Device for observing a sample using imaging

Abstract

A sample observation device (1) comprising a light source (10); an image sensor (35) comprising several pixels (36); a microlens array (30) comprising several converging microlenses (31), each microlens being positioned opposite a pixel, each microlens having an image focus, each microlens being configured to form, from light waves propagating at parallel incidence, a beam of light waves (13) converging towards the image focus of said microlens; the device being configured to hold the sample (20) between the light source and the microlens array; the device being characterized in that it comprises several attenuating elements (32, 32'), each attenuating element being associated with a microlens and a pixel, each attenuating element being configured to attenuate the light waves converging towards the image focus of the lens to which it is associated. Fig. 1A.

Description

Title of the invention: Device for observing a sample by imaging Technical field

[0001] The invention relates to a device for observing a sample by strioscopy. PREVIOUS ART

[0002] The observation of biological samples may be faced with a difficulty linked to the transparent nature of certain biological objects, for example cells, cellular structures or microorganisms. In order to address this problem, strioscopy imaging techniques can be implemented, aimed at obscuring light rays emitted by the light source. This forms a so-called dark field, or phase contrast, image. An example of implementation is described in EP2556164B1.

[0003] In the field of infrared, US patent 11313792 describes a lensless imaging device and method, in which a slide of a sample is exposed to infrared radiation.

[0004] The inventors propose a device allowing observation of an essentially transparent sample, that is to say comprising a large number of transparent particles. Statement of the invention

[0005] A first object of the invention is a device for observing a sample, comprising - a light source, configured to emit a light wave in a propagation direction, according to an emission wavelength; - an image sensor, comprising several pixels; - a plurality of converging microlenses, each microlens being arranged facing a pixel, each microlens comprising an image focus, each microlens being configured to form, from light waves coming from the light source and propagating parallel to the direction of propagation, a beam of light waves converging towards the image focus of said microlens; - the device being configured to hold the sample between the light source and the microlens array; - the device being characterized in that it comprises several attenuating elements, each attenuating element being associated with a microlens and a pixel, each attenuating element being configured to attenuate the lu waves miners converging towards the image focus of the lens with which it is associated, and in that: - each mitigating element is: • either an opaque mask at the emission wavelength; • either a phase shifting element, formed from a transparent material arranged to induce a phase shift, the phase shift being determined relative to a light wave propagating in the absence of the attenuating element; - each attenuating element extends between the microlens and the pixel with which it is associated;

[0006] such that each attenuating element attenuates each light wave propagating towards or from the image focus of a microlens.

[0007] According to a preferred embodiment: - the pixels are coplanar, and distributed according to a detection plane; - the respective image foci of each microlens are coplanar, and form a focal plane, parallel to the detection plane; - the attenuating elements extend in the focal plane or in a plane parallel to the focal plane.

[0008] The attenuating elements may in particular extend in a plane parallel to the focal plane, said parallel plane being arranged at a distance of less than 1 mm from the focal plane.

[0009] According to one possibility: - the pixels are coplanar, and distributed according to a detection plane; - each attenuating element extends, parallel to the detection plane, with a diagonal or larger diameter less than 2 times the emission wavelength.

[0010] According to one embodiment, each attenuating element is an opaque mask, at the emission wavelength.

[0011] According to one embodiment: - each attenuating element is a phase shifting element; - the phase shift generated by each phase shifting element is a quarter of the emission wavelength, or an odd multiple of a quarter of the emission wavelength.

[0012] According to one embodiment: - each attenuating element is a phase shifting element; - each attenuating element is formed from an inclusion of a first material in or on a support layer formed from a second material.

[0013] Each attenuating element may be integral with a support layer, the support layer being integral with the plurality of microlenses or arranged at a distance from the plurality of microlenses.

[0014] Each attenuating element can be integral with the image sensor.

[0015] The emission wavelength may be between 800 nm and 15 pm.

[0016] The distance between the sample and the plurality of microlenses may be less than 1 mm or 500 pm;

[0017] The distance between the plurality of microlenses and the image sensor may be less than 1 mm or 500 pm;

[0018] The distance between the sample and the image sensor may be less than 1 mm.

[0019] Another object of the invention is a method of observing a sample using of a device according to the first subject of the invention. The sample is placed between the microlenses and the light source. The sample may comprise transparent particles, for example biological particles.

[0020] The invention will be better understood upon reading the description of the exemplary embodiments presented in the remainder of the description, in conjunction with the figures listed below. FIGURES

[0021] [Fig. 1 A] represents a first embodiment of the invention.

[0022] [Fig.lB] shows a detail of the example shown in [Fig.lA].

[0023] [Fig.2] shows a second embodiment of the invention.

[0024] [Fig.3] shows a third embodiment of the invention.

[0025] [Fig.4] shows a fourth embodiment of the invention.

[0026] [Fig.5] shows a fifth embodiment of the invention. PRESENTATION OF SPECIAL EMBODIMENTS

[0027] [Fig. 1A] shows a first embodiment of a device 1 according to the invention. A light source 10, preferably monochromatic, or which can be considered as such, produces a light wave 11 propagating parallel to a propagation axis Z. The light source can in particular be a laser. In the example shown, the light wave is emitted according to an infrared emission wavelength, for example between 800 nm and 15 pm. However, the invention may not be limited to the infrared spectral range and may be implemented in the visible or ultraviolet spectral range.

[0028] The device is intended for the observation of a sample 20. The sample 20 is held on a holding element 21. In this example, the sample 20 is a biological sample that it is desired to characterize. The sample is deposited on a transparent slide 22 acting as a sample support. By thin thickness, we mean a thickness preferably less than 100 μm, and preferably less than 10 μm, typically a few micrometers. The sample preferably extends perpendicularly or substantially perpendicularly to the propagation axis Z. The term substantially perpendicular means perpendicular while admitting an angular tolerance of a few degrees, preferably less than 10° or 5° or 1°.

[0029] The sample may comprise transparent particles, the spatial distribution of which in the sample is to be evaluated. These may, for example, be biological particles, such as cells, for example of the white blood cell type, or microorganisms. The sample may be liquid, in which case the transparent slide 22 forms the bottom of a fluidic chamber.

[0030] Preferably, the sample does not include colored or fluorescent marking agents intended to attach to a predetermined type of particle. Observation of the sample is thus non-destructive.

[0031] The blade 22 is transparent to the incident light wave 11. When the emission wavelength is in the infrared, the blade 22 may comprise or be made of materials such as silicon, germanium, calcium fluoride (CaF 2), barium fluoride (BaF2). Preferably, the transparent blade 22 has an anti-reflective coating.

[0032] The sample is essentially transparent, or translucent, with respect to the incident light wave. By essentially transparent, it is understood that the sample absorbs only a small part of the incident light wave 11. The sample comprises microscopic objects of the cell or microorganism type which scatter or diffract the incident light wave 11, which forms a deflected light wave 14. The absorption of the incident light wave 11 by the sample is low, typically less than 50%, or even 10%. The main effect of the sample on the incident light wave is a deviation, by scattering, or a phase shift. The phase shift is induced by a variation in the refractive index of the microscopic objects relative to the rest of the sample, as well as by the thickness of said microscopic objects.

[0033] In [Fig. 1A], a light wave 12 transmitted by the sample is shown, as well as a light wave 14 resulting from the diffusion and / or refraction of incident light 11 by particles contained in the sample. By transmitted light wave is meant a light wave propagating, downstream of the sample, in the same direction of propagation as upstream of the sample, in this case along the Z axis.

[0034] The distance A between the light source and the sample, along the Z axis, is preferably greater than 1 cm. It is preferably between 2 and 30 cm. Preferably, the light reaches the sample in the form of plane waves, or which can be considered as such.

[0035] The light source 10 is preferably a laser source. It may in particular be a wavelength-tunable laser source, for example a QCL laser, acronym for Quantum Cascade Laser, meaning quantum cascade laser, in particular an external cavity laser. The spectral emission bandwidth of the light source is preferably less than 50 nm, or even 10 nm, or even 5 nm. A light source may comprise several elementary QCL laser sources, emitting respectively in different spectral bands.

[0036] The device comprises an array 30 of microlenses, comprising several converging lenses 31. In this example, the array 30 is a matrix of microlenses 31 arranged regularly. In the example shown in [Fig.1A], each lens is delimited by a hemispherical external surface 33s. The term microlens designates a lens whose diameter or largest diagonal is less than 1 mm or even 100 μm. Usually, the diameter or diagonal of each microlens measures a few tens of μm.

[0037] The device comprises an image sensor 35, comprising pixels 36. According to the direction of propagation of the light wave emitted by the light source, each pixel is aligned with respect to a converging microlens 31. Thus, each pixel 36 is associated with the microlens 31 with which it is aligned. In this example, the image sensor 35 is formed by a matrix of bolometers, each bolometer of the matrix having a detection spectral band in the infrared. Each bolometer forms a pixel 36. In the examples described below, each pixel is formed by a vacuum-encapsulated bolometer. Preferably, the light source 11 is configured so that the sample is illuminated by a light beam that is relatively homogeneous in intensity over the width of the sample analyzed (i.e. the size of the pixel matrix).

[0038] According to a variant, the image sensor 35 may be a matrix of CMOS pixels, when the emission wavelength is in the visible range. It may also be a matrix of cooled infrared pixels, made from HgCdTe or InGaAs semiconductors or IILV semiconductors.

[0039] Whatever the image sensor, the pitch p between two adjacent pixels is preferably less than 100 pm, or even 500 pm. It can be of the order of a few tens of pm or 1 pm.

[0040] Each microlens 31 defines an image focus and an optical axis A. The optical axis A of each microlens is parallel to the propagation axis Z of the light wave 11. Under the effect of exposure to the light wave 12 transmitted by the sample, propagating parallel to the propagation axis Z, each microlens focuses the light wave 12 towards its image focus. The focusing forms a converging wave 13 defining a cone of light, the point of convergence of which corresponds to the image focus of the microlens 31.

[0041] The device 1 comprises, at the level of each image focus, an attenuating element 32. By "at each image focus" is meant that the attenuating element is arranged either on the image focus, or upstream or downstream of the image focus, the distance between the attenuating element 32 and the image focus being small, typically less than 1 mm, or even less than 500 pm or 100 pm. Each attenuating element 32 is intended to block or attenuate the light 13 propagating towards or from the image focus of each microlens.

[0042] Preferably, the attenuating elements 32 are coplanar, and belong to the same focal plane P. The focal plane P is preferably perpendicular (or substantially perpendicular) to the propagation axis Z. In the focal plane P, the dimension of each attenuating element 32 corresponds to the dimension of the light cone 13 formed by each microlens 31. Note that each mask can be slightly offset relative to the focal plane, provided that the converging light wave 13 formed by each microlens 31 is masked. The dimension of each attenuating element is for example less than 2 or 3 times the emission wavelength. It is preferably less than or equal to the emission wavelength.

[0043] In the example shown in [Fig. 1A], each attenuating element 32 is a mask opaque to the emission wavelength. Each mask can be formed from a metal of the Titanium or TiN (Titanium nitride) or Gold type. The thickness of each mask can be between a few tens of nm and a few pm.

[0044] The function of the mask 32 is to absorb the light emitted by the light source and then transmitted by the sample before being focused by a microlens. Thus, the light that has not interacted in the sample is blocked. The image formed by the image sensor is an image acquired in dark background. The rays 14 scattered by the sample, then refracted by a microlens, propagate outside the attenuating elements 32 and reach the image sensor 35. Thus, the image formed by the image sensor corresponds to the light waves 14 deflected and possibly phase-shifted by the sample 20, according to the principles of strioscopy. The image formed by the image sensor comprises light points, each light point corresponding to a scattering particle of the sample.

[0045] In the example shown in [Fig.lA], the microlens array 30 is a SIL (Solid Immersion Lens) type array: each microlens 31 is arranged in contact with a transparent support plate 33. The support plate 33 extends between each microlens 31 and a lower face 33;. The lower face 33; comprises the focal plane P of each microlens. The masks 32 are arranged on the lower face 33; of the support plate 33.

[0046] The lower face 33 of the support blade 33 may be covered by an anti-reflection layer. The masks are either deposited on the anti-reflection layer or integrated into the latter.

[0047] The masks 32 can be deposited, on the lower face 33, by photolithography or by printing.

[0048] The pixels 36 of the image sensor 35 are preferably aligned along a detection plane P'. The detection plane P' is parallel or substantially parallel to the focal plane P. By substantially parallel is meant parallel to within an angular tolerance of less than 10°, preferably 5° or 1°.

[0049] The distance δ between the sample 20 and the lens array 30 is preferably less than 1 cm, and preferably less than 1 mm or 500 μm. The distance d between the microlens array 30 and the image sensor 35 is preferably less than 1 mm or 500 μm. According to one possibility, the attenuating elements 32 are in contact with the image sensor 35, being arranged at the interface between the image sensor 35 and the microlens array 30.

[0050] Whatever the embodiment, it is preferable for the sample 20 to be arranged as close as possible to the image sensor. However, it is advantageous to maintain a non-zero distance δ, so as to avoid transmission of evanescent waves at the upstream external surface 33s of the microlens array. By "upstream external surface" is meant the surface of the microlenses closest to the sample 20.

[0051] The upstream external surface 33s is structured to allow focusing of the light wave 12 transmitted by the sample, propagating parallel to the propagation axis Z. In the example shown in [Fig.lA], the upstream external surface 33s comprises curved parts, for example hemispherical, forming converging microlenses. According to another embodiment, shown in [Fig.2], the upstream external surface 33s is micro-structured, so as to form converging microlenses.

[0052] Figure 1B shows an example of dimensioning a microlens array. The maximum thickness F' of the support plate 33, measured along the Z axis between the apex of the microlenses and the lower face 33;, corresponds to the focal length of each microlens 31. The radius of curvature of each microlens 31 is designated by ?. The thickness of each microlens is designated h and is measured along the Z axis between its apex and a flat area 33f of the upper face 33s of the support plate. The half-width of each microlens along the X axis is designated a.

[0053] r_ M f ~ 2h V /

[0054] When the microlens is hemispherical, h — a and r “ a.

[0055] The minimum thickness F of the support blade 33 corresponds to the thickness of the blade between the flat area 33f of the upper face 33s of the support blade and the lower face 33; of the support blade, measured along the Z axis. If the index of the material forming the mi crolentille estn, and if the index of the external medium is equal to 1 (the external medium being air), then

[0056] F = ~ (2)

[0057] and

[0058] F=Fh (3)

[0059] The diameter 02 of each mask 32, in the focal plane, corresponds to the diameter of an Airy spot, which corresponds to the main diffraction spot.

[0060] $ = ««£_ (4) 2 nx(i>| X )

[0061] where

[0062] is the diameter of the microlens and 2 is the emission wavelength.

[0063] When the microlens is hemispherical, r = a = h and 0j = 2rz = 2r

[0064] and0 (5)

[0065] If the pitch p between two adjacent pixels is 35 pm, considering silicon microlenses (n = 3.4), and h = 5 pm, a = 15 pm, = 30 pm, the implementation of the previous equations leads to: r = 25 pm, F' = 35 pm, F = 30 pm.

[0066] Considering 2 = 5 / rm, we obtain 02 = 4.2 pm. Considering 2 = 10 pm, we obtain 02 = 8.4 pm. Thus, the diameter of each attenuating element 32 is of the order of magnitude of the wavelength, preferably being less than the latter.

[0067] Taking into account a hemispherical shape of the microlenses (h = a = 15 pm), F = 21 pm and 02=2.54 pm (2 — 5 pm) or 02=5.08 pm (2 — 10 pm).

[0068] [Fig. 2] shows a configuration according to which each microlens 31 has a microstructured surface to allow focusing, towards an image focal point, of the light wave 12 propagating, upstream of the lens, parallel to the propagation axis Z. The lens 31 is then formed of a layer 34 comprising an alternation of materials having different refractive indices. According to the possibility shown in [Fig. 2], the layer 34 comprises a microstructure forming a discretized Fresnel lens, symmetrical with respect to an optical axis A. Other types of diffraction gratings can be envisaged, such gratings being formed of a periodic alternation of materials having different refractive indices.

[0069] [Fig. 3] represents an embodiment in which the attenuating elements 32 are arranged on each pixel, preferably being centered relative to the optical axis A of a microlens 31 and relative to a pixel 36.

[0070] According to a variant, the attenuating elements are arranged on a support, separate from the matrix 30 of microlenses 31 and the image sensor 35. The distance between the attenuating elements and the image sensor can be adjustable.

[0071] In the examples shown in Figures 1A, 1B, 2 and 3, each attenuating element is an opaque mask. Figure 4 shows an embodiment in which each attenuating element 32' is transparent and is configured to produce a phase shift. The phase shift is preferably y (or kÿ, k being an odd integer) of a light wave passing through it, in a manner similar to a quarter-wave plate. Each attenuating element 32' is a phase shifting element. If n is the refractive index of an attenuating element 32', the thickness ( of the attenuating element, along the propagation axis Z, is such that:

[0072] t _ —i--- / 5) with n' > next 4(« '"ext ) \ /

[0073] where next is the refractive index of the medium external to the attenuating element 32'.

[0074] For example, if each attenuating element 32' is formed from the same material as the support plate 33 and the microlenses 31, for example silicon (n = n' = 3.4), r = 520 nm.

[0075] According to this embodiment, the converging wave 13 refracted by each microlens 31 is phase-shifted by y. In this embodiment, the phase defects introduced by the sample result in relative variations in the illumination of the image sensor. These variations are further sensitive to the sign of the phase shift of the light by the sample. This embodiment allows for improved image contrast compared to the first embodiment.

[0076] In the configuration shown in [Fig.4], each phase-shifting element 32 is obtained by adding material to the lower face 33 of the support blade 33.

[0077] [Fig. 5] represents a variant of the embodiment described in connection with [Fig. 4]. According to this embodiment, each phase-shifting element 32' is obtained by removing material from the lower face 33; of the support blade 33. The index of each phase-shifting element is therefore the index of the external environment, for example air. In this case, the thickness t of an element 32', along the propagation axis Z, is such that:

[0078] f— 4 (7) with n>next>, where n is the refractive index of the support plate 33. 4(^-^) \ /

[0079] Although described in connection with a light source emitting in the infrared spectral range, the invention can be implemented in the visible or UV spectral range.

[0080] Generally speaking, each phase shifting element 32' is formed by adding a first material to a support layer (33) formed from a second material (see [Fig.4], the first material being Si, which is also the second material), or by including a first material in the support layer (see [Fig.5], the first material being air)

[0081] The invention may be implemented for the observation of essentially transparent samples, comprising transparent particles or which may be considered as such. It can find applications in the field of biological samples, but also in other industrial fields, for example the control of samples in the environment or in the food industry. It applies to both liquid and solid samples.

Claims

Claims

1. Device (1) for observing a sample, comprising - a light source (10), configured to emit a light wave (11) in a propagation direction (Z), according to an emission wavelength; - an image sensor (35), comprising several pixels (36); - a plurality (30) of converging microlenses (31), each microlens being arranged facing a pixel, each microlens comprising an image focus, each microlens being configured to form, from light waves coming from the light source, propagating parallel to the direction of propagation, a beam of light waves (13) converging towards the image focus of said microlens; - the device being configured to hold the sample (20) between the light source and the microlens array; - the device being characterized in that it comprises several attenuating elements (32, 32'), each attenuating element being associated with a microlens and a pixel, each attenuating element being configured to attenuate the light waves converging towards the image focus of the lens with which it is associated, and in that: - each mitigating element is: • either an opaque mask (32) at the emission wavelength; • either a phase shifting element (32'), formed from a transparent material arranged to induce a phase shift, the phase shift being determined relative to a light wave propagating in the absence of the attenuating element; - each attenuating element extends between the microlens and the pixel with which it is associated; such that each attenuating element attenuates each light wave (13) propagating towards or from the image focus of a microlens.

2. Device according to claim 1 in which - the pixels are coplanar, and distributed according to a plane of detection; - the respective image foci of each microlens are co-planar, and form a focal plane (P), parallel to the detection plane; - the attenuating elements (32, 32') extend in the focal plane or in a plane parallel to the focal plane.

3. Device according to claim 2 in which the attenuating elements extend in a plane parallel to the focal plane, said parallel plane being arranged at a distance of less than 1 mm from the focal plane.

4. Device according to any one of the preceding claims, in which - the pixels are coplanar, and distributed along a detection plane; - each attenuating element extends, parallel to the detection plane, with a diagonal or a larger diameter less than 2 times the emission wavelength.

5. Device according to any one of the preceding claims, in which each attenuating element (32) is an opaque mask, at the emission wavelength.

6. Device according to any one of the preceding claims, in which - each attenuating element is a phase shifting element (32'); - the phase shift generated by each phase shifting element is a quarter of the emission wavelength, or an odd multiple of a quarter of the emission wavelength.

7. Device according to any one of the preceding claims, wherein - each attenuating element is a phase shifting element (32'); - each attenuating element is formed from an inclusion of a first material in or on a support layer (33) formed from a second material.

8. A device according to any preceding claim, wherein each attenuating element is secured to a support layer (33), the support layer being secured to the plurality of microlenses or disposed at a distance from the plurality of microlenses.

9. Device according to any one of the preceding claims, in which each attenuating element (32, 32') is integral with the image sensor.

10. A device according to any preceding claim, wherein the emission wavelength is between 800 nm and 15 pm.

11. Device according to any one of the preceding claims, wherein: - the distance between the sample and the plurality of microlenses is less than 1 mm or 500 pm; - and / or the distance between the plurality of microlenses and the image sensor is less than 1 mm or 500 pm; - and / or the distance between the sample and the image sensor is less than 1 mm.

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