Integrated apparatus for the analysis of biological samples

Abstract

The present invention relates to a large-field microscopy apparatus for the analysis of biological samples integrating a micro-fluidic control system.

Description

[0001] INTEGRATED APPARATUS FOR THE ANALYSIS OF BIOLOGICAL SAMPLES

[0002] DESCRIPTION

[0003] Technical field of the invention

[0004] The present invention relates to the field of the microscopy apparatuses for the analysis of biological samples.

[0005] The present invention, more specifically, relates to a large field of view microscopy apparatus integrating a micro-fluidic control system.

[0006] Background

[0007] In the biology field, the fluorescent light and transmitted light apparatuses and microscopy techniques evolve quickly. One first requirement is to increase to the maximum the spatial resolution of the images. Up to now, the best microscopy lenses allow to reach an optical resolution (OR) of about 0.25 pm, which can be further increased with super-resolution (SR) techniques. The advantage of having a high resolution lies in the possibility of studying the behaviour of sub-cellular structures, such as for example mitochondria-protein aggregates, DNA and micro-RNA, and it requires an adequate tool for acquiring the intensities measured with high “spatial sampling rate” or with a sampling with reduced pixel sizes (PS). A reduced PS, in fact, allows to acquire the same images with a greater number of pixels (NoP) of a “camera”-like image acquisition device and then to save a greater amount of information.

[0008] To the need for having a high resolution even the need for obtaining so-called Large Field (LF) images has added recently, that is to obtain an optical piece of information on an extended detection area (or region of interest), for example of the order of centimetre or even larger. The fact of achieving simultaneously one LF and high OR involves a series of challenges, both in the implementation of dedicated tools, and in terms of software for managing large amounts of data.

[0009] In order to obtain a Large Field low magnification optical lenses can be used which, in their most common versions, can produce a native Field Of View (FOV), up to two centimetres. This type of lenses is usually overlooked in the commercial tools.

[0010] The needs for having a Large Field derive from the fact that complex biological structures show their functional behaviour only if studied in their entirety. Examples of biological samples in which the functionality emerges on large scales are, for example, tumour organoids

[0001] , groups of nematodes for behavioural studies [2], organoids [3], printed cellular structures [4] and large, highly interacting cell cultures such as neural networks and neural rosettes [5].

[0011] The observation of these types of samples then requires the use of a Large Field microscope and, as in any image acquisition, the maximum OR together with an adequate NoP.

[0012] In the field of the cellular and biological microscopy, it is also known that the analysis of a living sample (live imaging) is associated to measurement modes allowing the best study of the biological samples with respect to the modes in which the sample is fixed, both since the fixing process modifies drastically the molecular content of the sample and since in the fixed samples any process is crystallized and there is no dynamic behaviour.

[0013] In this context, the live imaging has a series of additional difficulties.

[0014] In particular, any system of biological interest, especially in case of cellular cultures, requires a perfectly controlled environment to operate in which, for example, temperature, culture medium and / or CO2-Oxygen ratio in the microatmosphere have to be monitored and corrected continuously.

[0015] The biological experiments of this type then require the arrangement of a system known as “incubator”, having typically the extension of ten centimetres and allowing to reproduce an optimum environment. In the live imaging then it is important to monitor the samples over time to observe behaviour, evolutions, response to stimuli, conditions, stress, drugs and different reagents, growth and aging processes [6],

[0016] The study of these dynamic systems has extremely variable characteristic periods of time, going from the millisecond in case of the neural response to one stimulus, to days or months in case of growth and maturation of cell cultures of human origin. Therefore, the need arises to carry out prolonged and continuous measurements on a same experimental setup, capable to maintain the system under optimum conditions even for weeks. In order to meet these needs, it is known to use fluidic control systems, for example robotic syringe pumps, capable of performing procedures automatically, such as, for example, the culture medium exchange, which, otherwise, would disturb the balance of the sample in case of human intervention.

[0017] The automation is especially required in case of very long measurements, since repeated manipulations of the biological sample would lead to an optical misalignment of said sample with respect to the image acquisition system, to contaminations and, sometimes, they would require the human intervention in time and days in which the laboratories are hardly accessible (for example at night or on holidays).

[0018] The implementation of micro-fluidic chips [7], housing the biological system inside the incubator, is of particular interest for this type of measurement. The use of miniaturized inlets and outlets allows to control the sample with great precision, to spray different areas with different reagents or media, to create diffusion or pressure gradients which are useful to simulate macroscopic organic structures. The miniaturization of the micro-fluidic control tools further allows to integrate multiple approached inlets and, then, to obtain several automated activities working with different components, in particular the culture medium exchange, the collection of the waste solutions, the administration of reagents and / or markers, the deposition on the medium of cells and other biological elements prepared in suspension. The micro-fluidic chips, then, pave the way for the need for controlling, synchronizing and integrating a plurality of control tools of the sample.

[0019] From the point of view of the information content of the acquired images, it is known to use a so-called “stitching" technique (juxtaposition of images) to obtain, as mentioned above, Large Field images which simultaneously maintain high the OR [8], [9],

[0010] , Such technique adopts a microscope lens with smaller native FOV than the Large Field to be measured and provides a series of subsequent measurements (tiled acquisitions) in which the sample is translated by a determined number (Ns) of times so that subsequent translations allow to acquire images corresponding to different portions of the sample. By taking a number of

[0020] LF images Ns> and by juxtaposing them next to each other in the right order, a complete image of the entire region of interest (the whole Large Field) can be reconstructed.

[0021] The above-mentioned technique advantageously allows to maintain the native resolution (RN) of the selected microscope lens with very high resolution and at the same time to amplify the field of view at will.

[0022] Such technique, however, has several limitations.

[0023] Firstly, the translation system of the sample requires a bulky, but at the same time quick and very precise, motorisation.

[0024] As said, in case of living samples (live imaging) the actuators have to be capable of moving the whole incubator with high precision and repeatability of the motion. This type of translation motorisation usually is extremely expensive. Consequently, the handling time is generally high and makes longer the total duration of the measurement since the physical movement of the whole incubator cannot take place instantaneously, especially if one wants to maintain high motion precisions (motion precision lower than OR and PS).

[0025] An additional disadvantage due to prolonged motion time relates to the quality of the final image, especially in case of analysis of living and dynamic samples (live imaging with samples with motility such as, for examples, nematodes or cells with motility such as bacteria or spermatozoa). The motion of the motile biological sample during the various motions of the system-incubator will cause effects of “non-overlapping” between the edges of the images of distinct acquisitions.

[0026] Moreover, the fact of obtaining a perfectly uniform lighting of the sample requires an extremely sophisticated lighting apparatus, for example through laser coupled in single mode fibre, or a led source with several stages of spatial filtering. Both these solutions work at the expense of lighting power and often require a continuous maintenance in order to be obtained. In case of lack of uniform lighting, worsening of lighting performances, or aberrations in the field of view due to other factors (for example “vignetting” in the image acquisition process), the final result of the stitching will be strongly compromised, by producing periodic aberrations and a difficult juxtaposing of the acquisitions.

[0027] In order to increase the effectiveness of the juxtaposing process there are software solutions. However, the activity of post-processing on the acquisitions however requires a certain degree of image overlapping, so as to record perfectly the obtained images and to reduce possible errors due to the mechanical motion (repeatability or precision). In order to obtain images with a certain degree of overlapping, then, it is required that the single motions of the system-incubator are smaller than the native FOV. This involves the sacrifice of a certain percentage (typically the frame) of the acquired image, thus implementing a smaller “effective” native field of view than the native FOV of the lens.

[0028] Summary of the invention

[0029] The technical problem placed and solved by the present invention is then to provide a solution allowing to obviate one or more of the drawbacks mentioned above with reference to the known art.

[0030] Such problem is solved by a microscopy apparatus according to claim 1 .

[0031] Preferred features of the present invention are set forth in the depending claims. The invention concerns a solution in the field of microscopy, for example for diagnostic tests or in the experimental research and it relates to an apparatus which integrates means suitable to offer a wide range of functionality and a precise fluidic control.

[0032] In particular, the microscopy apparatus of the invention is characterized by a micrometric optical resolution and it works with a large field of view. Such apparatus, indeed, is configured for the observation of a detection area of the sample equal to at least 1.0 cm2and it integrates, in the same supporting structure or frame, an automated, dynamic, continuous and operator- programmable micro-fluidic imaging and control system.

[0033] The microscopy apparatus of the invention, in a preferred embodiment, is of the fluorescence type and it allows to obtain Large Field images of a dynamic biological sample, meant to include living biological samples (live imaging), intended to be contained inside a box-like body carried by the frame itself.

[0034] Said frame further carries a sample illumination unit with an incident signal, an optical unit for receiving a signal transmitted through the sample detection area and a detection group configured to acquire said transmitted signal and to generate an image of the sample corresponding to the detection area.

[0035] In the context of the present description the expression “detection area” is meant to designate a region of interest to be observed of the biological sample and it corresponds to the extension of the native field of view (FOV), as defined above and detected as characteristic parameter of the optical unit used for said observation.

[0036] The apparatus of the invention allows a more accurate and in-depth activity with respect to the known solutions and it opens new perspectives in the field of the biological research and the diagnostic medicine which will be evident in detail hereinafter in the present description.

[0037] In the applications which study the dynamic biological samples, the traditional microscopy systems are provided with a box-like body which comprises a containment unit (incubator) of the sample under controlled atmosphere conditions, in particular wherein temperature and oxygenation are constantly monitored. Even if they are sophisticated and performing, such traditional systems are generally provided with an external micro-fluidic unit, functionally and operatively connected to the box-like body in order to be able to manage the different flows of fluids interacting with the dynamic biological sample, especially in case of a timed administration of chemical agents (stimulating substances such as nicotine for example) or biological agents (micro-rna, proteins, hormones or suspension of bacteria or cells).

[0038] The criticality of manipulating very small volumes of fluid in controlled and timed way imposes the use of micro-fluidic units which are positioned at a distance, even though close, from the sample and which makes the set-up on the workbench complex, cumbersome and vulnerable to losses on the microscope or loss of the temperature and sterility conditions for the (bio)fluids.

[0039] Then, it will be appreciated that the apparatus of the invention integrates in the same bearing structure means for controlling a micro-fluidic flow entering and / or exiting the above-said box-like body and which allow, advantageously, the activities required to monitoring and the study of dynamic biological samples by providing, for example, the possibility of a real time control, a timing of stimulating agents, a discharge of waste liquids.

[0040] A control of the fluids integrated in the apparatus removes the constraints of an external control system, by simplifying the management of the apparatus by the user especially in case of long experiments. Advantageously, said configuration avoids a subsequent integration / synchronization of components, units, accessory groups which could be long, expensive and could require advanced technical capabilities.

[0041] The apparatus of the invention, moreover, provides that the box-like body of the apparatus of the invention results to be motionless during the acquisition of the signal transmitted through the sample.

[0042] In fact, as mentioned above, within the apparatuses and the microscopy techniques for analysing specifically dynamic biological samples, the known solutions face the need for implementing Large Field images through the stitching technique in which the motion of the incubator containing the sample, during the microscopic observation phase, is essential and it involves the above-mentioned problems.

[0043] Differently, the apparatus of the invention, in the various implemented embodiments, does not provide the use of the stitching, technique, it does not require the motion of the sample and integrates an optical unit characterized by a native Field Of View (FOV) already equal to or greater than the centimetre.

[0044] The above-mentioned devices and, in particular, the configuration of the apparatus which allows to do without means for moving the sample, are particularly useful in case of long measurements with dynamic biological samples, in which the stitching technique can produce low-quality images or with artefacts and the micro-fluidic control is required to keep the sample alive without the human intervention.

[0045] Other advantages, features and use modes of the present invention will result evident from the following detailed description of some embodiments, shown by way of example and not for limitative purposes.

[0046] Brief description of the figures

[0047] The figures of the enclosed drawings will be referred to, wherein:

[0048] ■ Figure 1 shows a schematic view of a microscopy apparatus according to a first embodiment of the invention;

[0049] ■ Figure 2 shows a schematic view of a microscopy apparatus according to an alternative embodiment of the invention;

[0050] ■ Figure 3 shows schematically two exemplifying modes for acquiring the image of the sample implemented by the apparatus of Figure 2;

[0051] Figure 4 shows an example of experimental characterization of the performances of the apparatus according to the embodiment of Figure 2.

[0052] Detailed description of preferred embodiments

[0053] The present invention will be described hereinafter by making reference to the above-mentioned Figures.

[0054] According to a general aspect, the invention provides a large field of view microscopy apparatus which integrates in its bearing structure means for controlling a micro-fluidic flow.

[0055] The apparatus of the invention finds application in the field of microscopy and diagnostics in the research laboratories. The apparatus of the invention offers a wide range of functionalities and allows to perform detailed analyses on biological samples of great sizes (at least of the order of the centimetre) and studies on micro-fluidic chips with a precise and timed fluid-dynamic control, by guaranteeing versatility and use safety.

[0056] By firstly referring to Figure 1 , a schematic view of the apparatus, the invention relates to, according to a first embodiment is shown.

[0057] The apparatus 100 comprises a frame carrying a box-like body 10 configured to contain the biological sample 1. In the context of the present description, the expression “biological sample” or “sample” mainly relates to dynamic or living biological samples, and then the structure of the box-like body is configured and shaped so as to allow a suitable containment of such type of sample. Preferably, the typical sample to be examined is arranged with a micro-fluidic chip containing biological material, such as for example a cell culture, organoid, 3D printed organoid, nematode and the like.

[0058] However, the apparatus 100 of the invention, and in particular said box-like body 10, can be suitably configured even to house, contain and analyse fixed biological samples.

[0059] In a preferred embodiment, the box-like body 10 comprises a chamber, or incubator, 11 with controlled atmosphere to house the micro-fluidic chip. The boxlike body 10 can house, for example, an incubator of the Okolab H301 mini type. Alternatively, a metal mask for the direct housing of a micro-fluidic chip with the standard sizes of a microscope slide.

[0060] Preferably the incubator 11 houses sensor means, for example a temperature sensor and / or a CO2 sensor. Advantageously, the box-like body 10 is provided with an access for the direct insertion of the sample 1 .

[0061] According to a preferred variant, the box-like body 10 comprises a (not shown) cover, preferably removable and not transparent to light. For example, a lid can be provided, suitable to close in a sealed manner the area of the incubator 11 in order to protect it during the experiment. The system for closing the cover can be a spring or a tilt system and it allows to insulate optically the incubator 11 and the optical measurement area from outside. This feature allows to perform measurements even in rooms with light or variable lighting, and not insulated optically from outside.

[0062] The frame further carries an illumination unit 20 configured to generate a signal 2 incident the sample and an optical unit 30 for receiving or collecting a signal transmitted through a detection area of said sample 1 . The incident signal 2 and the transmitted signal are preferably light signals.

[0063] In particular, the sample results to be interposed between the illumination unit 20 and the optical unit 30. Preferably, the box-like body 10 has an optical access configured so as to allow observation from the bottom of the sample 1. This advantageously allows to analyse the sample 1 inside the micro-fluidic chip itself, by easing the manipulation and simultaneous observation of the fluidic samples and of the biological structures inside thereof.

[0064] In the illustrated examples, the illumination unit 20 comprises in sequence a led source 21 , a condenser lens 22 and a beam splitter 23 to obtain the signal 2 incident the sample 1 . According to a preferred embodiment, the micro-fluidic chip can be illuminated with light having two selectable channels and adjustable intensity. The apparatus 100 of the invention preferably is a fluorescence microscopy apparatus and thus it incorporates a (second) source 2T of an additional (light) signal which follows a dedicated optical path - for example by providing a suitable filter 21 O’, lens 22’ and / or dichroic mirror 23’ - to generate a component of the incident signal 2 capable of exciting the fluorescence of particular molecules naturally present in the sample 1 or added thereto in a phase for preparing the latter. In this way the uniform lighting of the sample with two different wavelengths is allowed and, in a configuration of fluorescence type, the apparatus can include filtering means 24’ placed downstream of the sample 1 of the fluorescent component of the transmitted signal.

[0065] A detection group 40 acquires the transmitted signal and generates an image of the sample corresponding to the detection area.

[0066] As mentioned above, the detection area is meant to designate a region of interest to be observed of the biological sample 1 and corresponds to the extension of the native Field Of View (FOV) characteristic of the optical unit 30.

[0067] The native field of view and then the detection area of the optical unit 30 of the apparatus is equal to or greater than 1.0 cm2. Such feature advantageously allows a broad spectrum viewing of biological and fluidic samples and the simultaneous observation of most part of the biological cultures used for diagnostic tests or experimental research.

[0068] In a preferred embodiment, the detection group 40 comprises a high resolution camera, preferably equal to or higher than 25 Megapixel, allowing to display details up to few microns. This feature is particularly useful for the analysis of biological samples, since it allows to detect cellular structures and particles at detail levels not reached by microscopy apparatuses known in the reference field and with native field of view starting from 1 .0 cm2.

[0069] By virtue of the features of the optical unit 40 of the apparatus 100, which will be described in detail hereinafter, it is possible to obtain an image of the sample 1 without aberrations and without artefacts on the whole field of view.

[0070] The frame of the apparatus 100 further carries control means, designated as a whole with reference 50, of a micro-fluidic flow entering and / or exiting the boxlike body 10.

[0071] The micro-fluidic control means 50 preferably comprises independent infusion and / or suction channels F, preferably of the type comprising syringe pumps.

[0072] In the illustrated embodiments of the invention, said control means comprises four syringe pumps 51 guaranteeing a precise flow control from 0.1 microliters per minute up to 10 millilitres per minute and they are advantageously configured so as to be compatible with all types of commercially available syringes comprised in the range of 0.5 millilitres and 10 millilitres. This wide range of flows allow to perform different types of analysis and the manipulation of samples with various viscosities.

[0073] Each syringe pump 51 advantageously has its own independent motion which allows each channel to move a specific syringe in infusion or suction and with specific speed.

[0074] Even for the micro-fluidic control means 50 a covering element can be provided, comprising an access opening, analogously to what described above with reference to the box-liked body 10. In a preferred embodiment, an electronic unit is provided which allows the syringe plunger to be controlled thermostatically at a temperature comprised between 15 and 35°C.

[0075] The apparatus 100 is further characterized by the fact that the box-like body 10 results to be motionless during the acquisition of the transmitted signal.

[0076] Such aspect is particularly advantageous since it allows to free its operation from the need for moving the sample 1 to acquire an image of the detection area. In the state of art of the apparatuses working in the implemented applications of the invention, such motion is due to the fact that the implemented optics is characterized by fields of view whose order of magnitude is significantly lower than 1.0 cm2and the known apparatuses make use of the previously described stitching technique in order to obtain high resolution images.

[0077] In the embodiment of the invention illustrated in Figure 1 , the optical unit 30 of the apparatus 100 provides one single optical system comprising a relay lens 3a which collects the signal transmitted by the sample 1 and allows to reduce the spherical and chromatic aberrations on a field of view equal to at least 1.0 cm2. Differently, the known apparatuses under this profile provide a double optical system with a lens to collect the transmitted signal and a tube lens to form the image on the camera.

[0078] The possibility of using one single multi-element optical system will be therefore appreciated, that is consisting of at least ten individual optical elements (lenses) organized in one single monolithic block, both to implement the collection of the transmitted signal and to form the image on the detection group 40, by reducing manufacturing costs and reducing the number of optical interfaces (hardware simplification) in favour of better efficiency in collecting said signal, by obtaining more intense and contrasted images than the known systems with a greater number of optical interfaces.

[0079] According to this embodiment of the invention, the obtainable image of the sample 1 can be characterized by a real optical resolution equal to or higher than 3.4 micron, an effective pixel size equal to or higher than 2.740 micron and a surface area equal to at least 24 Megapixel.

[0080] Figure 2 shows a second embodiment of the apparatus 200 of the invention which will be described hereinafter by referring exclusively to the distinctive technical features with respect to the embodiment of Figure 1 .

[0081] In such variant, the optical unit 30 comprises a movable element 3b configured to steer the signal transmitted by the sample 1 in a plurality of sampling positions on the detection group 40.

[0082] In particular, with respect to the apparatus of Figure 1 , the relay lens 3a of the optical unit 30 is replaced by a more sophisticated system comprising a piezoelectrically-controlled movable mirror. Preferably, the optical unit 30 further comprises a so-called infinity corrected Large Field lens 31 and a Large Field tube lens 32. Said movable element 3b is placed in the area of the optical unit 30 in which the transmitted signal is a parallel beam signal, in particular between the infinity corrected lens 31 and the tube lens 32. It is specified that the tube lens 32 is placed downstream of the movable mirror 3b according to a forwarding direction of the signal transmitted towards the detection group 40.

[0083] Such configuration of the apparatus 200 allows to obtain optics, and then images, with higher quality with respect to the embodiment of Figure 1 by leaving unchanged the fluidic control performances.

[0084] The movable element 3b of the optical unit 30 substantially allows " to scan” the image associated to the (Large Field) detection area of the sample 1 on the camera of the detection group 40. A scan of some micro-radians allows to perform a shifting of the image on the plane P of the camera by 5.48 micron (equivalent to two pixels) along a first axis x and by the same distance along a second orthogonal axis y.

[0085] By additionally referring to Figure 3, in a first embodiment variant, such scan is “fast” and comprises sixteen motions of the movable element and, then, corresponding sampling positions obtainable in about one second (Figure 3a). In a second variant, such scan is slower and comprises thirty-six motions and, then, corresponding sampling positions obtainable in about two seconds (Figure 3b). In particular, the “fast” scan comprises four motions per axis (x, y) to form a squared grid having width of 5.48 x 5.48 micron. The slower scan is similar to the fast scan, but the sampling positions result to be closer, the scanned area surface being equal.

[0086] The plurality of sampling positions is associated with a respective plurality of images of the detection area of the sample 1 .

[0087] The apparatus 200 of the invention and, in particular the detection group 40, advantageously then comprises a control unit 60 carried by the frame of the apparatus 200 and configured to obtain a final image of the sample 1 starting from said plurality of images.

[0088] The control unit 60, however, is present in all embodiments of the illustrated examples and it is operatively connected to the microfluidic control means 50 to control the operation of the latter based upon the type of performed experiment or analysis, or to adjust in use the various operating parameters inside the boxlike body 10 containing the sample 1 under examination.

[0089] To this purpose the control unit 60 is preferably connected to a terminal 61 and to a control interface 62 carried by the frame of the apparatus 100, 200. Such interface 62 preferably comprises a display, for example touchscreen, which can be fixed or removable (connectable with cable or wi-fi) and it allows to monitor the activity of the apparatus and the control on the micro-fluidic control means 50.

[0090] The above-mentioned plurality of collected images is processed by an algorithm implemented by the control unit 60 to implement one single final image having a higher sampling frequency and a higher resolution and optical contrast (with respect to the image obtained with the apparatus 100 of the configuration of Figure 1 ). Preferably, the processing algorithm is implemented according to one or more of the techniques known as “Super-Resolution by subpixel shifted pictures" from publications

[0011] ,

[0012] ,

[0013] and integrally incorporated in the present description.

[0091] According to this embodiment of the invention, the apparatus 200 results to be provided with an optical unit having native Large Field higher than 1 .0 cm2and a “super resolution” system capable of improving the native optical resolution of the lens by a factor = and to increase significantly the sampling frequency. 2

[0092] In particular, the image of the sample 1 obtainable with such configuration can be characterized by a real optical resolution equal to or higher than 2.4 micron, a real size of the pixel equal to 0.658 micron and a surface area equal to at least 320 Megapixel (or 16,000x16,000 pixels).

[0093] Such configuration of the apparatus 200 and the described optical qualities allow to overcome the known problems associated to the stitching technique which has disadvantages especially with dynamic biological samples since it requires expensive engines for moving and positioning the sample with resolution and nanometric repeatability.

[0094] Figure 4 shows an example of experimental characterization of the performances of the apparatus 200 according to the embodiment of Figure 2 wherein an image of a biological sample 1 , consisting of microparticles having diameter of 15 micron with annular shape, is acquired.

[0095] Whereas an imaging using the final image without processing (Figure 4a) or a processing of the final image through a simple average (Figure 4b) does not succeed in reproducing the annular shape of the sample, that is of two rings placed side by side, the processing of the plurality of images through a super resolution algorithm succeeds in returning a final image of the sample (Figure 4c) showing clearly the two rings.

[0096] Figure 4d shows the comparison between the intensity profiles of the final image obtained through the average of the plurality of images and through the super resolution algorithm along the white line shown in Figure 4b.

[0097] The apparatus 100, 200 of the invention can optionally further integrate means for moving the box-like body 10. In this case, such means allows to shift the sample exclusively in a phase of the experiment in which the acquisition of one or more images related to the same detection area of the sample is absent. Said moving means advantageously allows to simplify the procedures for setting up the apparatus for subsequent measurements on the sample and to acquire an image corresponding, for example, to distinct detection areas of one same biological sample or distinct detection areas of distinct biological samples in case the box-like body 10 houses more than one of them.

[0098] It is highlighted that for implementing the means for moving the box-like body 10 a particular precision for the re-alignment of the sample 1 between a measurement and the subsequent one is not required, and said moving means does not have as objective the implementation of a stitching of the acquired images.

[0099] The implementation then can be made with standard precision and repeatability features. Then with reduced costs.

[0100] For example, the implementation can be of manual type and can allow to move the box-like body 10 in a plane. The maximum excursion of the box-like body 10 along mutually perpendicular axes contained in said plane can be equal to about 10 mm for a manual motion along a first axis and about 45mm along a second axis.

[0101] The present invention has been described sofar with reference to preferred embodiments. It is to be meant that other embodiments belonging to the same inventive core may exist, as defined by the protective field of the herebelow reported claims.

[0102] BIBLIOGRAPHY

[0103]

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Claims

CLAIMS1. A wide-field microscopy apparatus (100; 200) for the analysis of biological samples, which apparatus comprises a frame carrying:■ a box-like body (10) configured to contain a biological sample (1 ),■ an illumination unit (20) of the sample with an incident signal (2),■ an optical unit (30) for receiving a signal transmitted through a detection area of said sample (1 ),■ a detection group (40) configured to acquire this transmitted signal and generate an image of the sample (1 ) corresponding to said detection area, wherein said frame further carries control means (50) of a micro-fluidic flow entering and / or exiting said box-like body (10), wherein said box-like body (10) is motionless during the acquisition of the transmitted signal and said detection area is equal to or greater than 1 .0 cm2.

2. The apparatus (100) according to the previous claim, wherein said optical unit (30) includes a relay lens (3a).

3. The apparatus (100) according to the previous claim, wherein said image is characterized by an effective optical resolution equal to or greater than 3.4 micron, an effective pixel size equal to 2.740 micron, a surface area equal to 24 Megapixel.

4. The apparatus (200) according to claim 1 , wherein said optical unit (30) includes a movable element (3b) configured to steer said transmitted signal in a plurality of sampling positions on the detection group (40).

5. The apparatus (200) according to the previous claim, wherein said plurality of sampling positions is associated with a respective plurality of images of the sample (1 ) and wherein the detection group (40) further comprises a controlunit (60) configured to obtain a final image of the sample (1 ) starting from said plurality of images.

6. The apparatus (200) according to the previous claim, wherein said final image is characterized by an effective optical resolution equal to or less than 2.4 micron, an effective pixel size equal to or less than 0.658 micron, a surface area equal to or greater than 320 Megapixel.

7. The apparatus (100; 200) according to any one of the previous claims, wherein said box-like body (10) comprises a controlled atmosphere chamber (11 ) configured to house a micro-fluidic chip.

8. The apparatus (100; 200) according to the previous claim, wherein said chamber (11 ) houses sensor means, preferably a temperature sensor and / or a CO2 sensor.

9. The apparatus (100; 200) according to any one of the previous claims, wherein said box-like body (10) and / or said control means (50) comprise a respective cover, preferably removable, not transparent to light.

10. The apparatus (100; 200) according to any one of the previous claims, further comprising means for moving the box-like body (10) to acquire an image corresponding to distinct detection areas of the same biological sample or detection areas of distinct biological samples.

11. The apparatus (100; 200) according to any one of the previous claims, wherein said means (50) for controlling a micro-fluidic flow comprises independent infusion and / or suction channels, preferably of the type comprising syringe pumps.

12. The apparatus (100; 200) according to any one of the previous claims being of the fluorescence type.

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