by the fluorescent marker is collected and aligned by the first optical circuit, The second optical circuit forms an image of the sample on the sensor. The method according to any one of claims 19 to 25, further comprising:

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