WO2008132325A2

WO2008132325A2 - Fluorescence reading device

Info

Publication number: WO2008132325A2
Application number: PCT/FR2008/000306
Authority: WO
Inventors: Claude Weisbuch; Henri Benisty; Lucio Martinelli; Maxime Rattier; Georges-Olivier Reymond
Original assignee: Genewave
Priority date: 2007-03-09
Filing date: 2008-03-07
Publication date: 2008-11-06
Also published as: WO2008132325A3; FR2913499A1

Abstract

The invention relates to a device for reading the fluorescence emitted by chromophorous members associated with biological or chemical components at the surface of an object (10), that comprises means (12) for lighting the chromophorous members with an excitation light supplied by a laser source (16) and flowing into a wave guide formed by a spoke (18) of a transparent material, and means (14) for collecting and picking up the fluorescence from the chromophorous members, said means (14) including a large field optical system (34).

Description

Fluorescence reading device

The invention relates to a device for reading the fluorescence emitted by chromophoric elements associated with biological or chemical components, for the identification of these components and the measurement of the present amounts of these components.

DNA or protein biochips and microscope slides having a suitable surface coating are known examples of carriers on which biological components are immobilized after hybridization and are detectable by markers associated with them and fluoresce in the medium. a narrow band of wavelengths in response to light excitation in another narrow band of wavelengths. Other examples of sensors using the principle of fluorescence are the sensors sensitive to glucose and pH.

The collection and measurement of the fluorescence emitted by the markers makes it possible to detect and to count the components associated with these markers.

It has been proposed to use for this detection a scanning confocal microscope, the drawbacks of which are well known: slow measurement, need to move the biochip or the microscope in two dimensions quickly and accurately, which requires heavy robotization, bulky and expensive, etc.

Some disadvantages of this technique could be avoided by using a large-field optical system for the collection of fluorescence, but there is the problem of a sufficiently efficient illumination of the observed surface, which can not be solved by the use a conventional source of white light, whose brightness is too low in the excitation wavelength band of the chromophores, which forces to illuminate only very small surfaces, less than 1 cm ² , or to lengthen excessively the exposure time (more than 30 minutes), and which can also be solved by the use of a conventional laser beam, because of its temporal coherence and its section generally circular or oval, which respectively produce scab ("speckle") to the crossing of the optics used and a non-uniformity of the lighting, which forces to retain only the center of the task of lighting, 90% of the excitation energy being lost.

The subject of the present invention is a new technique for acquiring fluorescence emitted by components of the aforementioned type, enabling a fast and accurate identification and counting of fluorescent markers present in a given area, by virtue of the combination of efficient lighting. and free from scab ("speckle") and wide-field imaging.

To this end, it proposes a device for reading the fluorescence emitted by chromophoric elements associated with biological or chemical components on the surface of an object, comprising means for illuminating the chromophore elements with an excitation light and means for collecting and capturing a fluorescence emitted by the chromophore elements in response to their light excitation, characterized in that the illumination means comprise at least one waveguide interposed between a source of excitation light and the chromophore elements and formed by a bar of material transparent to the excitation light, this waveguide having an output section adapted to the shape and dimensions of the image of the capture means formed on the surface of the object by the collection means. The device according to the invention makes it possible to use an intense light source, such as for example a laser diode, which is spectrally thin and compact and whose light beam shaped by the bar of transparent material provides uniform illumination of the section. for example rectangular or square, adapted to the form of a matrix sensor type CCD or CMOS for example part of the fluorescence capture means. This lighting device is advantageously combined with fluorescence collection and capture means comprising a large-field optical system, that is to say having a field of view greater than 1 ° in at least one direction of a matrix sensor on which is formed the image of the observed area.

This technique allows rapid acquisition of fluorescence information, the dimensions of the area observed being for example of the order of 15x15mm ² or 15x25mm ² depending on the embodiments, with a resolution of the order of 10 .mu.m and a limit of detection of 1 to 10 chromophores / μm ² according to the embodiments.

The lighting device according to the invention has other advantageous characteristics among which:

the faces of the aforementioned bar are polished to reduce the losses of optical power, and in particular its exit face to obtain a perfectly uniform and faultless illumination pattern,

the light source is connected by an optical fiber to one end of the bar, which makes it possible to deport the light source in order to reduce the space requirement and promote the evacuation of heat, and also makes it possible to multiplex the lighting means, either by connecting the light source to several bars in parallel, or by connecting several light sources in parallel to the same bar,

the light source comprises a laser or a laser diode with a high temporal coherence and the optical fiber connecting this source to the bar is associated with a vibrating means which has a temporal decoherence effect, by averaging in the time and space of the random speckle ("speckle") generated by the laser beam,

the optical fiber is a multimode fiber, which promotes temporal decoherence of the light emitted by the laser source,

the illumination axis and the optical axis of the fluorescence collection means form between them an angle of between 0 and 180 °. When this angle is different from 0 ° and 180 °, the illuminated surface of the object has a shape different from the shape of the output section of the waveguide (the illuminated surface is for example trapezoidal while the output section has a square shape). To limit this phenomenon, the output end of the waveguide can be cut so that its output face extends substantially parallel to the illuminated surface of the object. It is also possible to anticipate this phenomenon in order to cancel it, for example by choosing a waveguide with a trapezoidal exit section while the area observed has a rectangular or square shape. The angle between the optical axis and the illumination axis is for example about 30 to 50 °, which allows illumination of the object in "dark field", the excitation light of the chromophores not penetrating not directly in the fluorescence collection means. This avoids the use of a filter element such as a dichroic plate, the optical system is more compact and the stresses of rejection of the excitation light by the filters traversed by the fluorescence collected are much lower.

Advantageously, the area of the object covered by the lighting is exactly adapted to that imaged on the fluorescence acquisition sensor, which allows maximum efficiency of the lighting.

Indeed, when the illuminated area is too large, a certain amount of light is lost outside the image area, which is lit less effectively. Conversely, if the illuminated area is too small, only part of the image area is illuminated and the optical collection means are unnecessarily oversized.

When the area illuminated and imaged is smaller than the usable area of the object under study, it is possible to provide means for moving the object with respect to the illumination and fluorescence collection axes, and / or to provide means for for example a moving mirror, scanning the surface of the object by the illumination light. It is also possible to provide several lighting means independent of each other, these means each illuminating a surface or area of interest of the object. These areas of interest may be distinct or may overlap or overlap at least partially, the device further comprising means for collecting and capturing the fluorescence emitted by the chromophore elements of each of the areas of interest of the object.

In a particular embodiment of the invention, the image-forming optical system of the collection means comprises two photographic objectives mounted head-to-tail on a module movable along the optical collection axis. These two objectives, when they are identical, provide a magnification equal to 1 of the image of the illuminated zone formed on the fluorescence acquisition sensor, the resolution of the imaging device then being equal to the size of the pixels of the sensor. for example 9 μm.

One of the objectives is placed directly above the studied object and the module carrying the objectives includes a detection camera and a filter for stopping the excitation light, this module being precisely guided in translation along a vertical axis which is the common optical axis of the two objectives.

In an application of the invention to the detection and identification of biological agents, the studied object is a biochip housed in a hybridization chamber of a cartridge placed on a fixed support which is equipped with connecting the cartridge to a fluid circuit and an electronic module that provides general power to the device, control of the device and communications with the fluorescence acquisition camera.

The device then constitutes an integrated system capable of hybridizing nucleic acids injected into the hybridization chamber of the cartridge, reading the fluorescence emitted, as well as analyzing the images acquired by virtue of its connection with a system. information processing system such as a portable microcomputer equipped with appropriate image analysis software. In another application of the invention, the studied object is a glass support such as a microscope slide of an appropriate type commercially available, on which are fixed DNA strands detectable by fluorescent markers. This blade has a useful surface of 23x73mm ² for example and is carried by a carriage movable in translation by a stepper motor. The area illuminated on the blade has for example dimensions of 15x25mm ² . The blade is movable under the collection and fluorescence capture means in five different positions spaced 15mm, for the acquisition of a complete image of this blade in a few minutes.

The lighting device may comprise, in this application, two light sources emitting for example at 645 nm and 532 nm respectively, which are each connected by an optical fiber to a bar of transparent material of the aforementioned type.

In all cases, the device according to the invention is completely covered, to ensure the complete black inside during fluorescence readings.

The invention will be better understood and other characteristics, details and advantages thereof will appear more clearly on reading the description which follows, given by way of nonlimiting example and with reference to the accompanying drawings, in which: Figure 1 is a schematic perspective view of a first embodiment of a device according to the invention;

- Figures 2 to 8 are schematic perspective views of other embodiments of the invention;

FIGS. 9 to 11 show schematically an integrated hybridization and fluorescence readout device;

- Figure 12 schematically shows the essential components of a fluorescence reading device according to the invention;

FIG. 13 is a graph showing the evolution as a function of time of the fluorescence obtained by successive hybridizations, on a single support, of several pairs of oligonucleotides with different biological targets, this support being washed after each hybridization.

The device shown in FIG. 1 is intended to collect and capture the fluorescence emitted in response to light excitation by chromophore elements located on a surface S of an object 10.

It comprises means 12 for illuminating the surface S by an excitation light of the chromophore elements and means 14 for collecting and capturing the fluorescence emitted by the chromophore elements in response to the light excitation. The lighting means 12 comprise a light source 16 emitting light in a narrow band of wavelengths, such as for example a high-light emitting diode (LED), emitting over a spectral width of about 20 nm, that can be coupled directly to the rear end or input of a bar 18 of transparent material at these wavelengths, or a laser diode that can be coupled to the rear end of the bar 18 by an optical fiber 20 comprising loops 22, this optical fiber advantageously being a multimode fiber associated with a vibrating device 24 such as a mobile phone vibrator for example, capable of causing a displacement of the optical fiber at a low frequency (typically comprised between 10Hz and 1kHz) with an amplitude of a few tenths of a millimeter or less.

The bar 18 of transparent material is section adapted to the shape of the surface S to be illuminated, this section may be rectangular, square or trapezoidal for example.

To reduce the optical power losses, the bar 18 is polished on all sides and in particular on its outlet face 26 to obtain a flawless and perfectly uniform illumination pattern.

Alternatively, it could be applied to the output face 26 of the bar treatment ensuring low light scattering (with a angle diffusion indicator below the beam opening) to perfect uniformity of illumination.

The lighting means may also comprise a lens 28 for forming an image of the exit face 26 of the bar on the object 10, to limit the space requirement in the vicinity of the object and to enlarge or reduce the illuminated area 30 on the object. This lens 28 may be slightly defocused so as to smooth the illumination pattern and eliminate its high spatial frequency fluctuations.

The means 14 for collecting and capturing the fluorescence essentially comprise a matrix sensor camera 32 of the CCD, CMOS or photodiode type, a large-field optical system 34 and a fluorescence filter 36 stopping the excitation light.

The surface S corresponds to the image of the sensor 32 in the object space and defines the field of the optical system. This system is wide field when the angle α that the main ray 38 (passing through the center of the entrance pupil 40 and the end of the image of the sensor 32 on the object 10) with the optical axis 42 of the system, is greater than one degree according to at least one direction of the image of the sensor 32.

The fluorescence filter 36 which stops the excitation light, can indifferently be located in front of or behind the optical system 34, inside this system as shown in FIG. 1, or directly on the matrix sensor 32.

The device of FIG. 1 operates as follows:

the light emitted by the source 16 is guided by the bar 18 according to the laws of geometrical optics, the multiple reflections on each of the faces of the bar creating as many multiple images of the light source formed by the end of the fiber 20 at the front end of the bar 18, these images being superimposed on the output face 26 of the bar. This results in a spatial averaging which uniformizes the lighting which is also adapted to the shape of the section of the bar, which makes it possible to have an illumination figure of corresponding shape, for example square or rectangular and therefore well adapted to the shape of the matrix sensor 32.

When the source 16 is a laser diode, the emitted light has a high temporal coherence, this coherence being at the origin of random scabs ("speckle") harmful to the uniformity of the illumination.

When the multimode optical fiber 20 is vibrated by the vibrating device 24, this vibration causes scabs to move and allows them to be averaged over time and space, this averaging effect being all the more important as the fiber is very multimode. In the best case, the illuminated area 30 on the object 10 corresponds to the surface S which is the image of the matrix sensor 32 on this object. There is then a lighting of maximum efficiency for the optical system 34 used.

Typically, the surface S can be 15x25mm ² . Alternatively, the device may comprise a waveguide formed solely by an optical fiber that is disposed between the light source and the object or lens 28, and whose output section is adapted to the shape of the surface. S of the object.

In the device of FIG. 1, the illumination is carried out in "dark field", that is to say that the excitation light does not penetrate directly into the optical system for collecting the fluorescence emitted, the axis optical lighting means 12 being for this inclined with respect to the optical axis 42 of the collection means 14.

In the variant of Figure 2, the lighting is also performed in "dark field" although the illumination axis coincides with the axis 42 of the optical fluorescence collection system. The lighting means 12 and the collecting means are on either side of the object 10 and, in this case, it is the object 10 itself which, by its structure, realizes the separation between the fluorescence emitted by the chromophore elements present in the surface S and the excitation light. For this, the object 10 may comprise a set of micro-holes in which the fluorescence phenomenon is concentrated.

In the variant of Figure 3, the lighting is always made in "dark field". The optical axis of the lighting means 12 is inclined with respect to the optical axis 42 of the collecting means 14, and the front end or exit end of the bar 18 is cut so that its exit face 26 is parallel to the illuminated surface 30 of the object. This illuminated surface then has a shape (square, rectangular) identical to that of the exit section of the bar 18. The optical axis of the lighting means is in this case broken to take into account the refraction of the light at the level of the exit face 26 of the bar. The aforementioned lens 28 may advantageously be replaced by an off axis mirror 29.

In the variant of FIG. 4, the lighting is made in "light field", which requires the installation of an additional filter element, such as a dichroic plate 44, in the optical system 34 for collecting fluorescence. , this dichroic plate being inclined at 45 °, for example, on the optical axis 42 and the illumination axis being perpendicular to this optical axis 42.

The connection of the light source 16 to the bar 18 by an optical fiber 20 also makes it possible to multiplex the lighting means, for example by connecting the same source 16 to several bars 18 in parallel as shown in FIG. 5 or by connecting the same bar 18 to several sources 16 in parallel as shown in Figure 6, these different sources 16 emitting at different wavelengths. When the image area S is greater than the surface illuminated on the object 10 by the illumination means 12, as shown in FIG. 7, the illumination beam can be made mobile, for example by means of a mirror mobile 46 installed between the lens 28 and the object 10 and which sweeps the entire surface S, the mirror 46 being pivotally mounted about two perpendicular axes or around a center of rotation. When the image area S is greater than the surface 30, it is also possible to use several light beams, these beams being obtained by several lighting means 12 'independent of each other, as can be seen in FIG. 8. The surface illuminated on the object 10 by each illumination means 12 'represents an area of interest Zi of the object, these areas of interest Zi being distinct and all of which can be imaged within the same area. field. The lighting means 12 'are adapted to the size of the corresponding zone Zi. This makes it possible to maximize the useful luminous energy by illuminating only the areas of interest Zi of the object, and also to introduce the concept of spatial multiplexing, each zone Zi being able to be addressed individually.

In another variant shown in part of FIG. 8, the zones of interest Zi "overlap at least partly with each other (and may possibly be combined), these zones of interest Zi" being each illuminated by means of FIG. 12 "independent lighting and each having clean fluorescent markers different markers of other areas of interest Zi". The excitation wavelength of each of the lighting means is then adapted to each marker for spectral multiplexing. These zones Zi "are also all imaged inside the same field.

An application of the invention to the detection and identification of biological agents is illustrated in Figures 9-11.

In this application, the object 10 is a cartridge comprising a hybridization chamber in which is housed a DNA microarray comprising probes that correspond to one or biological agents sought. This cartridge 10 is placed in a housing formed in a fixed support 50 of the device according to the invention. This device is subdivided into three modules M1, M2, M3, the first module M1 being fixed and comprising the aforementioned fixed support 50, the module M2 being fixed and comprising a fluidic block and an electronic block, the module M3 being mobile and placed at above the M1 module and comprising two objectives 52, 54 which are identical and which are oriented head to tail along a vertical axis, the lower lens 52 being located directly above the DNA chip housed in the cartridge 10. The module M3 further comprises a camera 56 comprising the matrix sensor 32 described with reference to FIG. 1 and placed above the upper objective lens 54.

The fluidic unit of the module M2 comprises a pump, solenoid valves and fluid circuits connected to the hybridization chamber of the cartridge 10 and to bottles 58 containing the liquids of the reagents necessary for the hybridization of the nucleic acids sought on the probes. of the DNA chip.

The mobile module M3 comprises a filter-carrying plate 58 receiving the fluorescence filter 36 described with reference to FIG. 1, and on which the lower end of the upper objective 54 and the upper end of the lower objective are fixed. 52, this plate being itself carried at its ends by two scissors supports 60 which are oriented perpendicularly to one another and which make it possible to constrain the displacement of the module M3 along a well defined vertical axis, which is the common axis of objectives 52 and 54.

The fixed module M1 further comprises, in addition to the components already mentioned, an optical alignment device which ensures the parallelism of the image plane and the plane of the matrix sensor of the camera 56, heating and temperature control means of the chamber of hybridization, as well as the illumination means 12 described with reference to Figure 1, and stirring means comprising an ultrasound generator for promoting mixing in the hybridization chamber. This ultrasound generator also promotes hybridization and / or washing inside the cartridge. The module M1 may further comprise optical enhancement means excitation and / or fluorescence.

In addition, the two modules M1 and M3 are hoods, to ensure total darkness in the closed position for the fluorescence measurement. The camera 56 is also connected to an information processing system such as a laptop, equipped with an image analysis software which makes it possible to identify the nucleic acids present and to estimate their quantities. In this device, the two photographic objectives 52 and 54 aligned head-to-tail are identical, so that the magnification of the optical system is equal to 1, its resolution being equal to the size of the pixels of the camera 56, ie 9 μm in an example of realization.

The excitation of the fluorescent markers of the probes of the DNA chip is carried out around 640 nm, the observed area being 15x15 mm ² . The bar 18 of the lighting means 12 is made of PMMA (polymethyl methacrylate) and has dimensions of the order of 20x2.5x2mm ³ in an exemplary embodiment. The laser diode used emits a power of 4OmW, which allows illumination of the DNA chip of about 10mW / cm ² .

The image acquisition speed of the observed area is three images per minute.

Another application of the invention has been illustrated schematically in FIG. 12. In this application, the support of the chromophore elements is a microscope slide 62 of a commercially available type, which has been specially prepared for the hybridization of strands. of nucleic acids on its upper surface. This blade 62, whose dimensions are for example 26x76.5mm ² with a usable area of 23x73mm ² , is placed on a carriage 64 movable in horizontal translation by a stepper motor M under all of the two objectives 52 and 54 above, between which is mounted a plate 66 filter holder. The camera 56 placed above the lenses 52, 54 is connected to a portable computer68.

Two lighting means 12 of the type already described are provided to illuminate the blade 62 at wavelengths of 645 nm and 532 nm respectively. The illuminated surface of the blade 62 has dimensions of 16x25mm ² so that five translations of the blade 62 under all the objectives 52 and 54 are sufficient to obtain a complete image of the useful surface of the blade. The acquisition speed is for example a complete image of the blade in three minutes.

In all embodiments of the invention, the angle of incidence of the illumination axis on the surface of the object 10 can be determined to produce constructive interference on the surface of the object; comprising a reflective layer. The device according to the invention makes it possible to carry out the hybridization, washing and melting procedures on one and the same support, and to identify and quantify in real time the biological or chemical components present on this support via aforementioned means for processing the information. This device also makes it possible to carry out conventional hybridizations in relatively short times, to carry out successive hybridizations on a support on which is fixed a matrix of biological probes (such as nucleic acids) organized into spots for monitoring or monitoring. samples without altering the intensity of the signals or the reproducibility of the results, in order to significantly reduce the cost of this monitoring. It also makes it possible to evaluate the hybridization behavior of the targets on the biological probes fixed on the support and thus to study the kinetics of hybridization.

The device according to the invention has many other advantages:

- time saving. Usually, hybridizations of nucleic acids on solid surfaces are performed "blind" for several hours (usually 16 to 20 hours). Thanks to the real-time measurement, the device allows the user to stop the hybridization reaction at any time when he considers that the intensity of hybridization is sufficiently high for its application, resulting in a gain of time important. For example, a standard hybridization of products derived from gene amplification or PCR (polymerase chain reaction) on slides comprising a matrix of oligonucleotides organized in spots can be carried out in one hour only instead of one night as is the case. case in the prior art. In addition, since the reading is concomitant with the hybridization, the results are obtained immediately.

an evaluation of the hybridization efficiency of the targets on the biological probes. The signal intensities are generally very variable for probes with similar length and percentage of guanine (G) and cytosine (C), indicating that the signal intensity generated by hybridization does not depend solely on the length of the signal. and the base composition of the probe, but also its melting temperature (Tm) which depends on the nucleic sequence of this probe. The development of matrix of biological probes organized into spots and containing a very large number of probes results in significant differences in the melting temperature (Tm) of the different probes present in the matrix, and gives rise to very large differences in the target hybridization efficiencies on the probes. This is very critical for both the sensitivity and the specificity of the chips. By visualizing the hybridization properties of each target on each immobilized probe in the form of spots on a slide, the device according to the invention makes it possible to quickly validate the design of the probes for a given experiment and to quickly determine the optimal hybridization temperature. for the complete probe matrix, so that each hybridization (target on probe) gives rise to the best compromise between intensity and specificity of the signal.

an analysis of the fusion profile and the kinetics of hybridization on a solid support. There are significant differences in the hybridization kinetics of a given sequence in solution and the same sequence immobilized on a solid support. For example, it has been found that this sequence does not have the same influence on the hybridization reaction when this reaction takes place on a solid support and when it takes place in solution. Data analysis from commercial biological probe matrices also suggested that hybridization on a solid support is thermodynamically disadvantaged compared to hybridization of the same sequence in solution. Thanks to its precise temperature control system, the device according to the invention makes it possible to measure the melting temperature (Tm) of probes immobilized on a solid support, as well as their kinetics of hybridization.

- a reduction in the cost of analysis. With a suitable procedure for dehybridizing the target samples of their specific probes, the same slide can be successively hybridized to a large number of distinct samples. FIG. 13 illustrates an example of successive hybridizations, on a single and same slide, of several pairs of oligonucleotides (probes) with different targets, this slide being washed after each hybridization.

In this example, eleven pairs of oligonucleotides each having between 19 and 24 nucleotides are immobilized on an AmpliSlide ™ slide. Each pair is present in twelve copies. The two oligonucleotides of each pair differ from each other by a single nucleotide. A positive control formed by Cy5-labeled Gaba oligonucleotides is also immobilized on the slide in twenty copies.

The device according to the invention makes it possible to measure the value of the parameter FB which is equal to the fluorescence (F) emitted by the marker minus the background noise (B). The hybridization is carried out with eleven oligonucleotide targets labeled with Cy5, each of these targets comprising a sequence complementary to one of the two oligonucleotides of each aforementioned pair. The first of these targets is deposited on the slide and will hybridize to an oligonucleotide of a first pair. The hybridization causes a fluorescence emission which is represented in FIG. 13 by a first curve 70. This curve presents a first part (t = 1 to 41 min) during which the fluorescence gradually increases, which means that the hybridization takes place, and a second part (t = 41 to 61 min) during which the fluorescence drops very sharply which corresponds to the wash the blade. The other targets are then deposited one after the other on the slide to hybridize to an oligonucleotide of each pair, and the slide is washed after each of these hybridizations, giving the curves 72 to 90. The last two curves 92 and 94 correspond to two successive deposits of the mixture of eleven targets on the slide to cause simultaneous hybridization of the eleven targets with the corresponding oligonucleotides. Curve 96 corresponds to the aforementioned positive control.

This technique makes it possible to reduce the cost of an experiment by significantly reducing the number of slides required, and on the other hand, to significantly accelerate the hybridization cycles by avoiding the production of new slides and the necessary treatments. for the preparation of these blades, which allows continuous sample tracking with very close readings.

Claims

1. A device for reading the fluorescence emitted by chromophoric elements associated with biological or chemical components on the surface of an object, comprising means (12) for illuminating the chromophore elements by excitation light and means ( 14) for collecting and capturing a fluorescence emitted by the chromophore elements in response to their light excitation, characterized in that the illumination means (12) comprise at least one waveguide interposed between a source (16) excitation light and the chromophore elements and formed by a bar (18) of material transparent to the excitation light, this waveguide having an output section adapted to the shape and dimensions of the image of the means capture (32) on the surface of the illuminated object (10).

2. Device according to claim 1, characterized in that the means (14) for collecting and capturing the fluorescence comprise a large field optical system (34).

3. Device according to claim 1 or 2, characterized in that the capturing means comprise a matrix sensor camera of the CCD or CMOS type or photodiode array.

4. Device according to one of the preceding claims, characterized in that the faces of the bar (18) of the lighting means are polished.

5. Device according to one of the preceding claims, characterized in that the bar (18) is rectangular, square or trapezoidal section.

6. Device according to one of the preceding claims, characterized in that the bar (18) is made of glass or plastic.

7. Device according to one of the preceding claims, characterized in that the light source (16) is connected by a multimode optical fiber (20) at one end of the bar (18).

8. Device according to one of the preceding claims, characterized in that the light source (16) comprises a light emitting diode or a laser, such as a laser diode.

9. Device according to claim 7 or 8, characterized in that the optical fiber (20) connecting the light source (16) to the bar (18) is associated with a vibrating means (24) for the decoherence of the light. excitation.

10. Device according to one of the preceding claims, characterized in that an imaging lens (28) is mounted between the bar (18) and the surface of the object (10).

11. Device according to one of the preceding claims, characterized in that the illumination axis and the axis (42) of the collection and fluorescence capture means between them an angle between 0 and 180 °.

12. Device according to one of claims 1 to 10, characterized in that the axis of the illumination means (12) and the axis (42) of the means (14) for collecting and capturing fluorescence are merged.

13. Device according to one of claims 1 to 12, characterized in that the output face (26) of the waveguide extends substantially parallel to the surface of the object (10) illuminated.

14. Device according to one of claims 1 to 13, characterized in that it comprises means (M) of stepwise displacement of the object relative to the optical axes of illumination and collection.

15. Device according to one of the preceding claims, characterized in that the lighting means (12) comprise means, for example mobile mirror, scanning the surface of the object (10) carrying the chromophore elements.

16. Device according to one of the preceding claims, characterized in that it comprises a plurality of lighting means (12 ') independent of each other, these means illuminating surfaces or separate areas (Zi) of the object (10). ), the device further comprising means (14) for collecting and capturing the fluorescence emitted by the chromophore elements of each of the zones of the object.

17. Device according to one of the preceding claims, characterized in that it comprises a plurality of lighting means (12 ") independent of each other, these means illuminating surfaces or zones (Zi") of the object (10). ) which overlap at least in part, the device further comprising means (14) for collecting and capturing the fluorescence emitted by the chromophore elements of each of the zones of the object.

18. Device according to one of the preceding claims, characterized in that the means (14) for collecting the fluorescence comprise two photographic objectives (52, 54) mounted head-to-tail on a module (M3) movable along the optical axis.

19. Device according to claim 18, characterized in that one of the objectives (52) is placed above the surface of the object (10) bearing the chromophore elements, and the mobile module (M3) comprises a camera ( 56) and a filter for stopping the excitation light.

20. Device according to claim 18 or 19, characterized in that the object carrying the chromophore elements is a biochip housed in a hybridization chamber of a cartridge placed on a fixed support (50) equipped with connection means from the hybridization chamber of the cartridge to a fluid circuit.

21. Device according to claim 20, characterized in that the cartridge comprises optical enhancement means excitation and / or fluorescence.

22. Device according to claim 20 or 21, characterized in that the fixed support comprises stirring means promoting mixing of the contents of the hybridization chamber.

23. Device according to claim 22, characterized in that the stirring means comprise an ultrasonic generator.

24. Device according to one of claims 20 to 23, characterized in that the fixed support carries means (60) for guiding the module (113) carrying the objectives along a vertical axis and support means means illuminator (12), the light guide bar (18) of which is oriented obliquely to the axis of the lenses (52, 54) of the collection means.

25. Device according to one of claims 20 to 24, characterized in that the fixed support comprises means for controlling and regulating the temperature in the hybridization chamber of the cartridge.