Three-dimensional sample scanning system

Abstract

The invention relates to a system for sample scanning, the system comprising: a first stage comprising a movable sample holder having a sample slot; a first displacement means coupled to the sample holder for moving the sample holder in a first direction; an optical arrangement comprising a light source for emitting light towards the sample slot, an element comprising: a first tube having a longitudinal axis along an optical path of the light emitted by the light source towards the sample slot; a second tube having a longitudinal axis for accommodating light scattered by a sample located in the sample slot, wherein the longitudinal axis of the first tube and the longitudinal axis of the second tube form an angle a of less than 90°, a second stage extending in a plane parallel to the first stage and comprising: a second displacement means coupled to the optical arrangement for moving the optical arrangement in a second direction; a third displacement means coupled to the element for moving the element in a direction parallel to the second direction; and a camera for receiving scattered light of a sample located in the sample slot, wherein the optical arrangement and the element are located between the first stage and the second stage, and the first direction is perpendicular to the second direction.

Description

Technical Field

[0001] This invention relates to the field of sample scanning systems. In particular, it relates to the field of portable systems for detecting light emission and fluorescence for scattered light. This invention is particularly applicable to the analysis of PCR (polymerase chain reaction) results and digital PCR results, where digital PCR results involve the generation of aqueous particles for nucleic acid amplification and analysis. Background Technology

[0002] Sample scanning systems can be used with samples in optically transparent materials to allow for optical excitation (fluorescence) or illumination (selective absorption) of such samples. Typically, a sample plate or microfluidic chip containing an array of samples to be analyzed is inserted into a system with an optical module that exposes the sample to excitation light and detects the scattered light. This allows for optical detection of the spectral properties of the scattered light from the sample, for example, within a microfluidic chip.

[0003] US Patent Application Publication No. US2016 / 0101418 applies this principle and discloses a method for quantifying nucleic acids in a sample. The method includes generating multiple droplets in oil within a microfluidic device, wherein at least some of the droplets contain nucleic acids, amplification reagents, and fluorescent probes or dyes contained therein. The droplets are delivered to a collection chamber to form an array of droplets. These droplets undergo multiple thermal cycles within the collection chamber to amplify nucleic acids within the droplets. The array of droplets is imaged during the multiple thermal cycles and at the end of the thermal cycles. The initial concentration of nucleic acids in the sample is calculated based on at least one of the ratio of aqueous droplets exhibiting fluorescence within the array at the end of the thermal cycles or the cycle threshold (Ct) of one or more aqueous droplets within the array.

[0004] This disclosure allows the array of droplets to be imaged during multiple thermal cycles and at the end of the thermal cycles. Changing the filter in this disclosure is time-consuming because the configuration does not allow for easy replacement of such a filter.

[0005] To improve movement accuracy, U.S. Patent 9,824,259 relates to an apparatus and method for microscopy of multiple samples, wherein the apparatus includes: a first optical detector configured to continuously employ multiple measurement positions and detect first image data of the sample at each measurement position with a first spatial resolution; an image data analysis device configured to determine, in each case, a region of the sample to be examined represented within the first image data for each sample; and a second optical detector coupled to the first optical detector such that the second optical detector tracks the first optical detector and thus employs measurement positions previously employed by the first optical detector. The second optical detector is configured to detect corresponding second image data from the region of the sample to be examined in the relevant sample at a spatial resolution higher than the first spatial resolution.

[0006] In this disclosure, the detector holder is arranged on an XY-coordinate moving stage. This disclosure improves spatial resolution, but the sample analysis position is directly related to the number of optical detectors. Therefore, the number of focusing positions is limited.

[0007] In European Patent EP 1 620 572, sample wells are scanned by moving a detection module and activating excitation / detection channels. The detection module is moved such that the excitation / detection channels are sequentially positioned to be in optical communication with each of a plurality of sample wells. The device includes a support structure attachable to a thermal cycler and a detection module movably mounted on the support structure. The detection module includes one or more channels, each having an excitation light generator and an emission light detector disposed within the detection module. When the support structure is attached to the thermal cycler and the detection module is mounted on the support structure, the detection module is movable and thus positioned to be in optical communication with the plurality of wells.

[0008] This disclosure allows for imaging of different samples using a thermal cycler; however, changing the filter in this disclosure is time-consuming because the configuration does not allow for easy replacement. Focusing requires human intervention. Furthermore, the thermal cycler within the system adds to its complexity.

[0009] Therefore, to overcome the shortcomings of existing technologies, an improved system is needed that allows for greater flexibility and increased accuracy in image scanning. Furthermore, greater flexibility is also sought in the use of filters. Summary of the Invention

[0010] Therefore, the present invention relates to a system for scanning samples, comprising:

[0011] - A sample holder configured to hold the sample;

[0012] - An optical device comprising a light source for emitting light toward the sample holder, an emission light filter including a first array of filters, and a scattering light filter including a second array of filters;

[0013] - A base element comprising a first housing extending toward the sample holder along an optical path of light emitted by the light source and a second housing extending along an optical path of light scattered by a sample held in the sample holder;

[0014] The emitted light filter is positioned along the optical path of the emitted light, and the scattered light filter is positioned along the optical path of the scattered light; and

[0015] Wherein, the first housing is fixed relative to the second housing;

[0016] - A first shifting device, coupled to at least one of the optical device and the base element, for moving the emission filter and the scattering filter of the optical device relative to the base element along a first direction; and

[0017] - A second shifting device, different from the first shifting device and coupled to at least one of the base element and the sample holder, for moving the base element and the sample holder relative to each other along the first direction, such that the optical path of the emitted light moves relative to the sample held in the sample holder.

[0018] In one embodiment, the system includes:

[0019] - The first stage, which includes:

[0020] a movable sample holder with a sample slot.

[0021] o A first shifting device, which is coupled to a sample holder for moving the sample holder in a first direction,

[0022] - Optical device, comprising a light source for emitting light toward the sample slot,

[0023] - An element comprising:

[0024] The first tube has a longitudinal axis along the optical path of light emitted from the light source toward the sample slot.

[0025] The second tube has a longitudinal axis for accommodating light scattered by the sample located in the sample slot.

[0026] The longitudinal axis of the first tube and the longitudinal axis of the second tube form an angle α of less than 90°.

[0027] - A second stage, extending in a plane parallel to the first stage, and comprising:

[0028] o A second shifting device, which is coupled to the optical device for moving the optical device in a second direction, and

[0029] a third shifting device, which is coupled to the element for moving the element in a direction parallel to the second direction.

[0030] - and a camera, which is used to receive the scattered light from the sample located in the sample slot,

[0031] The optical device and the element are located between the first stage and the second stage, and the first direction is perpendicular to the second direction.

[0032] In a preferred embodiment, a first shifting device is coupled to a sample holder for moving the sample holder in a first direction Y and is driven by a first motor, thereby improving accuracy and limiting human intervention.

[0033] Preferably, the first motor is connected to the sample holder via a cable.

[0034] In a preferred embodiment, the second shifting device is coupled to the optical device for moving the optical device in the second direction X, and is driven by a second motor to improve accuracy, thereby limiting human intervention.

[0035] More preferably, the third shifting device is driven by a third motor, which is coupled to the element for moving the element in a direction parallel to the second direction X, thereby improving accuracy and limiting human intervention.

[0036] In a particular embodiment, the first shifting device is made of a first guide rail system and at least one U-shaped component and is coupled to a sample holder, the first guide rail system being configured to guide at least one U-shaped component in a first direction Y.

[0037] Compared to other possible displacement devices, this first guide rail system is easier to implement. It improves the accuracy of displacement in the first direction Y, thereby maintaining the accuracy of perpendicularity in the second direction X and the third direction Z.

[0038] In another specific embodiment, the second shifting device is made of a second guide rail system and at least one second U-shaped component, and is coupled to an optical device, the second guide rail system being configured to guide the second U-shaped component in a second direction X.

[0039] Compared to other possible displacement devices, this second guide rail system is easier to implement. It improves the accuracy of displacement in the second direction X, thereby maintaining the accuracy of perpendicularity in the first direction Y and the third direction Z.

[0040] In yet another specific embodiment, the third shifting device comprises a third guide system and at least one third U-shaped component, and is coupled to the element. The third guide system is configured to guide the at least third U-shaped component in a second direction X. This third guide system is easy to implement and improves the accuracy of displacement in the second direction X, thereby allowing the optical device and the element to move in very similar directions.

[0041] Preferably, the movable sample holder includes at least two sample slots.

[0042] In a preferred embodiment, the optical device according to the invention includes a main board extending in a plane perpendicular to the second stage and extending perpendicularly from the second stage in the same direction:

[0043] i. Light source

[0044] ii. Light emission filter, and

[0045] iii. Light-scattering filter plate,

[0046] The motherboard is configured such that:

[0047] - The light-emitting filter is configured to be located in the optical path of the light emitted by the light source, and

[0048] - The light-scattering filter is configured to be located in the optical path of the light scattered by the sample.

[0049] This configuration allows the emitted light filter and the scattered light filter to move together, thereby improving their positioning accuracy.

[0050] In an even more preferred embodiment, the emitted light filter includes an array of emitted light apertures, and the scattered light filter includes an array of scattered light apertures to form pairs of apertures for light emitted by the light source and light scattered by the sample, respectively. This allows multiple pairs of filters to be used immediately while reducing the risk of contamination. It also reduces image analysis time because filter replacement is simple and automatic.

[0051] In one embodiment, the emission light filter and the scattering light filter are arranged on one or more disks (such as turntables) connected to the emission light filter plate and the scattering light filter plate, which allows the filter to be easily selected by the rotation of the disks caused by a motor.

[0052] In another embodiment, the emission filters are arranged linearly in the emission filter plate, and the scattering filters are arranged linearly in the scattering filter plate. This allows for easy selection of the filters by translational movement along the linear shifting device. Preferably, the two linear arrangements are parallel. This configuration is particularly suitable for the translational movement-based environment of the present invention.

[0053] In another preferred embodiment, the emission filter and the scattering filter are arranged on one or more linear plates that extend from the main board in the same direction. This configuration requires only one shifting device and allows for more precise alignment of an emission filter with a pair of scattering filters when the filters are in a fixed relative position.

[0054] In another embodiment, the element includes a first slit (located in a first tube) having a cross-sectional shape complementary to that of the emission light filter and a second slit (located in a second tube) having a cross-sectional shape complementary to that of the scattering light filter, such that the emission light filter 36 and the scattering light filter can slide within the first and second slits to align the paired emission and scattering light apertures with the optical paths of the emitted and scattered light. This configuration improves the integrity of the filters configured to receive different filters. Therefore, the optical device is movable within the element to change the filter slots, and thus potentially change the filters, and this is used for different positions of the sample and / or the filters, since the element and the sample slot are movable. This configuration reduces analysis time.

[0055] In a particular embodiment, the system further includes a bright field light source coupled to the second tube and configured to emit bright light toward a sample located in a sample slot. In a preferred embodiment, the bright field light source is positioned substantially symmetrically with respect to the longitudinal axis of the second tube and the longitudinal axis of the first tube. This results in two symmetrical excitation paths, thus resulting in identical scattered light paths, each preferably forming a similar angle α of approximately 5°. This configuration allows for bypassing the emission filter of the bright field light source.

[0056] Preferably, the sample holder is further coupled to a fourth motor for focusing adjustment in a third direction Z perpendicular to the first direction Y and the second direction X.

[0057] Even more preferably, the system according to the invention further includes an electronic control unit for individually or in combination controlling the first motor, the second motor, the third motor, and the fourth motor.

[0058] In a preferred embodiment, a sample mask is located between the element and the sample slot. The sample mask is preferably a flat metal component with a rectangular aperture to guide excitation light and sample-scattered light. The sample mask faces the area to be imaged and avoids irradiating and degrading other areas of the sample by, for example, photobleaching. Preferably, the exposure duration for a given sample area is less than 20% of the total time required for image acquisition. More preferably, this time is less than 15% of the total time required for image acquisition (i.e., during which the sample is irradiated), even more preferably, this time is less than 10%, and still even more preferably, this time is less than 5% of the total time.

[0059] In a preferred embodiment, the present invention relates to a system for sample scanning, the system comprising:

[0060] - A first stage, which includes a sample slot movable in a first direction Y.

[0061] - An optical device comprising a light source for emitting light toward a sample slot, the optical device being configured to be displaceable in a second direction X.

[0062] - An element comprising a first tube and a second tube, the first tube having a longitudinal axis along the optical path of light emitted from a light source toward a sample slot, and the second tube having a longitudinal axis along the optical path of light scattered from a sample in the sample slot toward a camera, wherein the element is configured to be displaceable in a second direction X.

[0063] - A second stage, extending in a plane parallel to the first stage, and including means for moving optical devices and elements in a second direction X, and

[0064] - A camera, used to receive light scattered by the sample located in the sample slot.

[0065] The optical devices and components are located between the first and second stages, with the first direction perpendicular to the second direction. In one particular embodiment, the system is used for digital PCR and does not include a thermal cycler. In this particular embodiment, the thermal cycler is located in a separate device to provide separate and independent image analysis and thermal cycling functions.

[0066] definition

[0067] In this invention, the following terms have the following meanings:

[0068] - “Stage” refers to the base plate or flat horizontal surface of the system according to the present invention.

[0069] - "Optical path" refers to the direction of light emitted by a light source; it also includes changes in direction after scattering. - "Angle less than 90°" does not include angles of 0 degrees within the scope of this invention.

[0070] - The "vertical and parallel" features according to the invention include error tolerances for the tools used to measure these features.

[0071] - The "guide rail system" according to the present invention may include more than one guide rail.

[0072] - According to the present invention, a "pair of filters" refers to two filters located on the paths of emitted light and scattered light, respectively.

[0073] - According to the invention, a "shell" refers to a container configured to permanently house an element in a space. The shell may include a hollow object whose shape is configured to surround an optical path of light. The walls of the shell need not be solid and may include openings that allow airflow and light passage, as the scanning system can be used in a dark, enclosed space. However, in one embodiment, the shell may be opaque to prevent external light from entering and disturbing the light propagating within it. Furthermore, the interior of the shell may be treated or geometrically shaped to avoid scattering and reflection of light propagating within the shell. The shell may be cylindrical or have any other suitable shape.

[0074] The terms “digital PCR” or “dPCR” refer to a partial PCR analysis of a sample to determine the presence / absence, concentration, and / or copy number of a nucleic acid target in the sample based on how much of the sample portion supports the amplification of the target. Digital PCR can (or can not) be performed as endpoint PCR. For each partition, digital PCR can (or can not) be performed as real-time PCR. Theoretically, PCR results in the exponential amplification of nucleic acid sequences (analytes) in the sample. The initial concentration of nucleic acids can be theoretically calculated by measuring the number of amplification cycles required to reach a threshold level of amplification (as in real-time PCR). However, in practice, many factors make the PCR process non-exponential, such as varying amplification efficiencies, low copy numbers of the starting nucleic acids, and competition with background contaminant nucleic acids. Digital PCR is generally insensitive to these factors because it does not rely on the assumption that the PCR process is exponential. In digital PCR, individual nucleic acid molecules are spaced apart from each other and then amplified to a detectable level. Each partition then provides numerical information about the presence or absence of each individual nucleic acid molecule within that partition. When this technique is used to measure a sufficient number of partitions, the digital information can be combined to perform a statistically relevant measurement of the initial concentration of the nucleic acid target (analyte) in the sample. The concept of digital PCR can be extended to other types of analytes besides nucleic acids. In particular, signal amplification reactions can be used to allow the detection of a single copy of an analyte molecule in a single droplet, enabling data analysis of droplet signals of other analytes (e.g., using Poisson-based algorithms). Exemplary signal amplification reactions that allow the detection of single copies of other types of analytes in droplets include enzyme reactions. Attached Figure Description

[0075] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, the system is shown in a preferred embodiment. However, it should be understood that this application is not limited to the precise arrangement, structure, features, embodiments, and aspects shown. The drawings are not drawn to scale and are not intended to limit the scope of the claims to the depicted embodiments. Therefore, it should be understood that where feature reference numerals follow those mentioned in the appended claims, the inclusion of these numerals is solely for enhancing the comprehensibility of the claims and is in no way intended to limit their scope.

[0076] The features and advantages of the present invention will become clear from the following description of system embodiments, which is given by way of example only and with reference to the accompanying drawings, wherein:

[0077] Figure 1A This is a perspective view of the first stage of the system according to an embodiment of the present invention.

[0078] Figure 1B yes Figure 1A Exploded perspective view of the first stage.

[0079] Figure 2 This is a schematic diagram of an optical device according to an embodiment of the present invention.

[0080] Figure 3A and Figure 3B These are perspective and front views of a base element according to an embodiment of the present invention, the base element comprising a first housing and a second housing for accommodating emitted and scattered light.

[0081] Figure 4 This is a front sectional view of an optical device and support assembly including a first housing and a second housing according to an embodiment of the present invention.

[0082] Figure 5 This is a global perspective view of the system according to an embodiment of the present invention.

[0083] Figure 6 This is a perspective view of a system according to an embodiment of the present invention, showing a partial view of an optical device, a base element, and a second shifting device according to an embodiment of the present invention.

[0084] While various embodiments have been described and illustrated, the detailed description should not be construed as limiting thereto. Those skilled in the art can make various modifications to the embodiments without departing from the true spirit and scope of this disclosure as defined by the claims. Detailed Implementation

[0085] To improve the flexibility and accuracy of scanning systems, the present invention proposes a system for sample scanning, comprising a sample holder 11 configured to hold a sample 13 (preferably a microfluidic chip), an optical device 3, a base element 4 configured to receive the optical device 3, and shifting devices 20 and 21 configured to move the optical device 3 and the base element 4 relative to each other and to move the base element 4 and the sample holder 11 relative to each other.

[0086] Figure 1A and Figure 1B The first stage 1 of the system according to an embodiment of the present invention is shown in perspective and exploded view. Figure 1A The double arrow Y indicates the ability of the sample holder 11 to move along the longitudinal direction Y. The sample holder 11 is configured to hold a sample 13, such as a microfluidic chip. This movement occurs along the longitudinal direction Y to allow longitudinal scanning of the microfluidic chip 13.

[0087] In one embodiment, the sample holder 11 includes at least one sample slot 12 configured to hold a sample 13. In a preferred embodiment, the size and shape of the sample slot 12 are complementary to the size and shape of the sample 13 to be scanned, such that the sample 13 fits perfectly within the sample slot 12. Therefore, the sample slot 12 can be rectangular or have any shape suitable for accommodating a sample.

[0088] The sample holder 11 may include multiple sample slots 12, such as two or three sample slots 12. Having multiple sample slots 12 facilitates scanning a large number of samples 13 while reducing handling and thus reducing the risk of sample contamination.

[0089] Optionally, the sample holder 11 includes grippers configured to hold one or more samples 13.

[0090] like Figure 1A As shown, sample 13 itself may include multiple regions to be analyzed.

[0091] The movement of the sample holder 11 along the Y direction can be caused by the movement of the first shifting device 14 (see...). Figure 1B Motor 110 (in) Figure 1A China stated that, and Figure 1B (Drawn in the middle) Drive. The first shifting device 14 is a sliding link, which may include a first guide rail system 14a and a U-shaped component 14b. Figure 1A In this embodiment, the sample holder 11 is movable back and forth along the longitudinal direction Y. In one embodiment, the sample holder 11 rests on the holder support 10. In the illustrated embodiment, both the sample holder 11 and the holder support 10 are flat, rectangular elongated parts that extend in the same longitudinal direction Y for the motor displacement of the sample holder 11.

[0092] exist Figure 1A In this configuration, a flat cap 16 is connected to a sample holder 11 and a holder support 10. The sample holder 11, and thus the sample 13 configured to be held by it, is also capable of moving in the vertical direction Z, which is perpendicular to a first plane comprising the sample holder 11 and the longitudinal direction Y. This movement along the vertical direction Z can be driven by a motor 17.

[0093] The sample holder 11 is capable of moving longitudinally in the first plane.

[0094] Figure 2 This is a separate view of an example of the optical device 3. The optical device 3 includes a main plate 32 extending in a second plane perpendicular to the first stage 1. Therefore, the second plane is perpendicular to the first plane.

[0095] The optical device includes a light source 30, an emission light filter 36, and a scattering light filter 37. The light source 30, the emission light filter 36, and the scattering light filter 37 extend from the main board 32 along the same direction X, perpendicular to the second plane. Preferably, the light source 30, the emission light filter 36, and the scattering light filter 37 are fixed relative to each other.

[0096] The light source 30 can extend from the center of the motherboard 32. In one embodiment, the light source 30 includes a vertical array of light-emitting diodes (LEDs) 35.

[0097] The light emission filter 36 can extend from the edge 33 of the main board 32. The light emission filter 36 includes a plurality of filters 36a-f, which are located in corresponding filter slots and configured to be located in the optical path P1 of the light emitted by the light source 30.

[0098] The light-scattering filter 37 can extend from the free end of the arm 34 extending from the self-extension plate 32. The light-scattering filter 37 also includes a plurality of filters 37 af, which are located in corresponding filter slots and configured to be positioned within the optical path P2 of the light scattered by the sample 13 held by the sample holder 11 (see [reference]). Figure 1A Filters 37 af and 36a-f work in pairs. Therefore, the number of filters 37a-f is equal to the number of filters 36a-f. Optionally, the number of LEDs is also equal to the number of filters 36a-f and filters 37a-f.

[0099] In one embodiment, the emitting light filter 36 includes an array of six filters 36a-f, and the scattering light filter 37 includes an array of six filters 37a-f. Optionally, the light source 30 may include six LEDs.

[0100] The light source 30, the emitted light filter 36, and the scattered light filter 37 are integral. In other words, these three elements 30, 36, and 37 are solid and move as a whole. Therefore, the optical device is movable relative to the sample holder 11 and includes an array of filters 36a-f and filters 37a-f to allow different filters to be applied to the emitted and scattered light. More specifically, pairs of filters 36a-37a, 36b-37b, 36c-37c, 36d-37d, 36e-37e, and 36f-37f can be placed one pair at a time in the paths P1 and P2 of the light emitted from the light source 30, scattered onto the sample 13, and received by the camera capture device.

[0101] The base element 4 is configured to receive the optical device 3 and positions the emission light filter 36 and the scattering light filter 37 relative to the sample holder 11. For this purpose, the base element 4 may include: a first housing 41 extending along the longitudinal axis 4A, which is configured to be aligned with the optical path P1 of the light emitted from the light source 30 to the sample holder 11; and a second housing 42 extending along the longitudinal axis 4B, which is configured to be aligned with the optical path P2 of the light scattered by the sample 13 toward the camera 43.

[0102] In one embodiment, the angle α between the longitudinal axis 4A of the first housing 41 and the longitudinal axis 4B of the second housing 42 is less than 90°, preferably between 25° and 60°, more preferably between 30° and 45°, and even more preferably between 35° and 45°.

[0103] In the illustrated embodiment, camera 43 is housed in base element 4. However, this is exemplary and not limiting.

[0104] The first housing 41 and the second housing 42 can have any suitable shape and cross-section. For example, the first housing 41 and the second housing 42 can each include a tube, which can be a column with a square, rectangular, or circular cross-section. The tube can have solid walls to prevent light from passing through it. Alternatively, housings 41, 42 can include any structure capable of receiving and positioning the filter 36, the diffuser filter 37, and the light source 30 relative to the sample holder 11.

[0105] The first housing 41 includes a first slit 411 having a rectangular cross-section complementary to the cross-section of the light emission filter 36, such that the light emission filter 36 can slide relative to the base element 4 within the first slit 411.

[0106] The second housing 42 includes a second slit 421 having a rectangular cross-section complementary to the cross-section of the scattering light filter 37, allowing the scattering light filter 37 to slide within the second slit 421. This configuration of the optical device 3 and the base element 4 allows the paired emission and scattering light apertures to be aligned with the optical paths P1 of the emission and scattering light. It should be understood that these apertures are configured to receive the filter slots of the pairs of filters 36a-37a, 36b-37b, 36c-37c, 36d-37d, 36e-37e, and 36f-37f. In a preferred embodiment, the configuration of the optical device 3 and the base element 4 allows the pairs of emission and scattering light filters 36af and 37af to be aligned with the optical paths of the emission light P1 and the scattering light P2.

[0107] Camera 43 is configured to capture light scattered by sample 13. Therefore, a second slit 421 is located between sample holder 11 and camera 43. Thus, camera 43 is positioned after the second slit 421 in the scattering optical path P2. This optical assembly allows for image analysis.

[0108] The light emitted by the light source 30 first passes through the first housing 41 along the emitted light path P1, then through the emitted light filter 36, and then reaches the sample 13, which scatters the light. The scattered light then travels along the scattered light path P2, which first passes through the second housing 42, then through the scattered light filter 37, and then reaches the camera 43.

[0109] exist Figure 3B In the illustrated embodiment, the system further includes a bright-field light source 6 positioned to illuminate the sample 13. In one embodiment, the bright-field light source 6 may be fixed to the end portion of the second housing 42 opposite to the camera 43 and positioned to illuminate the sample 13. The bright-field light source 6 is configured to emit light toward the sample 13 held by the sample holder 11 and may be used, for example, to phototreat the surface of the sample 13. More specifically, by using anti-photobleaching fluorophores and photobleaching-sensitive fluorophores, two targets can be distinguished in a given monochromatic channel after exposure to light emitted by the bright-field light source 6. Thus, the bright-field light source 6 can create virtual color channels by selectively altering the fluorescence properties of the chemicals contained in the sample 13.

[0110] Advantageously, the same system can be used to scan sample 13 and perform photobleaching. In practice, the bright-field light source 6 can be easily fixed to the system, typically to the second housing 42 of the base element 4, such that the bright-field light source 6 illuminates the same area of ​​sample 13 as the light source 30. For this purpose, the bright-field light source 6 and the light source 30 are symmetrical with respect to the longitudinal axis 4B, and therefore symmetrical with respect to the plane containing the region of sample 13. For example, Figure 4 The optical axes of the bright field light source 6 and the light source 30 form the same angle with the plane containing the sample 13 and the camera 43. This angle is essentially equal to (+ / -5°) the angle α between the longitudinal axes 4A and 4B. This configuration greatly simplifies the system, as no motor or any other additional device is required to perform photobleaching.

[0111] In a preferred embodiment, the light emitted by the bright field light source 6 is low-power bright field light, such as an LED. "Low" should be understood as meaning that it substantially does not cause bleaching of fluorophores contained in the sample during its illumination. The light emitted by the bright field light source 6 can be detected by one of filters 37a, 37b, 37c, 37d, 37e, and 37f located inside the light-scattering aperture.

[0112] Figure 4A movable assembly including an optical device 3 and a base element 4 is shown. This movable assembly can be supported by a second stage 2.

[0113] exist Figure 4 The diagram shows a front view of the base element 4 having a first housing 41. The longitudinal axis 4A of the first housing 41 is aligned with the optical path P1 of the light emitted by the light source 30 toward the sample 13. The second housing 42 of the base element 4 is also shown in a front view. The longitudinal axis 4B of the second housing 42 is aligned with the optical path P2 of the light scattered by the sample 13.

[0114] The second stage 2 forms a support for the base element 4 and the optical device 3. The optical device 3 is movable relative to the second stage 2 in the X direction. For example, the optical device 3 can be connected to the second stage 2 via a shifting device 20, which includes a first sliding link 20 extending in the X direction perpendicular to the Y and Z directions. The first sliding link 20 may include a pair of U-shaped members 20b slidably associated with a pair of guide rails 20a. In the illustrated embodiment, the U-shaped members 20b are fixed to the optical device 3 and the guide rails are connected to the second stage 2. The U-shaped members 20b and the corresponding guide rails form a first guide rail system 20. Obviously, an equivalent reverse configuration is also considered, in which the pair of U-shaped members 20b are connected to the second stage 2 and the guide rails are fixed to the optical device 3. Furthermore, the base element 4 can be connected to the second stage 2 via a shifting device 21, which includes a second sliding link 21 extending in the X direction. The second sliding link 21 may include a pair of U-shaped elements 21b slidably associated with a pair of guide rails 21a. The U-shaped elements 21b and the corresponding guide rails form a second guide rail system 21. In the illustrated embodiment, the U-shaped elements 21b are fixed to the base element 4, and the guide rails are connected to the second stage 2. An equivalent reverse configuration is also obviously considered, in which the paired U-shaped elements 21b are connected to the second stage 2 and the guide rails are fixed to the base element 4. The two pairs of guide rails 20a, 21a are parallel to the direction X.

[0115] The movement of the optical device 3 is driven by a corresponding motor 310, while the movement of the base element 4 is driven by a corresponding motor 410, which is different from the motor 310. Both movements are along the direction X.

[0116] Figure 4 An electronic control unit 5 is shown for controlling motors 110, 310, and 410 individually or in combination. This electronic control unit 5 is also capable of controlling the movement of sample holder 11 in a direction Z perpendicular to the plane containing sample holder 11. See also... Figure 1A Motor 17 is used to perform control along the Z direction.

[0117] The interaction between the optical device 3 and the base element 4 through slits 411 and 421 Figure 4 As shown, the emission light filter 36 appears within the first slit 411, and the scattering light filter 37 appears within the second slit 421. Due to this configuration, independent control of the displacement of the optical device 3 and the base element 4 can be performed, allowing light emitted by the light source and light scattered by the sample 13 to pass through different filter pairs (shown herein, but not limited to, 36a-37a, 36b-37b, 36c-37c, 36d-37d, 36e-37e, 36f-37f) by moving one of the optical device 3 and the base element 4 along sliding links 20 or 21 while the other remains fixed. In the illustrated configuration, the optical device 3 moves relative to the base element 4 along guide rail 20a, which is fixed relative to the second stage 2 and the sample 13. Therefore, only the filters (and optionally the light source) placed on the emission light path P1 and the scattering light path P2 are changed to switch the analysis channels. More specifically, the region of the sample 13 being analyzed remains unchanged. This helps improve measurement accuracy, as the size of the object being analyzed can be very small (approximately 100 µm droplets), making any movement of the sample detrimental to the measurement when the filter is changed. Since sample 13 is fixed when the filter is changed, measurement accuracy is even improved: in fact, any movement of sample 13 necessarily implies a tiny movement of the droplets contained within it. Therefore, by simply moving the filter, the optical paths P1 and P2 can be kept fixed relative to sample 13.

[0118] Using the system of this invention, measurement accuracy can be better than 25 µm when recording two consecutive sample images in two different optical channels. Therefore, it is possible to distribute droplets from one channel to another, and, for example, allow multiplexing of multiple biological items within a single droplet.

[0119] Furthermore, the base element 4 can move relative to the first stage 1 and the second stage 2 along the second sliding link 21. In the illustrated configuration, the base element 4 moves relative to the second stage 2 along the guide rail 21a, which is fixed relative to the sample 13. This movement allows for imaging of another area of ​​the sample 13 by scanning it with the camera 43.

[0120] Now will be passed Figure 5 and Figure 6 The accompanying drawing is a perspective view of such a system, intended to explain the advantages of the system and kinematics according to the invention.

[0121] According to the present invention and reference Figure 5The sample 13 to be imaged is housed in the sample holder 11. In the illustrated embodiment, the sample 13 is placed within one of the three sample slots 12 of the sample holder 11. Figure 5 In this configuration, three samples 13 can therefore be analyzed. In other embodiments of the invention, different numbers of samples 13 can be analyzed and different types of sample holders 11 can be used.

[0122] When light is emitted by the light source 30, before being scattered by the sample 13 and passing through the corresponding scattered light filters 37a, 37b, 37c, 37d, 37e, and 37f, the light passes through filters 36a, 36b, 36c, 36d, 36e, or 36f of the emitted light filter plate 36 to excite the sample 13 at a given wavelength. Finally, the scattered light is captured by the camera 43.

[0123] Due to the second sliding link 20, the filters and wells through which the emitted and scattered light pass can be altered. In one embodiment, the light source 30 is changed simultaneously. For example, in Figure 6 In the middle, filters 36 and 37 have been moved out of the light paths P1 and P2. This movement is motorized in the X direction along sliding link 20 (see...). Figure 6 (Motor 310 in the image). During this movement, the emitting light filter 36 slides within the first slit 411 of the base element 4, while the scattering light filter 37 slides within the second slit 421 of the base element 4. In one embodiment, the array 35 of light-emitting diodes (LEDs) slides simultaneously. Thus, different filter pairs can be used for the same sample area to be scanned. Their longitudinal arrangement reduces congestion within the system and simplifies the assembly between the optics 3 and the base element 4 compared to another arrangement.

[0124] In addition, in order to change the area of ​​the sample 13 to be scanned, motor 110 (see Figure 1A The sample holder 11 can be driven along the Y direction by the first guide rail system 14. In this case, the sample 13 moves along the first Y direction and can image different areas. For this new area to be imaged, different filters can also be used by shifting the optical device 3 along the sliding link 20 as described above.

[0125] Alternatively or additionally, in order to change the region of the sample 13 to be scanned in the X direction, the motor 410 can move the base element 4 along the second sliding link 21. In this case, the second stage 2 and the first stage 1 (including the sample 13) remain fixed. However, due to the translation of the optical element 4, the light source 30 emits light toward different regions to be imaged. The optical device 3 can be moved simultaneously along the X direction by the motor 310 together with the base element 4. Then, as previously described, different filters and LEDs can also be used by moving the optical device 3 relative to the base element 4.

[0126] This stage allows for imaging of a two-dimensional region, enabling scanning of each region of sample 13 and changing the filters used. As previously mentioned, all of these possibilities are motorized.

[0127] Finally, the sample holder 11 can be moved by the motor 17 along the Z direction, which is perpendicular to the Y and X directions, for focus adjustment.

[0128] Given the above, three types of movement are possible:

[0129] The first motion in the X direction and the second motion in the Y direction are used for scanning a two-dimensional region image.

[0130] The third motion in the Z direction for each of these positions measured in the three-dimensional reference space.

[0131] Furthermore, the emitted light filter and the scattered light filter can be changed through associated pairs.

[0132] Those skilled in the art will understand that the arrangement according to the invention provides flexibility in image scanning, sample positioning, and filter selection. The invention is particularly suitable for digital PCR.

[0133] Figure label:

[0134] 1 First Platform

[0135] 10 Retaining Support

[0136] 11 Sample holder

[0137] 12 Sample slots

[0138] 13 samples

[0139] 14 Sliding Link

[0140] 14a Guide Rail System

[0141] 14b U-shaped component

[0142] 16 covers

[0143] 17 motors

[0144] 110 motor

[0145] Y First direction

[0146] X Second Direction

[0147] Z Third Direction

[0148] 2 Second Platform

[0149] 20 First sliding link

[0150] 21 Second sliding link

[0151] 20a, 21a Second and Third Guide Rails

[0152] 20b, 21b U-shaped components

[0153] 3. Optical devices

[0154] 30 Light Sources

[0155] 310 motor

[0156] 32 motherboard

[0157] 33. Motherboard edge

[0158] 34 Motherboard Extension Arm

[0159] 35 LED

[0160] 36. Light Emitting Filter Plate

[0161] 36A, 36b, 36c, 36d, 36e, 36f emission filters

[0162] 37. Light-scattering filter plate

[0163] 37A, 37b, 37c, 37d, 37e, 37f Scattering light filters

[0164] 4. Base components

[0165] 41 First Shell

[0166] 410 motor

[0167] 411 First Slit

[0168] 42 Second shell

[0169] 421 Second Slit

[0170] 4A First shell longitudinal axis

[0171] 4B Second shell longitudinal axis

[0172] 43 cameras

[0173] 5 Electronic Control Unit

[0174] 6 Bright Field Light Source

[0175] α is the angle between the longitudinal axis 4A of the first shell and the longitudinal axis 4B of the second shell.

Claims

1. A system for scanning a sample (13), comprising: - Sample holder (11), which is configured to hold sample (13); - Optical device (3), which includes a light source (30) for emitting light toward the sample holder (11), an emission light filter plate (36) including filters (36a-f) of a first array, and a scattering light filter plate (37) including filters (37a-f) of a second array. - Base element (4), which includes a first housing (41) extending toward the sample holder (11) along the optical path (P1) of the light emitted by the light source (30) and a second housing (42) extending along the optical path (P2) of the light scattered by the sample (13) held in the sample holder (11). The emitted light filter (36) is positioned along the optical path (P1) of the emitted light, and the scattered light filter (37) is positioned along the optical path (P2) of the scattered light; and The first housing (41) is fixed relative to the second housing (42); - A first shifting device (20) configured to move the emission filter (36) and the scattering filter (37) of the optical device (3) relative to the base element (4) along a first direction (X); and - A second shifting device (21), which is different from the first shifting device (20) and is configured to move the base element (4) and the sample holder (11) relative to each other along the first direction (X), such that the optical path (P1) of the emitted light moves relative to the sample (13) held in the sample holder (11).

2. The system according to claim 1, wherein, The first housing (41) extends along a first longitudinal axis (4A), and the second housing (42) extends along a second longitudinal axis (4B), the first longitudinal axis (4A) and the second longitudinal axis (4B) forming an angle (α) of less than 90°.

3. The system according to claim 2, wherein, The angle is between 30° and 45°.

4. The system according to claim 1, wherein, The first shifting device and the second shifting device (20, 21) include sliding links that are parallel to each other and extend along the first direction (X).

5. The system of claim 1, further comprising an additional shifting device (14) coupled to the sample holder (11) to move the sample holder (11) relative to the base element (4) in a second direction (Y).

6. The system according to claim 5, wherein, The second direction (Y) is perpendicular to the first direction (X).

7. The system according to claim 1, wherein, The first shifting device (20) is configured to move the optical device (3) relative to the base element (4), and the second shifting device (21) is configured to move the base element (4) relative to the sample holder (11).

8. The system according to claim 1, wherein, The sample holder (11) includes at least one sample slot (12).

9. The system according to claim 1, wherein, The optical device (3) further includes a main board (32) extending in a plane perpendicular to the sample holder (11), and the light source (30), the emission filter (36) and the scattering filter (37) extending from the main board along the first direction.

10. The system according to claim 9, wherein, The first housing (41) includes a first slit (411) extending along the first direction and configured to receive the emission light filter (36), and the second housing (42) includes a second slit (421) extending along the first direction and configured to receive the scattering light filter (37), such that the emission light filter (36) and the scattering light filter (37) slide within the first slit (411) and the second slit (421) to align the pairs of emission light filters (36a, 36b, 36c, 36d, 36e, 36f) and scattering light filters (37a, 37b, 37c, 37d, 37e, 37f) with the optical path (P1) of the emission light and the optical path (P2) of the scattering light.

11. The system according to claim 1, wherein, The emission light filter (36) includes multiple emission light filters (36a, 36b, 36c, 36d, 36e, 36f), and the scattering light filter (37) includes the same number of scattering light filters (37a, 37b, 37c, 37d, 37e, 37f).

12. The system of claim 1, further comprising a bright field light source (6) coupled to the second housing (42) and configured to emit light toward a sample held in the sample holder (11).

13. The system according to claim 12, wherein, The optical axis of the bright field light source (6) and the optical axis of the light source (30) are symmetrical with respect to the optical path (P2) of the scattered light.

14. The system according to claim 1, wherein, The optical device (3) and the base element (4) are connected to the second stage (2) via the first shifting device (20) and the second shifting device (21).

15. The system according to claim 14, wherein, The optical device (3) and the base element (4) are located between the sample holder (11) and the second stage (2).

16. The system according to claim 1, further comprising an electronic control unit (5) for controlling the movement of at least one of the optical device (3), the base element (4) and the sample holder (11).

17. The system of claim 1, further comprising a camera (43) for receiving light scattered by the sample held in the sample holder (11).

18. The system according to claim 1, wherein, The emitted light filter is fixed relative to the scattered light filter and the light source.

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