Infrared imaging system and related method

There are provided infrared imaging systems and methods for imaging a sample with fluorescent markers. The system includes a light source configured to illuminate a sample-contacting surface. The light source includes first and second illumination modules, each configured to project a corresponding first and second infrared illumination beam towards a sample holder, the infrared illumination beams interacting at an imaging plane to define an illumination area having a rectangular and homogeneous power profile. The system also includes a control unit operatively connected to a motor assembly and to an optomechanical mechanism. The control unit is configured to superimpose the sample plane and the imaging plane at any of the multiple locations within the enclosure. The system includes a detector configured to receive light emitted by the fluorescent markers of the sample upon illumination of the same in the imaging plane when the sample plane is superimposed with the imaging plane.

TECHNICAL FIELD

The technical field generally relates to imaging systems and related methods and more particularly concerns an infrared imaging system and related method.

BACKGROUND

Many preclinical imaging systems are available on the market. Nonlimitative examples includes the IVIS® Spectrum in vivo imaging system from Perkin Elmer, the Lago from Spectral Instruments Imaging, the Pearl Trilogy® from Li-Cor, the iBox® from UVP and the Newton from Vilber. These commercially available solutions typically rely on silicon-based detectors and, as a result, are intrinsically limited in terms of applications, as 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., about 400 nm to about 1000 nm).

Light absorption and scattering of living tissues is much weaker in the second near-infrared window (NIR-II i.e., about 1000 nm to about 1700 nm) portion of the electromagnetic spectrum, in comparison with the visible or NIR-I portions of the electromagnetic spectrum, meaning that small animals would be more transparent in the NIR-II imaging window.

There is thus a need for techniques, methods, systems and devices that addresses or at least mitigate at least some of the challenges presented above.

SUMMARY

In accordance with one aspect, there is provided an infrared imaging system for imaging a sample with fluorescent markers. The infrared imaging system includes an enclosure, a sample holder, a light source, a motor assembly, an optomechanical mechanism, a control unit and a detector. The sample holder is mounted in the enclosure. The sample holder has a sample-contacting surface and a sample plane. The light source is configured to illuminate the sample-contacting surface and includes a first illumination module and a second illumination module, each being configured to project a corresponding first and second infrared illumination beam towards the sample holder. The first and second infrared illumination beams interact at an imaging plane to define an illumination area having a rectangular and homogeneous power profile. The motor assembly is configured to move the sample holder at multiple locations within the enclosure. The optomechanical mechanism is configured to orient the first and second infrared illumination beams to move the illumination area within the enclosure. The control unit operatively is connected to the motor assembly and to the optomechanical mechanism, the control unit and is configured to superimpose the sample plane and the imaging plane at any of the multiple locations within the enclosure. The detector is configured to receive light emitted by the fluorescent markers of the sample upon illumination of the same in the imaging plane when the sample plane is superimposed with the imaging plane.

In some embodiments, the enclosure defines an internal volume, the enclosure further including a door or a drawer for accessing a content of the internal volume.

In some embodiments, the sample plane is vertically offset from the sample-contacting surface.

In some embodiments, the sample plane is vertically offset from the sample-contacting surface by a value corresponding to a thickness of the sample or a fraction thereof.

In some embodiments, the sample plane coincides with the sample-contacting surface.

In some embodiments, the sample-contacting surface is made from a black powder coated steel sheet.

In some embodiments, the infrared imaging system further includes one or more anesthesia ports, said one or more anesthesia ports being configured for the injection of an anesthesia gas in the enclosure and for the collection of the anesthesia gas from the enclosure.

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

In some embodiments, the infrared imaging system further includes a barrier mounted to the sample holder, the barrier projecting upwardly from the sample plane.

In some embodiments, each one of the first illumination module and the second illumination module includes one or more laser diodes.

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

In some embodiments, the illumination area has an illumination power density included in a range extending from about 1 mW/mm2to about 3 mW/mm2.

In some embodiments, each one of the first and second illumination module includes a Köhler integrator.

In some embodiments, the first and second illumination modules are symmetrically disposed on both sides of the detector.

In some embodiments, the first and second illumination modules are calibrated based on calibration data, the calibration data mapping a plurality of orientations of the first and second illumination modules with a corresponding plurality of illumination power densities of the first and second infrared illumination beams and with a corresponding plurality of positions of the sample holder within the enclosure.

In some embodiments, the detector includes a InGaAs camera.

In some embodiments, the detector includes:a sensor;a first optical circuit configured to collect and collimate the light emitted by the fluorescent markers; anda second optical circuit configured to form an image of the sample on the sensor.

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

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.

In accordance with another aspect, there is provided method for imaging a sample with fluorescent markers. The method includes providing the sample on a sample holder, the sample holder having a sample-contacting surface and a sample plane; generating first and second infrared illumination beam towards the sample with first and second illumination modules, the first and second infrared illumination beams interacting at an imaging plane to define an illumination area having a rectangular and homogeneous power profile; moving the sample holder at multiple locations within the enclosure; orienting the first and second infrared illumination beams to move the illumination area within the enclosure; superimposing the sample plane and the imaging plane at any of the multiple locations within the enclosure; and collecting light emitted by the fluorescent markers of the sample upon illumination of the same by the illumination beam light in the imaging plane when the sample plane is superimposed with the imaging plane.

In some embodiments, the method further includes vertically offsetting the sample plane from the sample-contacting surface.

In some embodiments, the sample plane is vertically offset from the sample-contacting surface by a value corresponding to a thickness of the sample or a fraction thereof.

In some embodiments, the sample plane coincides with the sample-contacting surface.

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

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

In some embodiments, the method further includes conditioning each one of the first and second infrared illumination beams with a Köhler integrator.

In some embodiments, the method further includes calibrating the first and second illumination modules based on calibration data, the calibration data mapping a plurality of orientations of the first and second illumination modules with a corresponding plurality of illumination power densities of the first and second infrared beams and with a corresponding plurality of positions of the sample holder within the enclosure.

In some embodiments, the method further includes:collecting and collimating the light emitted by the fluorescent markers with a first optical circuit; andforming an image of the sample on a sensor with a second optical circuit.

Other features and advantages of the method and system described herein will be better understood upon a reading of preferred embodiments thereof with reference to the appended drawings. Although specific features described in the above summary and in the detailed description below may be described with respect to specific embodiments or aspects, it should be noted that these specific features can be combined with one another unless stated otherwise.

DETAILED DESCRIPTION

In the present description, similar features in the drawings have been given similar reference numerals. To avoid cluttering certain figures, some elements may not have been indicated if they were already identified in a preceding figure. It should also be understood that the elements of the drawings are not necessarily depicted to scale, since emphasis is placed on clearly illustrating the elements and structures of the present embodiments. Furthermore, positional descriptors indicating the location and/or orientation of one element with respect to another element are used herein for ease and clarity of description. Unless otherwise indicated, these positional descriptors should be taken in the context of the figures and should not be considered limiting. More particularly, it will be understood that such spatially relative terms are intended to encompass different orientations in the use or operation of the present embodiments, in addition to the orientations exemplified in the figures.

Unless stated otherwise, the terms “connected” and “coupled”, and derivatives and variants thereof, refer herein to any structural or functional connection or coupling, either direct or indirect, between two or more elements. For example, the connection or coupling between the elements may be mechanical, optical, electrical, thermal, logical, or any combination thereof.

The terms “a”, “an” and “one” are defined herein to mean “at least one”, that is, these terms do not exclude a plural number of items, unless stated otherwise.

Terms such as “substantially”, “generally” and “about”, that modify a value, condition or characteristic of a feature of an exemplary embodiment, should be understood to mean that the value, condition or characteristic is defined within tolerances that are acceptable for the proper operation of this exemplary embodiment for its intended application or that fall within an acceptable range of experimental error. In particular, the term “about” generally refers to a range of numbers that one skilled in the art would consider equivalent to the stated value (e.g., having the same or equivalent function or result). In some instances, the term “about” means a variation of ±10 percent of the stated value. It is noted that all numeric values used herein are assumed to be modified by the term “about”, unless stated otherwise.

Likewise, the terms “superimposition”, “superimpose”, “superimposed” and “superimposing” are intended to refer herein to a condition in which two elements are either the same position or within some predetermined tolerance of each other in terms of spatial alignment. That is, these terms are meant to encompass not only “exactly” or “identically” superimposing the two elements but also “substantially”, “approximately” or “subjectively” superimposing the two elements, as well as providing a higher or best superimposition among a plurality of superimposition possibilities.

In the present description, the expression “based on” is intended to mean “based at least partly on”, that is, this expression can mean “based solely on” or “based partially on”, and so should not be interpreted in a limited manner. More particularly, the expression “based on” could also be understood as meaning “depending on”, “representative of”, “indicative of”, “associated with” or similar expressions.

In the present description, the terms “light” and “optical”, and variants and derivatives thereof, are used to refer to radiation in any appropriate region of the electromagnetic spectrum. The terms “light” and “optical” are therefore not limited to visible light, but can also include, without being limited to, the infrared and ultraviolet regions. For example, in some implementations, the present techniques can be used with electromagnetic signals having wavelengths ranging from about 400 nm to about 1700 nm, for example between 1000 nm and 1700 nm. However, this range is provided for illustrative purposes only and some implementations of the present techniques may operate outside this range. Also, the skilled person will appreciate that the definition of the ultraviolet, visible infrared and near-infrared ranges in terms of spectral ranges, as well as the dividing lines between them, can vary depending on the technical field or the definitions under consideration, and are not meant to limit the scope of applications of the present techniques.

In the present description, the expressions “illumination beam spectrum”, synonyms or derivatives thereof are used to broadly refer to the spectral power distribution of an illumination beam. The illumination spectrum can represent the distribution of power radiated per unit area and per unit wavelength or frequency over a spectral region of the electromagnetic spectrum.

The present description generally relates to infrared imaging system(s), associated method(s) and technique(s) for preclinical imaging purposes. In the context of the current description, the infrared imaging system can sometimes be referred to as an “IR VIVO instrument”. It is to be noted that the expression “preclinical imaging” is herein understood as techniques that allow the visualization and inspection of living animals (e.g., small animals, such as mice, rats and the like). Preclinical imaging techniques can be particularly useful for research purposes (e.g., drug development).

The infrared imaging system that will be described in greater detail below is a fluorescence-based imaging instrument. Such an instrument generally includes a light source that uses an excitation light to excite fluorescent probes inside the sample, which can be, in the context of preclinical imaging systems, small animals. The instrument also includes a detector that is configured to detect the fluorescence signal generated by these probes. Other optical component can be provided between the sample and the detector, such as, for example and without being limitative, imaging lens(es), spectral filter(s), a dichroic element (e.g., for separating the output signal into two spectral bands, which may be detected by two different cameras), and other optical components.

Now turning to the Figures, different embodiments of the infrared imaging system and method will be presented.

With reference toFIG.1, there is shown an infrared imaging system20for imaging a sample22. Of note, the sample22may include one or more animals. As such, the expression “sample” is not limited to refer to only one animal and is not intended to be limitative. Fluorescent markers (not illustrated) are provided in the sample22. For example, and without being limitative, the fluorescent markers can be injected inside the small animal being imaged. It is to be noted that the expressions “fluorescent marker(s)” and “fluorescent probe(s)” will be used interchangeably throughout the description. Nonlimitative examples of fluorescent markers are quantum dots (e.g., PbS, Ag2S), organic molecules like indocyanine green (ICG) and IR800 dye molecules, single wall carbon nanotubes, rare earth nanoparticles and the like.

Enclosure and Sample Holder

The infrared imaging system20includes an enclosure24(sometimes referred to as a “chamber”). The enclosure24includes walls defining an internal volume26in which can be mounted at least some of the other components of the infrared imaging system20. The enclosure24generally includes a door or a drawer (not shown inFIG.1) for accessing the internal volume26(i.e., the content thereof) when required, for example, and without being limitative for setting up a preclinical test or preparing the sample22. In some embodiments, the whole enclosure24can be light-tight. In some embodiments, the door or the drawer are light tight. It is to be noted that the enclosure24may be equipped with components such as, for example and without being limitative, anesthetic gas manifold(s), gas pipe(s) and thermal plate(s), as it will be described in greater detail below.

The infrared imaging system20also includes a sample holder28. The sample holder28is positioned in the enclosure24and has a sample-contacting surface30. The sample-contacting surface30has a sample plane32. It is to be noted that, in some implementations, the sample plane32may be vertically offset from the sample-contacting surface30by a value corresponding to the thickness (or a height) of the animals being imaged, or a fraction of the thickness (or the height) of the animals being imaged. Alternatively, the sample plane32may coincide or substantially coincide with the sample-contacting surface30. The sample22can be placed onto the sample-contacting surface30in a such a way that the sample plane32intersects with the sample22or at least a portion thereof. As it will be described in greater detail below, the sample holder28can be adjusted in translation along three dimensions, e.g., an x-axis, a y-axis and a z-axis. As such, the position of the sample plane32can change or be adjusted.

In some embodiments, the sample-contacting surface30is made from a black powder coated steel sheet. In other embodiments, the sample-contacting surface30can be made from aluminum. Alternatively, the sample-contacting surface30could be made from any type of material having the appropriate characteristics for preclinical tests or related medical applications as long as it has low reflection and fluorescence in the infrared and is relatively easy to clean with an ethanol solution or bleach (e.g., anodized aluminum, heated glass, and the like). It is to be noted that the material forming the sample-contacting surface30can be selected on various chemical (e.g., composition) and/or physical properties (e.g., optical and magnetic properties).

The infrared imaging system20generally includes components that are found in typical preclinical instruments. For example, the infrared imaging system20may generally include one or more anesthesia ports. In some embodiments, the infrared imaging system20includes three anesthesia ports, meaning that three samples22can be placed on the sample-contacting surface30. It will be noted that the number of anesthesia ports may be different from three, and that the fact that the infrared imaging system20may include three anesthesia ports serves an illustrative purpose only and should therefore not be considered limitative. For example, the infrared imaging system20may include one, two, three, four, five or more anesthesia ports. Similarly, the number of animals forming the sample22may also be different from three. For example, and without being limitative, the sample22may include one, two, three, four, five or more animals. In some embodiments, the number of anesthesia port(s) may be identical to the number of animal(s) forming the sample22. In other embodiments, the number of anesthesia port(s) may be proportional or at least related to the number of animal(s) forming the sample22. For example, and without being limitative, the ratio between the number of anesthesia port(s) and the number of animal(s) forming the sample22may be 1:1, 1:2, 1:3, or any other ratios that allow the anesthesia port(s) to achieve their function. In this regard, it is to be noted that the anesthesia ports allow for the injection and collection of the anesthesia gas, for inputting/outputting the anesthesia gas in/out the enclosure24. Anesthesia gas is generally useful to keep the sample22immobile during the imaging of the same.

The infrared imaging system20may also include a thermal plate or similar device(s). It is to be noted that the thermal plate can either maintain the whole internal volume26at a given temperature or only a portion thereof (e.g., the sample-contacting surface30). The thermal plate is generally useful to maintain the sample22at a given temperature. Indeed, in the case of small animals or mammals in general, the average body temperature tends to decrease under anesthesia. The thermal plate can therefore mitigate this consequence. The infrared imaging system20can also include a barrier or a fence mounted to or onto the sample holder28. This feature can be particularly useful in the rare occasions wherein the sample22(e.g. the small animal) wakes up while being under anesthesia, or when a failure occurs in the anesthesia process.

The dimensions and geometrical configuration of the sample holder28can vary. However, the dimensions of the sample holder28are preferably such that the sample holder28substantially fits in the field of view of the imaging system20, as it will be described in greater detail below. In one example of implementation, the field of view has the following dimensions: about 15.6 cm×about 12.5 cm.

In some embodiments, the sample holder28can include a lower platform and an upper platform. The lower platform can span the entire floor (i.e., width and depth) of the enclosure24. The upper platform can be smaller and can have the following dimensions: 300 mm×250 mm. The upper platform can be mounted onto the lower platform. In some embodiments, the upper platform can be mechanically connected to the lower platform by a two-dimensional translation stage. In the context of the current description, the two-dimensional stage is configured to translate the upper surface along an X-axis and a Y-axis, to move the sample22sideways, along these two axes and relative to the field of view of the camera.

Light Source

The infrared imaging system20includes a light source34configured to illuminate the sample-contacting surface30. The light source34includes a first illumination module36and a second illumination module38. The first and second illumination modules36,38are each configured to project a corresponding first and second infrared illumination beam40,42towards the sample holder30. The first and second illumination modules36,38can each generate a relatively high-power infrared illumination beam. It has to be noted that the first and second illumination modules36,38may each include one or more laser diodes, each laser diode being associated with corresponding optical characteristics (e.g., intensity and/or spectral profile). The relatively high power is useful to generate a fluorescence signal that is strong enough to be detected (about 0.05 to about 3 mW/mm2). It has to be noted that the fluorescent markers in the NIR-II portion of the electromagnetic spectrum typically have a relatively low efficiency relative to the fluorescent marker that can be used in the visible portion of the electromagnetic spectrum. It also has to be noted that detectors configured to operate in the NIR-II portion of the electromagnetic spectrum are generally less sensitive relative to detectors configured to operate in the visible portion of the electromagnetic spectrum. It has to be noted that the wavelength of the infrared illumination beams40,42emitted by the first and second illumination modules36,38can be selected or varied. The selection can be manual (e.g., by a user) or automatic (e.g., each module36,38could sequentially and/or automatically select an infrared illumination wavelength). This feature can be useful for exciting fluorescent markers of different nature. It will be noted that the illumination with different wavelengths is generally performed sequentially and not simultaneously. Nonlimitative examples of wavelengths that may be used are 750 nm, 808 nm, 860 nm and 980 nm.

The first and second infrared illumination beams40,42interact at an imaging plane44to define an illumination area46. The imaging plane44extends along the X-axis and the Y-axis. The illumination area46has a rectangular and homogeneous power profile (sometimes referred to as an “illumination profile” or a “power density profile”), and also extends along the X-axis and the Y-axis. The power or illumination density in the illumination area is representative of a cumulative power of the first and second infrared illumination beams40,42. In some embodiments, the power or illumination density is about 1 mW/mm2, for a total power of about 20 W. These values are relatively close to, but lower than the limit of power or illumination density for living tissue (i.e., about 3 mW/mm2).

One skilled in the art would note that the illumination in existing visible preclinical imagers is generally provided by halogen white lamps. These lamps are typically optically coupled with filters (e.g., a filter wheel) for excitation filtering. Existing visible preclinical images can also use LED sources and/or laser sources. In these cases, the illumination density is smaller from the illumination density that can be obtained using the first and second illumination modules36,38included in the technology being herein described. Indeed, the illumination density achievable with the technology presented in the current disclosure is an order of magnitude greater than existing technologies relying on halogen and LED devices.

The light generated by each laser diode included in the first and second illumination modules36,38is optically structured by optical components included in the first and second illumination modules36,38. This allows the definition of the illumination area46, having a substantially homogeneous rectangular shape, on the sample22. It will be noted that the illumination area46generally has the same dimensions as the field of view of the infrared imaging system20. One skilled in the art would understand that homogeneous illumination is important in order to ensure that every part of the sample22receives the same illumination density. For example, in the context wherein three mice are being imaged and placed in the field of view, they must all be illuminated with the same power density, so that the imaging results can be compared one with another. It is also advantageous to restrict the illumination area46to the field of view of the infrared imaging system20, in order to avoid wasting laser power. Indeed, if too much laser power is lost, then more powerful lasers would be needed, which would add to the costs of the system, as well as adding to the complexity of the thermal management of the first and second illumination modules36,38. Moreover, one would note that projecting a portion of the first and second infrared illumination beams40,42would contribute to adding unwanted stray light in the infrared imaging system20, which could be detected by the detector, and would then reduce the overall sensitivity of the infrared imaging system20.

Now turning toFIG.2, the first and second illumination modules36,38structure the light emitted by the laser diodes using a Köhler integrator design. InFIG.2, only the first illumination module36is illustrated, but it will be readily understood that the description also applies to the second illumination module38. The Köhler integrator design is known in the art, and includes the use of a collimation lens48, a first fly-eye lens50, a second fly-eye lens52, a projection lens54and a fold mirror55. These elements define an optical path therebetween, the optical path extending along an optical axis56. In some embodiments, the projection lens54may be substituted by an optical assembly (not illustrated in the Figures). The optical assembly may include a plurality of optical elements. Such optical elements include, but are not limited to lenses, mirrors, filters, and other suitable reflective, refractive and/or diffractive optical components.

In the illustrated embodiments, the first and second illumination modules36,38are positioned to project light (i.e., the first and second infrared illumination beam40,42) from above the sample22. Now referring back toFIG.1, it can be seen that the first and second illumination modules36,38are located on both sides of the detector58. In some embodiments, the first and second illumination modules36,38are symmetrically disposed on both sides of the detector58. It will however be understood that the first and second illumination modules36,38may be disposed at other positions with respect to the detector and/or may be asymmetrically positioned with respect thereto without departing from the scope of protection.

The first and second illumination modules36,38, and so the first and second infrared illumination beams40,42are generally not parallel to the Z-axis, but rather form a slight angle relative to the Z-axis. When the sample22includes a plurality of animals (i.e., two or more animals), it has to be noted that in order to prevent the animals from casting a shadow on each other, the angle between the first and second illumination beams and the Z-axis is kept as small as possible.

As it will described in greater detail below, the first and second illumination modules36,38can be rotated, which allows the projection of the first and second infrared illumination beams40,42at or near the center of the field of view of the infrared imaging system20, for example when the sample holder28moves up and/or down. The rotation of the first and second illumination modules36,38allows keeping a homogeneous illumination in the illumination area46.

Motor Assembly

A block diagram illustrating the operational connection between some of the components included in the infrared imaging system20is shown inFIG.5. The infrared imaging system20includes a motor assembly60configured to move the sample holder at multiple locations within the enclosure24. The motor assembly60can include one or more motors. The motor can be of any types or designs.

In some embodiments, the sample holder28can be moved or translated along two axes (e.g., X-axis and Y-axis) by the motor assembly60. The motor assembly60can be configured to translate the sample holder28in the X-axis and the Y-axis sequentially or simultaneously. For example, the sample holder28could be sequentially translated in a direction parallel to the X-axis, and then in a direction parallel to the Y-axis, or vice-versa. Alternatively, the sample holder28could be configured to be simultaneously adjustable along the x-axis and the y-axis. It will be noted that the motor assembly60can monitor or record the displacement of the sample holder28along each axis. The monitored or recorded information are included in calibration data.

In some embodiments, the motor assembly60may include two motors, each one of the motors being configured to move the sample holder28along a respective direction (e.g., X-axis or Y-axis).

The sample holder28can also be moved or translated in the Z-axis. The displacement of the sample holder28is generally performed once the sample holder28has been aligned or positioned in the X-axis and the Y-axis in the enclosure24. Movement of the sample holder28along this direction can be provided by one or more motors.

It will be appreciated that moving the sample holder28with the motor assembly60allows passing from a “wide view mode” towards a “close view mode”. Switching between those two view modes may be useful when the sample22includes a plurality of animals. For example, and without being limitative, the motor assembly60may allow passing from a first field of view encompassing all of the animals of the sample22to a second field of view encompassing only one animal forming the sample22, or a portion thereof. In some embodiments, the field of view may be adjusted to simultaneously image a portion of each of the animals forming the sample22, which may be useful in the context of a comparative characterization of a specific portion of the animals.

In some embodiments, the motor assembly60is manual. In these embodiments, the translation of the sample holder28includes two steps. In a first step, a spring-loaded normally-on brake, which prevents the upper platform from sliding, is released. It can be released momentarily by depressing a button, or semi-permanently by depressing and locking the same button. Once the brake is released, the upper platform can be pushed relatively smoothly “side to side” (e.g., along the X-axis), as well as “forwards and backwards” (e.g., along the Y-axis). In other embodiments, the motor assembly60is automatic.

With reference toFIG.5, the infrared imaging system20includes an optomechanical mechanism62configured to orient the first and second infrared illumination beams40,42to move the illumination area46within the enclosure24. Orienting the first and second infrared illumination beams40,42generally includes changing a spatial configuration (by rotation, translation or a combination thereof) of the first and second illumination modules36,38. More particularly, the adjustment of the orientation of the first and second infrared illumination beams40,42by the optomechanical mechanism62, combined with the motor assembly60, can be used to control the size of the illumination area46.

In some embodiments, and now referring toFIG.2, orienting the first and second illumination modules36,38includes rotating the same about the optical axis56. The rotation of the illumination modules36,38from an initial position towards a subsequent position results in the first and second infrared illumination beams40,42interacting from a first imaging plane (associated with the initial position of the modules36,38) to a subsequent imaging plane (associated with the subsequent position of the modules36,38) to define a subsequent illumination area, also having a substantially rectangular and substantially homogeneous power profile, as it will be described in greater detail with reference toFIG.4. It is to be noted that the first imaging plane and the subsequent plane are generally not at a same position along the Z-axis. For example, the subsequent imaging plane is generally higher or lower than the first imaging plane. As such, orienting the first and second illumination modules36,38results in a change in the position of the imaging plane44within the enclosure24(e.g., along the Z-axis). In other embodiments, only the fold mirror55could be rotated. It is to be noted that in addition to its orientation, the power of the first and second infrared illumination beams40,42can be also be altered (i.e., modified or changed). The alteration or adjustment of the orientation of the first and second infrared illumination beams40,42to move the illumination area46within the enclosure is generally referred to as “illumination modulation”.

It has to be noted that the first and second illumination modules36,38are generally calibrated. The calibration data include, but are not limited to a mapping between a plurality of orientations of the first and second illumination modules36,38and corresponding optical properties of the first and second infrared illumination beams40,42. As such, once the position of the sample holder28in the enclosure24is known, the orientation of the first and second illumination modules36,38to be achieved can be determined, either because this information is included in the calibration data or by calculations based on calibration data, e.g., interpolation, extrapolation and other techniques. The interpolation may be linear, polynomial (Lagrange, Newton and the like), a spline, or the like. In one example, the calibration step is useful to maintain a relatively constant power density comprised between about 1 mW/mm2and about 3 mW/mm2at any location in the enclosure24, i.e., even if the distance between the sample holder28and the detector58changes. More specifically, a change in the orientation of the first and second infrared illumination beams40,42allows at least partially, approximately or substantially preserving the rectangular and homogeneous power profile at any locations within the enclosure24. In some embodiments, the calibration data may further include information on illumination powers that need to be provided or generated in order to keep the power density constant or to a desired value. Of note, the power density generally increases as the sample holder28rises, i.e., when the distance between the sample holder28and the first and second illumination modules36,38. The rectangular and homogeneous illumination becomes smaller as the sample holder28raises. In some embodiments, the power density of the infrared illumination beams40,42may be controlled in order to maintain the power density of the illumination area relatively constant. In some embodiments, the power density of the illumination modules36,38may be constant, which would allow increasing the power illumination density of the illumination area, when the platform rises.

Control Unit

As illustrated inFIG.5, the infrared imaging system20includes a control unit64. The control unit64is operatively connected to the motor assembly60and to the optomechanical mechanism62. The control unit64is configured to superimpose the sample plane32and the imaging plane44at any of the multiple locations within the enclosure24. When the sample plane32and the imaging plane44are superimposed, the two planes are substantially at the same position along the Z-axis. It is to be noted that when the sample plane32is vertically offset from the sample-contacting surface30, for example when it is required to accommodate for the thickness of the sample22(or a fraction thereof), the control unit64may be configured to position the sample plane32and the imaging plane44according to this vertical offset. In some embodiments, the value of the vertical offset may be automatically determined by the infrared imaging system20. In other embodiments, the value of the vertical offset may be provided by a user and may be, for instance, manually provided. In yet other embodiments, the value of the vertical offset may be obtained from a database. Such a database may, for example and without being limitative, associate an average value of the thickness of the animal(s) forming the sample22with a corresponding position of the sample holder28within the enclosure24.

For example, and without being limitative, the control unit64can 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. The programmable computer can, in some embodiments, execute computer programs that allow controlling the motor assembly60and the optomechanical mechanism62. The control unit64is configured to keep track, monitor or record the position of the sample holder28in the enclosure24. More particularly, the control unit64receives as an input the position of the sample holder28in the enclosure24, and, based on the calibration data, outputs a signal that is sent to the first and second illumination modules36,38.

Now referring toFIG.4, there is illustrated an example in which the sample holder28starts in an initial or first position (left portion of the image) and is then moved to a subsequent or second position (right portion of the image).

In the initial position, the sample holder28is positioned such that the distance between the detector58and the sample holder28is approximately 400 mm (in the Z-axis). As such, the sample plane32is distanced by approximately 400 mm from the detector58. In the initial position, the first and second illumination modules36,38are oriented such that the illumination area46covers the sample22. The imaging plane44and the sample plane32are superimposed.

In the subsequent position, the sample holder28is positioned such that the distance between the detector58and the sample holder28is approximately 200 mm (in the Z-axis), meaning that the sample holder28has been brought up closer to the detector58. As such, the sample plane32′ is distanced by approximately 200 mm from the detector58. In the subsequent position, the orientation of the first and second illumination modules36,38are changed with respect to the initial position. It will be noted that, in the subsequent position illustrated in the nonlimitative embodiments ofFIG.4, the first and second illumination modules36,38are oriented such that the illumination area46′ covers the sample22. The imaging plane44′ and the sample plane32′ are superimposed.

It has to be noted that shape and dimensions of the illumination area46may change upon a change in the orientation of the first and second illumination modules36,38and/or a change in the position of the sample holder28in the enclosure24.

Detector

As mentioned above, the infrared imaging system20includes a detector58, see for exampleFIGS.1and3. The detector58is configured to receive light emitted by the fluorescent markers of the sample22upon illumination of the same by the first and second infrared illumination beams40,42light in the imaging plane46when the sample plane32is superimposed with the imaging plane44. In the depicted embodiments, the detector58includes a InGaAs camera. Such a camera allows relatively precise localization of fluorescence originating from within the sample22, yielding precious information for biologists.

As better illustrated inFIG.3, the detector58includes two optical circuits66,68, separated by a filter wheel70. The detector58also includes a sensor72. The first optical circuit66collects the light emanating from the sample22and approximately collimates it. The resulting light then passes through the double filter wheel70. The filter wheel70generally includes a plurality of filters. In some embodiments, the filter wheel70includes a bandpass filter and edgepass filters. In some embodiments, the first optical circuit66may include one or more detection lenses, and/or any other optical elements. For example, in one embodiment, the optical circuit66may include two lenses. The second optical circuit68forms the image of the sample22on the sensor72. In some embodiments, the second optical circuit68may include one or more lenses, and/or any other optical elements. In some embodiments, the detector58is provided with a motorized focus mechanism74. The motorized focus mechanism74is positioned between the second optical circuit68and the sensor72and can vary the distance between both in order to adjust the focus.

The filters included in the filter wheel70are typically dielectric interference filters, for which the wavelength of transmission is angle dependent. In some embodiments, the optical design of the first optical circuit66is such that the light passes through the filter wheel70as close as possible to normal incidence (i.e., parallel to the Z-axis). The light coming from different field points on the sample22hits the filter at different angles. As such, optimizing the optical design of the first optical circuit66to allow the light to pass through the filter close to normal incidence ensures that the detected wavelengths will be close for every point in the field of view.

It is also to be noted that the positioning of the filter wheel70between the two optical circuits66,68also prevents stray light from hitting the filters far from normal angle, which would result in unwanted light not being blocked by the filter and cause undesirable artefacts in the image formed on the sensor72.

Changing the working distance (i.e., the distance between the sample22and the sensor72) and adjusting the focus (e.g., the motorized focus) of the infrared imaging system20, in combination with the operation of the motor assembly60and the optomechanical mechanism62can allow imaging an area (i.e., the illumination area46) measuring about 156 mm by about 125 mm to an area (i.e., the illumination area46) measuring about 50 mm by about 40 mm, meaning that the field of view of the detector58can be controlled. In the first configuration, up to three mice (or other similar samples22) can be imaged. In the second configuration, about a third of the body of a single mouse (or similar sample22) can be imaged in an area of 50 mm×40 mm (at a Z distance of 200 mm).

The combination of the illumination modulation, the X-Y translation and the FOV adjustment, allows the infrared imaging system to capture an image of the sample22anywhere in a 156 mm×125 mm×50 mm three-dimensional space with a spatial sampling of 80 μm per pixel when the field of view is about 50 mm by 40 mm.

Method

In accordance with embodiments, there is also provided a method for imaging a sample with fluorescent markers.

The method can include a step of providing the sample on a sample holder, the sample holder having a sample-contacting surface and a sample plane.

The method can include a step of generating first and second infrared illumination beam towards the sample with first and second illumination modules, the first and second infrared illumination beams interacting at an imaging plane to define an illumination area having a rectangular and homogeneous power profile.

The method can include a step of moving the sample holder at multiple locations within the enclosure.

The method can include a step of orienting the first and second infrared illumination beams to move the illumination area within the enclosure.

The method can include a step of superimposing the sample plane and the imaging plane at any of the multiple locations within the enclosure.

The method can include a step of collecting or receiving light emitted by the fluorescent markers of the sample upon illumination of the same by the illumination beam light in the imaging plane when the sample plane is superimposed with the imaging plane.

In some embodiments, the method further includes vertically offsetting the sample plane from the sample-contacting surface.

In some embodiments, the sample plane is vertically offset from the sample-contacting surface by a value corresponding to a thickness of the sample or a fraction thereof.

In some embodiments, the sample plane coincides with the sample-contacting surface.

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

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

In some embodiments, the method further includes conditioning each one of the first and second infrared illumination beams with a Köhler integrator.

In some embodiments, the method further includes calibrating the first and second illumination modules based on calibration data, the calibration data mapping a plurality of orientations of the first and second illumination modules with a corresponding plurality of illumination power densities of the first and second infrared beams and with a corresponding plurality of positions of the sample holder within the enclosure.

In some embodiments, the method further includes collecting and collimating the light emitted by the fluorescent markers with a first optical circuit and forming an image of the sample on a sensor with a second optical circuit.

Several alternative embodiments and examples have been described and illustrated herein. The embodiments described above are intended to be exemplary only. A person skilled in the art would appreciate the features of the individual embodiments, and the possible combinations and variations of the components. A person skilled in the art would further appreciate that any of the embodiments could be provided in any combination with the other embodiments disclosed herein. The present examples and embodiments, therefore, are to be considered in all respects as illustrative and not restrictive. Accordingly, while specific embodiments have been illustrated and described, numerous modifications come to mind without significantly departing from the scope defined in the appended claims.