Imaging system comprising microlenses and associated device for detecting a sample

The invention relates to an imaging system and an associated device for detecting a sample (4). The imaging system comprises a matrix (24) of photosensors (26), a first lamina (28) disposed opposite the matrix (24) of photosensors defining a face (28A) for supporting the sample, and a set (30) of optical elements, disposed between the matrix (24) of photosensors and the first lamina (28). Each microlens (34) is disposed above a photosensor (26) of the matrix (24) of photosensors. The set (30) of optical elements comprises a matrix (32) of microlenses (34). The set (30) of optical elements comprises an optical medium (36) disposed between the matrix (32) of microlenses and the first lamina (28), the refractive index of the optical medium (36) being substantially between 1 and the refractive index of the microlenses (34). The distance between the face supporting the sample (28A) and the apex of the microlenses (34) is substantially between 0 and 1500 μm as measured along the optical axis (Z) of the photosensors (26).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. nationalization under 35 U.S.C. §371 of International Application No. PCT/FR2010/051076, filed Jun. 2, 2010, which claims priority to French application no. FR0953631, filed Jun. 2, 2009. The disclosures set forth in the referenced applications are incorporated herein by reference in their entireties.

The present invention relates to an imaging system comprising microlenses and an associated device for the detection and optical characterization of a sample.

The invention relates to the field of miniaturizing measuring instruments for chemical or biological analysis.

In biology, cell observation generally requires the use of an optical microscope in order to magnify the image of the cells and separate the details of that image. Similar equipment is used to view other types of biological objects, such as bacteria, yeasts, fungi, pollens, algae, liposomes, as well as synthetic particles. To that end, a microscope includes optical lenses (objective, eyepiece), a light source and at least one photo sensor. The microscopes used daily by researchers are often expensive and restrictive, in particular due to their spatial bulk. Generally, they do not make it possible to produce images continuously over time or at predefined moments.

One of the miniaturization routes currently considered is to eliminate the magnification lenses of the microscope and produce the image of the shadow or the diffraction pattern created on the sensitive surface of the photo sensors. The image formed by the shadow or diffraction pattern of the observed object in contact with the photo sensors is called photogram. A priori the resolution, defined as the capacity to distinguish two very close disks, is in this type system limited by the size of the pixels and the distance between the object and surface of the optical sensor. In a traditional microscope, it is limited by the diffraction in the lens system of the objectives.

Systems exist describing a system for imaging a sample comprising a matrix of photosensors, a first lamina disposed opposite the matrix of photosensors defining a face for supporting the sample, and a set of optical elements, disposed between the matrix of photosensors and the first lamina, the set of optical elements comprising a matrix of microlenses, a microlens (34) being situated above each photosensor (26) of the matrix (24) of photosensors.

However, the images acquired by such an imaging system do not show the morphology or the shape in the cases where the cells of the sample to be analyzed are adhesive. In fact, the suspended or adhered cells do not correspond to the same challenge: cells are objects having a low contrast with the culture medium because the optical indices are similar. When they are adhered, they have a flat and elongate shape, which makes them more difficult to image.

The aim of the invention is to propose a smaller device able to image cells, in particular adhesive cells, to locate and identify the cells, number them and analyze their morphology. The smaller size of the device makes it possible to insert it directly into an incubator in order to allow the proliferation of the cells of the sample.

To that end, the invention relates to a system for imaging a sample of the aforementioned type, characterized in that the set of optical elements comprises an optical medium disposed between the matrix of microlenses and the first lamina, the refractive index of the optical medium being substantially between 1 and the refractive index of the microlenses, and in that the distance measured along the optical axis of the photosensors between the face supporting the sample and the apex of the microlenses is substantially between 0 and 1500 μm.

According to specific embodiments, the system for imaging a sample includes one or more of the following features:the distance measured along the optical axis of the photosensors between the support face of the sample and the apex of the microlenses is smaller than or equal to −150×F×n+400×F, where F is the focal length of the microlenses measured in the air and n the refractive index of the optical medium and in that the refractive index of the optical medium is substantially between 1 and 1.64;it comprises a light source intended to light the sample, the light source being disposed above the sample so as to produce a transmission image;it comprises a light source intended to light the sample, the light source being disposed so as to light in the first lamina of the imaging system perpendicular to the optical axis of the imaging system;the light source can light with a wavelength comprised between 200 nm and 800 nm;the light source is a white light;the optical medium is a liquid;the imaging system comprises a cuvette containing the liquid and the sample;it comprises a second lamina disposed so that the sample is situated between the first lamina and the second lamina;the support face of the sample of the first lamina is disposed opposite the matrix of photosensors;the first lamina includes a slide and a removable lamina alongside the slide and defining the support face of the sample;the imaging system comprises means for adjusting the distance, measured along the optical axis of the photosensors between the support surface of the sample and the apex of the microlenses;the first lamina comprises a plurality of microchambers or microchannels;the photosensors can detect a light emitted between 200 nm and 800 nm by bioluminescence, chemiluminescence or fluorescence;the sample comprises cells and the photosensors associated with the microlenses are arranged so that a cell is covered by at least two adjacent photosensors in a first direction and two other adjacent photosensors in a second direction perpendicular to the first direction;the opening diameter of each microlens is at least two times smaller than the smallest lateral dimension of a cell and in that the distance between the optical axes of two closer neighboring microlenses is smaller than at least 30% of the opening diameter of a microlens, the opening diameter of a microlens being the diameter of the intersection section of the flat diopter and the convex diopter forming the microlens, and the smallest lateral dimension of a cell being the smallest dimension of the cell passing through the center of gravity thereof and measured in a plane parallel to the support face (28A) of the sample; andthe opening diameter of each microlens is substantially comprised between 0.7 and 10 μm.

Thus, this smaller imaging system can produce cell images continuously over time or at predefined times to quantify the proliferation of the cells, individually monitor their movement and deduce their trajectory and speed therefrom, detect the divisions of the cells and establish relationships, show and quantify changes in morphology, identify cellular events of interest (rare events, etc.).

The invention also relates to a device for detecting and characterizing a sample characterized in that it comprises an imaging system as described above, an electronic controller and a computer system intended to control the imaging system.

According to specific embodiments, the device for detecting and characterizing a sample includes one or more of the following features:it comprises at least two imaging systems as described above according to the invention and steered in parallel by the electronic controller;the first lamina is shared by at least two adjacent imaging systems; andit comprises an incubation chamber in which the imaging system and the sample are placed.

The invention relates to a device2for detecting and characterizing a sample4, one embodiment of which is illustrated inFIG. 1. The sample4comprises objects5to be detected and imaged in order to study their characteristics (morphology, size, etc.). The objects5are micrometric-sized objects (bacteria, yeasts, fungi, pollens, algae, liposomes, etc.) and preferably cells.

The device2comprises at least one imaging system6intended to detect and characterize the sample4and an electronic controller8able to control the imaging system6. The electronic controller8comprises a support able to fix the imaging system6, for example to plug it in if it comprises connection plugs.

The imaging system6is described in detail hereafter.

The device2also includes a computer system10intended to steer the electronic controller8to which it is connected.

The computer system10comprises a man/machine interface12intended to enter information to steer the electronic controller8and display information concerning the sample4.

The computer system10also includes a computation unit able to perform processing on the images and a storage unit to record the acquired images of the sample.

The device2comprises an incubation chamber or incubator14and means16for monitoring and regulating the CO2level, humidity and temperature inside the incubation chamber. For example, the incubator14is a standard incubator for cellular biology, with dimensions in the vicinity of one cubic meter in a known manner, which may potentially contain several imaging systems6, or an incubator miniaturized to the dimensions of an imaging system6. In the latter case, the device2is easily transportable to be used, for example, near a sample collection site.

The imaging system6, the electronic controller8and the sample4are disposed inside the incubation chamber14.

The imaging system6includes an optical system17intended to analyze the sample4and, preferably, the imaging system6includes at least one light source18intended to light the sample4, the latter being arranged between the light source18and the optical system17.

The light source18is situated opposite the optical system17so as to produce a transmission image of the sample and at a controlled distance, for example a distance of several centimeters. The light source18is situated at the vertical of the optical system17.

According to one alternative, the light source18is offset relative to the vertical of the optical system17, for example at a distance of several centimeters according to an arc of circle around the optical system17.

Furthermore, the light source is slightly or not at all collimated with a critical light angle smaller than 20°, for example 10°.

Nevertheless, depending on the operator's needs and in a known manner, the light source can be collimated by a system of lenses, diaphragms and/or filters, directional or not, polychromatic or monochromatic, polarized or not, extended or periodic.

The light source18can light with a wavelength comprised between 200 nm and 800 nm, i.e. emitting in the visible and/or near ultraviolet (UV). Preferably, it is a white light, i.e. a light emitting a continuous wavelength spectrum, in particular in the visible region (400 nm to 800 nm).

For example, the light source18comprises a light-emitting diode (LED), for example a white LED, an array of LEDs, an incandescent bulb, or a light-emitting sheet or flat LED such as an XLamp marketed by Cree (Durham, United States).

According to one alternative, the light source18includes an illumination device of the Kölher type, i.e. an imaging system commonly used in optical microscopes to illuminate the object or sample.

A means20for controlling the light source18is incorporated into the device2and connected to the computer system10.

According to one alternative, the light source18is positioned above the optical system17so that it lights the sample at a specific angle (lighting angle) monitored by the operator or by the computer system10.

The light source18lights the object or sample at an intensity and/or during periods and/or at moments specified by the operator or computer system10.

Depending on the operator's needs, the light source18can be shared by several samples or only light a single sample.

The operation of the device2for detecting and characterizing a sample4according to the invention will now be described.

In order to study and analyze the sample4, an operator places it in the imaging system6. The imaging system6is connected to the electronic controller8, for example by plugging the optical system17onto the support of the electronic controller8.

If necessary, the assembly comprising the optical system17, the sample4, the electronic controller8and the light source18is placed in the incubation chamber14, in particular to allow the normal proliferation of the cellular population. The incubator14regulates the temperature and CO2level, and maintains a high humidity level owing to the monitoring and regulation means16adjusted beforehand by the operator.

Furthermore, this assembly (imaging system6including the light source18, sample4and electronic controller8) is preferably placed in a darkroom in order to reduce parasitic noise related to the ambient light.

The operator then enters information via the man/machine interface12to adjust the or each light source18via the light control means20and to steer the optical system17of the imaging system6via the electronic controller8. The operator thus checks remotely, using the computer system10, the or each imaging system6, including the or each light source18.

During operation, the or each light source18lights the sample4. The light or optical signal (photons) transmitted by the sample4is converted by the optical system17of the imaging system6into an electric signal. The electric signal is then transmitted by the electronic controller8to the computer system10.

The data acquired by the optical system17of the imaging system6are recorded in the storage unit of the computer system10. Then, the computation unit reconstructs the image or image sequence of the cells5of the sample4from the data. In fact, the device records, in a known manner, dynamic events, either by performing a continuous acquisition (video mode) to obtain a film, or by acquiring images at regular intervals (photo mode) to obtain a sequence of images, and not only the intensity of the optical signal detected during transmission of the sample.

Lastly, the operator controls, via the man/machine interface12, the computation unit to perform other processing of the image or image sequence in order to characterize the sample4. For example, the computation unit may spatially identify the cells5of the sample, identify the cells5based on an analysis of their morphology and/or fluorescent signals and/or specific luminescents, number the cells, compare their position with their earlier position, record the trajectory and speed of migration of each cell, quantify the proliferation of the cells, characterize the morphology of each cell (computation of the perimeter and area covered by each cell, circle symmetry criterion of each cell, texture of the contour), show morphological changes on individual cells or a population of cells, segment the image to trace the junctions between the cells and compute the number of neighboring cells, detect events of interest such as cellular divisions or differentiations or calcareous bursts, establish relationships, and/or locate organelles (nuclei, etc.) in the cells. Furthermore, it can eliminate background noise, for example by producing, for each experiment, an image with a light background (parasitic light) and with a dark background (dark current). It can also reconstitute films of the cellular culture on all or part of the image.

According to another embodiment for a sample4emitting light, for example a bioluminescence or a chemiluminescence, the device2is used without any light source18. In that case, the analyses must be done in total darkness (in a darkroom). The intensity of the luminescence is integrated for a particular period by the operator in order to increase the sensitivity of the measurement, for example the integration time of the luminescence is 5 minutes.

FIG. 2is a diagrammatic cross-sectional view of the optical system17of the imaging system6intended to detect and characterize the sample4. For example, the sample4comprises objects, for example cells5and preferably adhered biological cells22in an aqueous medium, in a known manner a culture medium.

The optical system17of the imaging system6comprises a matrix24of photosensors26, regularly arranged, whereof the photosensitive area of each photosensor26is for example made from silicon.

The optical system17also includes a first transparent lamina28disposed opposite the matrix24of photosensors26defining a support face28A of the sample4support. The face28A is preferably flat and transparent.

Preferably, the first lamina28has a specific surface treatment to capture and/or favor the adhesion of the cells22on the support face28A of the sample4, in a known manner through a functionalization method with appropriate proteins, preferably proteins of the extracellular matrix, for example fibronectin, or with an oxygen plasma.

Furthermore, another surface treatment prevents adhesion on the face28B opposite the face28A, support for the sample, for example by using polylysine-PEG (polylysine-polyethylene glycol), which gives it hydrophobic properties.

For example, the support face28A of the adhered cells22has a surface treatment to increase the adhesion of the cells thereon and the other face28B has a surface treatment to reduce or eliminate the adhesion of the cells22.

For example, the first lamina28serves as support for the cellular culture.

The first lamina28has good qualities in terms of optical transmission, for example it is made from glass or polystyrene.

The optical system17of the imaging system6also comprises a set30of optical elements disposed between the matrix24of photosensors26and the first lamina28.

The set30of optical elements includes a separator element37situated opposite and in contact with the matrix24of photosensors, and a matrix32of microlenses34disposed between the separator element37and the first lamina28.

The matrix32of microlenses34is positioned above the matrix24of photosensors26so that a microlens is situated above each photosensor26of the matrix24of photosensors. Thus, each microlens34is associated with a photosensor26. The optical axis of each microlens34is substantially combined with the optical axis of a photosensor26.

For example, the optical axis of a microlens34and the optical axis of a photosensor26can be slightly offset, the offset (angular or in distance) being less than or equal to 20%. This offset is either involuntary as the result of a difficulty in aligning the microlens matrix with the photosensor matrix, or deliberate so as to reorient an oblique incident optical beam toward the photosensor owing to the microlens.

The separator element37is formed by one or more optically transparent materials, for example filters or passivation layers.

The function of the separator element is to space the matrix24of photosensors26away from the matrix32of microlenses34so that the light radii are concentrated or focused toward the photosensors.

The set30of optical elements also comprises a first medium36, with refractive index or optical index n, disposed between the microlens matrix32and the first lamina28. The first optical medium36is intended to modify the focal distance of each microlens34. In fact, in the first medium36, with optical index n, the focal distance of each microlens34is

Fn=nlens-nairnlens-n×F,
where F is the focal distance of each microlens34measured in the air.

The refractive index of the first optical medium36is substantially comprised between 1 and the refractive index of the microlenses34. Preferably, the medium36is a gel or a liquid, for example an oil.

The optical system17of the imaging system6also includes a second lamina40disposed so that the cells22are situated between the first lamina28and the second lamina40in a second medium42delimited by the two laminas28,40.

Advantageously, the second lamina40has a surface treatment from among those already described for the first lamina28.

The second medium42comprises a liquid, for example a biological liquid susceptible to cellular culture such as, traditionally, DMEM (Dulbecco's Modified Eagle's Medium) or a saline solution such as a phosphate buffered saline (PBS).

The level of the liquid of the second medium is such that it covers the support face28A of the sample4and the objects22.

According to one alternative, the first medium36is a liquid similar to that of the second medium42.

The optical system17of the imaging system6comprises a tight cuvette38in which the matrix30of optical elements, the first lamina28, and the sample4are placed so as to contain the liquids or gels of the first medium36and/or the second medium42.

The bottom of the cuvette38can coincide with the surface of the photosensors26. For example, the cuvette38is pierced at the bottom and adhered with the matrix24of photosensors26in order to guarantee sealing owing to a biocompatible glue.

The optical system17of the imaging system6also includes separating means43able to separate the first medium36from the second medium42if the two are different. This separating means43is fixed to the walls of the cuvette38or to the first lamina28.

According to another alternative, the distance measured along the optical axis Z between the first slide28and the second slide40is adjusted so as to obtain, in a known manner, a configuration between slide and lamina of the sample4. In this configuration the second lamina40is in contact with the sample4.

The second lamina40can favor gaseous exchanges through the second lamina40, and/or prevent evaporation through the second lamina40of part of the second medium42, for example of the culture medium and the sample4.

The optical system17also comprises means44for adjusting the distances measured along the optical axis Z between the support face28A of the sample of the first lamina28and the face of the photosensors26disposed opposite the first lamina28.

Preferably, this adjusting means44includes at least one piezoelectric ceramic. The adjusting means44can be mechanically fixed to the bottom or on the edges of the cuvette38or on the support of the matrix24of photosensors26.

According to another alternative, the first lamina28is fixed at a fixed height to the walls of the cuvette38of the imaging system6by a mechanical, electrostatic, magnetic or equivalent means.

The first lamina28, and therefore the sample4and the objects5, preferably adhered cells22, can be removed from the optical system17of the imaging system6, depending on the operator's needs, for example for characterization of the sample on another instrument or for additional treatment of the sample (dying, marking with a fluorophore, etc.). The first lamina28, and the sample or object, can also be repositioned in the imaging system6.

The operation of the imaging system6according to the invention will now be described.

During the method for analyzing a sample by the device2described above, the manufacturer or operator of the imaging system chooses the separator element37and the first lamina28beforehand according to their optical characteristics (materials, refractive index, thickness, etc.) and the desired use/application.

Then, the matrix32of microlenses34is chosen taking into account the angle of the acceptance cone of the photosensors26, which is controlled by the presence of the microlenses34, in particular by the nature (refractive index) and shape (curve radius) of each microlens34.

Each microlens34comprises a flat diopter and a convex diopter. The upper face opposite the sample of the microlenses is the convex diopter, which can be likened to a spherical bowl. Preferably, the microlenses are made from polystyrene, polyamide, AZ4562 photoresin or a similar material, poly(dimethylsiloxane), SU-8, silica, boron phosphorous silicate glass (BPSG), or a transparent thermoplastic material such as poly(methyl methacrylate) (PMMA) or polycarbonate.

The focal length of each microlens34is less than 25 μm, preferably less than 10 μm, so as to limit optical cross-talk phenomena between the microlenses and the underlying associated photosensors.

Preferably, the opening diameter of each microlens is at least two times smaller than the smallest lateral dimension of a cell. The smallest lateral dimension of a cell is the smallest dimension of the cell passing through its center of gravity and measured in a plane perpendicular to the optical axis of the imaging system, i.e. in a plane parallel to the face28A of the lamina serving as support for the cell.

Moreover, the distance between the optical axes of two closer neighboring microlenses is preferably less than at least 30% of the opening diameter of a microlens. As a reminder, the opening diameter of a microlens is the diameter of the intersection section of the flat diopter and the convex diopter forming the microlens.

The photosensors26associated with the microlens34are arranged so that an object5, preferably a cell, is covered by at least two adjacent photosensors in a first direction and at least two other adjacent photosensors in a second direction perpendicular to the first direction.

Thus, the image of a cell is done by at least a 2×2 matrix of adjacent photosensors.

For example, the opening diameter of each microlens is substantially comprised between 0.7 and 10 μm, preferably between 0.7 and 3.0 μm, to image objects such as cells whereof the diameter is substantially between 20 and 30 μm. These values make it possible to avoid a discontinuity in the image of the objects, even until a same portion of the cell contributes to the light intensity of two neighboring photosensors. Over-sampling of the object5is preferable to under-sampling in order to produce good-quality images of the cells.

Each cell is thus shown in the image by at least 2 pixels per side, preferably by at least 6 pixels per side, so as to facilitate the subsequent computer processing on the images of the cells: the more the cells are represented by a large number of pixels, the more the contrast improvement, filtering and intensity thresholding operations carried out during the computer processing of the images will correspond to reality.

For example, the distance between the optical axes of two closer neighboring microlenses is less than 3.0 μm for microlenses with an opening diameter of 10 μm, less than 900 nm for microlenses with an opening diameter of 3.0 μm, less than 210 nm for microlenses with an opening diameter of 700 nm.

Then, the manufacturer or operator chooses the first medium36as a function of its refractive index n in order to modify the focal distance of the microlenses34and thus the acceptance angle of the photosensors26. In fact, the light beams are deflected at the optical interface between the first optical medium36and each microlens34according to Descartes' law. Thus, the index of the first medium36also makes it possible to adjust the angle of the acceptance cone of the photosensors.

More specifically, the selection of the refractive index of the first medium36controls the position of the intersections of the acceptance cones of neighboring or adjacent photosensors. Thus, the angle of the acceptance cone of the photosensors26is increased, and consequently the position of the intersections of the acceptance cones of neighboring photosensors is decreased, by increasing the refractive index n of the medium36, and vice versa.

For example, for a microlens34made from polystyrene with a refractive index 1.61, it suffices to replace the medium36, initially air (i.e. n=1.0003), with an oil having a refractive index 1.55 to magnify the acceptance cone of the photosensors26.

The refractive index of the first medium36is substantially comprised between 1 and the refractive index of the microlenses34, preferably between 1 and 1.64.

In fact, any optically active material, situated between the matrix of photosensors and the sample, participates in modifying the acceptance cone of the photosensors. In particular, this is the case for the separator element37and the first lamina28. For the latter, the light beams are deflected at the optical interface between the first lamina28and the first optical medium36according to Decartes' law. As a result, the choice of the first lamina also adjusts the acceptance cone of the photosensors. Nevertheless, it is also chosen for its optical qualities (high transmission) and is used as support for the objects5and preferably the adhered cells22to be imaged.

Then, owing to the adjustment means44, the distance, measured along the optical axis Z, between the support face28A of the sample4of the first lamina28and the apex of the microlenses34, is adjusted, and consequently the thickness of the first medium36is as well. The distance is adjusted so as to be comprised between 0 and 1500 μm, and preferably between 100 and 500 μm.

Preferably, this distance is smaller than or equal to −150×F×n+400×F, where F is the focal distance of the microlenses34measured in the air and n is the refractive index of the first optical medium36.

H denotes the distance measured along the optical axis Z, between the face of the first lamina28opposite the microlenses and the apex of the microlenses. Adjusting the distance H then makes it possible to obtain a more or less clear and/or more or less contrasted image of the cells5, preferably of the cells22adhered to the sample4depending on what the operator wishes to observe as details of the cells5.

In fact, varying the distance D between the object5and the surface of the photosensors results in:modifying the size or magnification of the image of the object, i.e. spreading over a variable number of adjacent photosensors: the further the object is from the matrix24of photosensors26, the more it is imaged by a large number of photosensors26, and thus the larger the object appears on the final image produced,showing the diffracted contour of the object5, preferably an adhered cell22, from a certain height. Close to the surface, the diffraction pattern on the contour of the object is not shown because the diffraction pattern is concentrated on a single photosensor, which does not make it possible to reveal the spatial details of the diffraction pattern. As one moves gradually further away from the surface, the diffraction pattern on the contour of the object spreads over 2, 3, 4, etc. adjacent photosensors and is better and better observed. Nevertheless, far from the surface, the diffraction pattern is diffused on too large a number of photosensors (it is then not possible to establish a usable image of the object if the image of a point of the object spreads over more than 200 photosensors), andmodifying the contrast of the contour of the object.

The optical properties of the faces28A and28B of the first lamina28can be optimized to reinforce the diffraction, diffusion, interference and dispersion effects making it possible to view the contour of the cells22.

In a second embodiment of the optical system17of the imaging system6illustrated inFIG. 3, the face28A of the first lamina is situated opposite the matrix24of photosensors26; the sample4comprising cells5, preferably adhered22to the face28A of the first lamina, is therefore disposed between the first lamina28and the matrix32of microlenses34.

This embodiment makes it possible to do away with the thickness of the first lamina28in adjusting the distance between the objects to be imaged and the apex of the microlenses. This configuration has the advantage of offering very small, practically zero, distances between the objects22and the microlenses34, and making images as close as possible to the photosensors26.

According to a third embodiment shown inFIG. 4, the first lamina28is placed on the apex of the microlenses34. The distance H between the apex of the microlenses and the face28B opposite the microlenses of the first lamina28is then zero.

In that case, adjusting the distance between the support face28A of the sample and the apex of the microlenses amounts to choosing a thickness E of the first lamina28, adapted so that the measured distance, along the optical axis Z of the photosensors26, between the support face of the sample28A and the apex of the microlenses34, is substantially comprised between 0 and 1500 μm, and preferably between 100 and 500 μm.

Nevertheless, it must be noted that the first medium36is still present in the interstices between the microlenses34and the first lamina28, and consequently still acts on the acceptance cones of the photosensors26.

According to a fourth embodiment shown inFIG. 5, the first lamina28includes a slide45, with thickness E1, and a removable lamina46, with thickness E2, alongside the slide45and defining the support face for the sample; the sum of the thicknesses E1and E2is consequently equal to the thickness E of the first lamina28. This alternative makes it easier to move the sample without altering the distance H between the first lamina28and the apex of the microlenses34previously adjusted.

The presence of the second lamina40, for example made from glass, is especially necessary in the case of a use in fluorescence, for example to image fluorophores or quantum dots. In this embodiment, the first and second laminas28,40are preferably optical filters.

Preferably for this use, the light source18, then excitation source, is monochromatic.

The second filter40is not necessary if the excitation wavelength is filtered by the first filter28. For example, by eliminating the second lamina40and using a first glass lamina28as filter, the absorbance of the glass in the ultraviolet (UV) filters the excitation light if it is in the UV. A second example consists of a first lamina28whereof the face28A or the face28B is covered with a filtering stack, made by stacking dielectric layers, which absorbs the excitation wavelength.

According to one alternative, the optical system17of the imaging system6comprises a filter, or an array of dedicated filters, situated between the matrix24of photosensors26and the matrix32of microlenses34. For example, this filter, or array of filters, is integrated into the separator element37. The filters can be different under adjacent microlenses in order to characterize a same sample4according to at least two different wavelengths.

According to another alternative, the second lamina40is a directional filter so as to compensate for the absence of a collimation system for the light source18, which causes distortions in the image.

According to another embodiment illustrated inFIG. 6, the light source18is arranged so as to make the light pass into the first lamina28perpendicular to the optical axis Z so as to light the sample4or the objects22from inside the lamina.

According to another alternative, either one of the laminas28,40is a polarizer, or both laminas28,40are cross-polarizers. In the first case, the light source18emits rectilinearly polarized light. For example, the light source18is a liquid crystal display (LCD). In the second case, the light source18does not emit polarized excitation light, and the excitation light is then polarized by the second polarizer40. In both cases, the excitation light is stopped by the first polarizer28while the fluorescent emission light coming from the sample4is polarized and transmitted by the first polarizer28. The intensity is lower in this configuration, but the signal to noise ratio is increased relative to a direct transmission of the excitation light. Preferably, the second polarizer40is in direct contact with the liquid making up the second medium42to avoid depolarization of the excitation light at the air/liquid interface.

According to another embodiment illustrated inFIG. 7, the first lamina28comprises a plurality of microchambers (or wells)48that may or may not be in communication with one another. The microchambers48are preferably arranged in a matrix, for example the microchambers48are formed on or etched in the first lamina28. The thickness E participating in adjusting the distance between the face28A of the first lamina28and the apex of the microlenses34is then the distance measured along the optical axis Z between the bottom of the microchambers48and the face28B of the first lamina28.

According to the alternative illustrated inFIG. 8, the microchambers48are formed in the entire thickness E2of the removable lamina46previously described, and the thickness participating in adjusting the distance H between the face28B of the first lamina28and the apex of the microlenses34is then the thickness E1of the slide45.

According to one alternative, the plurality of microchambers48forms a plurality of flow areas or microchannels for the sample, intended to circulate a fluid, for example the sample4, using means for actuating the fluid incorporated into the optical system17of the imaging system6. In a known manner, the actuating means are micropumps, microvalves, etc.

The plurality of microchambers48or microchannels is used, for example to perform screening by parallelizing observations on the same optical system17of the imaging system6. In a known manner, parallel observations make it possible to reproduce experiments, study several cell types and/or several cell densities, vary the spatial organization, reagent concentrations, culture conditions, etc.

One then records the optical signal coming from each microchamber48or microchannel in order to reconstruct the image of each microchamber48or microchannel.

Advantageously, the images of each microchamber48or microchannel correspond to the entire lower surface of each well or study area48contrary to the observations done in traditional optical microscopy transmission.

Another embodiment intended to perform parallel studies is shown inFIG. 9; the device then comprises a plurality of imaging systems6steered in parallel by a same electronic controller8. In that case, the computer system10manages the signals coming from each imaging system6in a synchronized or alternating manner.

According to one alternative, the device comprises a plurality of imaging systems6steered in parallel by several electronic controllers8. Using a limited number of electronic controllers8makes it possible to centralize the interface function between the imaging systems according to the invention and the computer system10.

Preferably, the imaging systems6are mounted on a same electronic circuit8and/or assembled in a housing or on a rack, in order to simplify handling by the operator and limit the space taken up by the device2in the incubator14.

According to two embodiments illustrated inFIGS. 10 and 11, the first lamina28is respectively the bottom of a well plate or the wall of a bottle intended for cell cultivation.

Advantageously, the walls of the well plates (or microplate) and bottles for cell cultivation are sterile and have a surface treatment favoring the adhesion of the cells, for example a deposition of proteins of the extracellular matrix or an oxygen plasma. The sterility and the surface treatment improve the attachment, spread and growth of cells on these supports, in particular on the support surface28A of the objects5, preferably the adhered cells22.

FIG. 10illustrates the embodiment of a device for characterizing and detecting a sample according to the invention wherein the first lamina28is the bottom of a well plate or microplate50.

The microplate50comprises a base52, a support54having a plurality of wells56and a lid58. The lid is placed on the support54, which in turn is placed on the base52. The lid prevents the sample4from evaporating, in particular the cell culture medium.

The support54of the microplate50comprises a lower extension68. The total height of the support54, with the lower extension68, is larger than the height of a well56. Thus, the lower extension68makes it possible to raise, for example by at least 1 mm, the bottom60of the wells56relative to the base52of the microplate. This available space between the bottom60of the wells56and the base52is intended for the imaging system6and the electronic controller8.

Furthermore, the four outer corners of the lower extension68of the support54of the microplate50are rounded with a curve radius of 3.18±1.6 mm so that handling robots of the microplates50can grasp the microplate50by the corners.

The device also comprises a plurality of imaging systems6according to the invention in order to carry out parallel studies on the samples contained in the different wells56.

In this embodiment, the first lamina28of each imaging system6is shared by all of the imaging systems placed under the support54of the microplate. The face28A of the first lamina28intended to support the objects5, preferably adhered cells22, forms the bottoms60of the set of wells56of the microplate50. The bottoms60are preferably flat and transparent to the light radiation transmitted by the objects5of the sample4.

To that end, the first lamina28is made from glass or plastic and adhered under the wells56using a biocompatible glue, such as a class 6 USP glue used in biomedical devices, in order to form the bottom60of the wells56.

Metrology markings are deposited or etched on the first lamina28making up the bottom60of the wells56, preferably on the lower face28B opposite the microlenses. The metrology markings are preferably 30 μm crosses with a line thickness of 4 μm, spaced apart every millimeter. These marks appear on the produced images. The metrology markings provide a reference scale for measuring the dimension of the cells and facilitating reframing of the images during image processing operations using the computer system10.

The walls of the microplate50(base52, support54, walls of the wells56and lid58) other than the bottom of the wells are made from polystyrene or polypropylene.

Furthermore, all or part of these walls are transparent to the light radiation transmitted by the objects5of the sample4. In that case, the microplate50and the imaging systems6assembled under the microplate50are placed in a darkroom to prevent disruptions from outside light.

According to one alternative, all or part of these walls are opaque with a black or white color. In a known manner, the microplates having opaque and white walls are more particularly used for luminescence experiments.

Preferably, the walls of the microplates50are opaque and black in order to prevent outside light disruptions, with the exception of the first lamina28. This first lamina28is transparent to the light radiation transmitted by the objects5of the sample, for example made from glass or plastic.

When the walls are opaque and black, it is not necessary to place the microplate50covered with its lid in a darkroom for imaging of the objects5. In fact, the walls of the microplate50with the lid58act as a darkroom. Such microplates50are particularly suitable for fluorescence experiments.

If the lid is completely black, the light source(s)18are fixed on the lid58opposite the samples contained in the wells56of the microplate50.

In the embodiment shown inFIG. 10, each imaging system6comprises a light source18that is therefore associated with a single well in order to illuminate the sample inside it.

For example, the light source18is a light-emitting sheet or a flat LED, such as an XLamp marketed by Cree (Durham, United States).

The light source(s)18can also be placed on the outer face if the lid is transparent, or if it is opaque and black with transparent windows above each well56, so as to illuminate the inside of each well56.

In general, light sources18giving off little heat are selected to limit the heating and evaporation of the sample4.

A matrix24of photosensors26topped by a matrix30of optical elements is positioned under each well56of a microplate50in order to view the objects5disposed on the bottom60of each well56of the microplate50, in particular so as to be able to produce an image of the sample4, preferably cellular, in each well56. The thickness E is then the thickness of the first lamina28, as in the embodiment shown inFIG. 4.

Thus, several experiments are carried out and visualized at the same time in the different wells56of the microplate50.

Each imaging system6is assembled on an electronic board62or printed circuit making it possible to produce the interface between the imaging systems and the electronic controller(s)8of the device. The electronic board62is shared by several imaging systems and one or more electronic controllers8.

In a configuration of this embodiment shown inFIG. 10, the imaging systems are connected to a single electronic controller8by the electronic board62, in order to centralize the interface function between the imaging systems according to the invention and the computer system10.

The electrical trails connecting the various electronic components of the electronic board62are preferably buried in the printed circuit or covered with a protective varnish in order to protect them from the humid atmosphere.

The electronic board62is connected to the computer system10via an electronic port64, connected on the board62, and a cable66, connect to the port on the one hand and to the computer system10on the other. An opening70is pierced in the lower extension68of the microplate50for passage of the cable66connecting the connection port64and the computer system10.

For example, the imaging systems6are connected on one face of the electronic board62opposite the wells, the port64and the electronic controller8being on the opposite face.

According to another configuration, each imaging system6is connected/assembled on an electronic daughterboard connected by a connector to an electronic motherboard on which the electronic controller8and the connection port64are assembled.

The electronic board(s)62are secured to the microplate50using mechanical fastening means and bearing points disposed between the microplate50and the electronic board(s)62.

In a known manner, to ensure the high transmission speed of the images to the computer system10, the connection port64is a USB2, FireWire, Ethernet Gigabit or CamLink port.

According to another alternative, the two-way communications between the computer system10and the electronic board62are done by transmitters and receivers, and the power supply for the imaging system(s)6is done by a cell or battery close to the device.

The dimensions of the matrix24of photosensors26make it possible to cover all or only part of the bottom60of the well56opposite which it is disposed. Furthermore, the dimensions of the matrix30of optical elements are substantially equal to those of the matrix24of photosensors26opposite which it is disposed.

For example, each matrix24of photosensors26has an area of 3.6 mm×2.7 mm, placed under a circular well with a diameter of 9 mm.

In another example, the matrix24of photodetectors26has an area of 6.4 mm×4.6 mm placed under a well with a diameter of 18 mm.

For each imaging system6, the center of the matrices24and30and that of a well56are aligned.

Using a matrix24of photosensors26and a matrix30of optical elements with dimensions smaller than the diameter of the well makes it possible to image the cells situated on the edge of the well whereof the behavior is often considered non-representative of the cell population.

In a known manner, the dimensions of the microplate50verify the primary specifications ANSI/SBS 1-2004 to 4-2004 recommended by the Society for Biomolecular Screening (SBS). More specifically, the microplates have a length of 127.76±0.5 mm and a width of 85.48±0.5 mm.

The total height of the support54of the microplate50equipped with the imaging systems6is comprised between 14.35 mm and 35.00 mm, i.e. the distance, measured along the optical axis Z of the photosensors26, between the end of the wells56opposite the end in contact with the first lamina28and the end of the extension68in contact with the base52.

The first lamina28is for example a glass plate measuring 175 μm or 210 μm thick or a transparent polystyrene film measuring 125 μm thick.

The microplates50integrated with the imaging systems can be used inter alia for cell proliferation studies, toxicology studies, morphological analyses, motility analyses, chemotaxis analyses, viral infection analyses, cancerology studies, pharmaceutical screening analyses or research on molecules influencing cell behavior.

The electronic board62is 115 mm long and 75 mm wide so as to be inserted into the lower extension68of the support54of the microplate50. Thus, no component of the electronic board touches the base52of the microplate50. Furthermore, this base52protects the electronic board62from the humidity in the incubator14.

Imaging systems according to the invention can also be disposed against the outer wall of one or more bottles in order to characterize the objects disposed on the inner wall of one or several bottles. The thickness E is then the thickness of the bottle wall.

In light ofFIG. 11, a cell culture bottle80is equipped with an imaging system6according to the invention in order to monitor the proliferation of the cells in the culture bottle.

In a known manner, the culture bottle80is a bottle with flat walls made from polystyrene or polypropylene that preferably have a surface treatment favoring cell adhesion.

Preferably, the imaging system6is placed under the culture bottle80so as to image the objects, preferably cells22adhered on the lower wall84of the bottle80, the lower wall84being the wall opposite the imaging system6. It forms the first lamina28of the imaging system6. Thus, the lower wall84or only part thereof has a thickness E adapted to perform cellular imaging according to the invention.

According to another alternative, the culture bottle is made from polystyrene, with the exception of all or part of the lower wall84, which is a lamina or plate made from glass or plastic with thickness E. Said lamina, or glass or plastic plate, is adhered to the polystyrene walls of the culture bottle using a biocompatible glue such as a class 6 USP glue.

As in the embodiment shown inFIG. 10, the imaging system6is connected to the electronic controller8via an electronic board86. An electronic port90connected to a cable92is assembled to the electronic board86in order to connect the electronic controller8to the computer system10of the device.

Furthermore, the electronic board86is secured to the lower wall84of the bottle80using mechanical fastening means88A,88B and bearing points disposed between the lower wall and the electronic board.

Furthermore, the light source18is fixed on the upper wall of the bottle80. Furthermore, the bottle comprises a stopper82to prevent the sample4from evaporating, for example the cell culture medium.

It is possible to stack the culture bottles80, the imaging systems6and the light sources18vertically to save space inside the incubator14. A very flat light source such as a light-emitting sheet is then chosen to illuminate the sample of the culture bottle, which makes it easier to stack the bottles and imaging systems. If another type of light source is chosen, a space for the light source is reserved above each culture bottle using fastening means and bearing points.

The images collected by the computer system10can be viewed remotely using a computer server. The user accessing the images of the cells remotely may or may not decide to change the culture medium of the cells or to relocate the cells in a new culture bottle.

Microbeads with a calibrated size can be introduced into the sample so as to measure the temperature of the medium from the produced images. In fact, in a known manner, the microbeads appear on the images and the movement of a microbead between two successive images depends on the Brownian movement, i.e. the temperature. Thus, the measurement of the movement of a microbead between two successive images makes it possible to deduce the temperature by using the Stokes-Einstein equation:

T=3⁢π⁢⁢r⁢⁢η⁢〈d2〉kt
with r the radius of the microbead, η the viscosity of the medium, <d2> the distance to the square traveled by the microbead during time t, and k Boltzmann's constant (k=1,3806504.10−23J.K−1).

The precise measurement of the position of the beads on the image is done by using the metrology markings deposited or etched on the lamina28as previously indicated as reference, irrespective of the embodiments of the imaging system (microplate, culture bottle, etc.).

The imaging system and the associated device are intended to detect and characterize the images optically, in particular the cells.

The microlenses focus the light coming from the areas of the sample in the corresponding sensitive areas of the silicon in order to collect as much light as possible. The light intensity is integrated by each photosensor. The matrix of microlenses modifies the division (discretization) of the space done by the matrix of photosensors.

Furthermore, the first lamina28creates diffraction and interference effects that favor the visualization of low-contrast objects such as cells adhered to the surface of the first lamina, in particular the contour of those objects.

The field of vision of the imaging system6corresponds to the dimensions of the matrix24of photosensors26. As an example, the field of vision is widened relative to a traditional microscope over a rectangle of several mm2and up to several cm2of area. Currently the obtained images correspond to a ×4 magnification in traditional microscopy, but over a larger field of about 3.5×4.5 mm2relative to a traditional digital opening objective. The magnification of the images differs according to the distance between the face28A, support for the sample, of the first lamina28and the apex of the microlenses34. In fact, if the sample4or the objects22are spaced away from the matrix24of photosensors26, the image of the sample or of the objects is spread over an increasing number of photosensors26.

The resolution depends on the size of the pixels, but it also depends on the focal distance of the microlenses, the distance between the sample4or the objects22and the matrix of photosensors, and the refractive index n of the medium36.

The device has smaller dimensions, preferably that of a cube with 10 cm sides, and is easy to install in an incubator.

The reduced dimensions of the device have the advantage of the great portability of the device, while considering applications to the patient's bed, such as blood cell counting, or being able to be placed directly in an incubator while adding only the presence of a computer in the cell culture room.

The invention has been described in the context of biological applications with samples comprising cells. However, it is applicable to other fields, for example to view bacteria, yeasts, fungi, pollens, algae, liposomes, synthetic particles, and/or to detect optical responses to physicochemical phenomena such as viewing the formation of crystals in reservoirs on the first lamina28.