FR2852394A1 - System for fibre optic high-resolution imaging, especially confocal, fluorescence imaging, used especially in in-vivo imaging in-situ, uses several thousand optical fibres which collect return fluorescent signals - Google Patents

Abstract

The method consists in deflecting the excitation signal at a speed corresponding to acquisition of a number of images per second sufficient for real time use and in detecting the fluorescence signal at a detecting frequency corresponding to a minimum frequency for sampling the fibers one by one. The method uses an image guide consisting of several thousands of optical fibers, an excitation signal being emitted by a source, deflected and injected by turns into the optical fibers of the guide, each excitation point of the tissue in the fiber output emitting in return a fluorescence signal collected by the fiber, then detected and digitized to form an image element. The method also provides for the focussing of the beam in the fiber output to excite a subsurface plane to produce a confocal image. The method also provides for the production of a divergent beam in the fiber output capable of exciting a micro-volume of the tissue from the surface.

Description

- 1

  The present invention relates to a method and an apparatus for high resolution fibered fluorescence imaging.

  The fields of application of the invention are in particular the analysis of 5 biological tissues in vivo on humans or animals, external for example in the field of dermatology, or internal and accessible in particular by endoscopy, for example in the field of neurology or gynecology. The present invention also relates to the ex-vivo analysis of tissue samples from biopsy samples, and the in-vitro analysis of cultures in cell biology.

  According to a second object, the present invention relates to a high resolution fluorescence imaging method and apparatus suitable for in vivo analysis, in situ, in real time, ie capable of providing a sufficient number of images. per second without being dependent on the subject and practitioner shakes 1 5 to allow in particular a fairly rapid examination.

  The fluorescence observed can come from an exogenous compound (typically an injected marker) or endogenous (naturally present in the cell or transfected) from a biological tissue.

  The present invention relates more particularly to a fluorescence apparatus using a transport using flexible optical fibers of the excitation signals and of the fluorescence signals emitted, this allowing in particular to access the site to be observed, externally as an articulated or endoscopic control arm, in particular via the operating channel of an endoscope, by moving the excitation source away.

  In the prior art, fluorescence imaging devices are known intended for surface observation of a site and comprising an image guide comprising an optical fiber for illuminating the site. The end of the optical fiber is placed at a distance from the site to be observed and produces an excitation beam which is divergent to form a zone of excitation on the largest possible surface of the tissue. As a variant, it is known to use instead of the single optical fiber a bundle consisting of several flexible optical fibers, or even several thousand flexible optical fibers, which are excited simultaneously. The fluorescence signal is detected using a CCD video camera placed either at the proximal end of the optical fiber (on the side - 2 of the manipulator), or at the distal end of the optical fiber (on the side of the tissue observed).

  This apparatus provides a macroscopic video image of the surface of the tissue.

  More recently, fluorescent confocal fibration imaging devices have been developed, intended for the observation of a subsurface plan of a site, using an image guide made up of a bundle of thousands of flexible optical fibers. which is provided at its end with a focusing optical head, this time having to be placed in contact with the site.

  Each optical fiber is used in turn to convey both the excitation signal and in return to convey the fluorescence signal emitted, so as to form in a subsurface plane a point-to-point scan of the site to be observed. The apparatus further comprises, on the proximal side of the image guide, an excitation source, means for scanning the fibers in turn, means for detecting each fluorescence signal emitted and means for signal processing. to reconstruct a digitized image of the site from each image element carried by a fiber.

  The first type of device has a so-called macroscopic lateral resolution because in general of the order of 100 μm, making it possible to view only elements located on the surface, while the second type of device called confocal imaging device has a microscopic lateral resolution which can be of the order of 2 prm allowing the visualization of nuclei located in a plane at a depth of between 20 and 100 pm. In addition, a confocal imaging device of this type is advantageously characterized by a very good axial resolution, of the order of 10 μm, thanks to the focusing of the excitation beam 25 at the output of each fiber which makes it possible to select a section plane in the tissue, also thanks to spatial filtering of the fluorescence signal emitted using the same excitation optical fiber and possibly also thanks to a filtering hole placed in front of the detector.

  These two types of fluorescence imaging apparatus therefore make it possible to observe tissue in two different aspects either on the surface with macroscopic lateral resolution, or in a subsurface plane with very good lateral resolution on the microscopic scale imparted by the confocal character of the apparatus.

  The primary object of the present invention is to provide yet another type of fluorescence imaging method and apparatus.

  It proposes a high resolution fluorescence imaging method using an image guide made of several thousand optical fibers, an excitation signal being emitted by a continuous source, deflected and injected in turn into one of the fibers. optics of said image guide and a fluorescence signal emitted in reaction being collected by the same optical fiber, then detected and digitized to form an image element. The method according to the invention is characterized in that the end of the fibers is placed bare directly in contact with the surface of the tissue, each fiber being adapted to produce a divergent bundle making it possible to excite a micro-volume of tissue located from the surface to a maximum depth dependent in particular on the core diameter of said optical fiber.

  Unlike the state of the art, the method according to the invention does not envisage scanning a signal which is focused at the output of each fiber but scanning a signal which is divergent at the output of each fiber. This goes against what it was customary to do to obtain a fluorescence image of the interior of a tissue which is of the best possible quality, that is to say coming from a plane of chopped off. However, it turns out that, by virtue of the method of the invention, the non-focusing of the signal at the output of the fibers makes it possible to obtain images of a volume located just below the surface of the tissue which are usable and interesting from a point from a medical point of view. These images are not "confocal" because they do not come from a subsurface sectional plane scanned point to point, but images which one can however describe as "highly resolved" because coming from the scanning in turn of microvolumes located directly below the surface and spatial filtering of the fluorescence signal emitted by each microvolume by the same fiber as that used for the excitation.

  The main advantage arising from the present invention resides in the fact that for an endoscopic application, the diameter of the endoscopic probe can be very small depending solely on the diameter of the image guide and therefore on its number of optical fibers. By way of example, for an image guide of 6000 fibers generally have a diameter of 330 μm while current endoscopic probes provided with an optical head have a diameter of a few mm. We can then aim, thanks to the invention, to the fields of - 4 applications, such as for example the field of neurology, where the size of the endoscopic probe is a critical factor in overcoming the problems inherent in miniaturization of the head. focusing optics.

  According to the invention, the size of the excited microvolumes and therefore the depth of integration of the fluorescence signal (maximum depth below the surface), depend in particular on the core diameter of the fibers used, the inter-core distance, and their numerical aperture (ON) which will be preferred as large as possible to collect the maximum of photons. Preferably, a core diameter of between 1 and 4 μm is chosen, generally corresponding to an inter-core distance of between 2 and 8 μm, allowing an integration depth of between 5 and 25 μm. A signal processing performed on the fluorescence signal obtained for each microvolume makes it possible to reconstruct an image whose axial resolution varies from 5 μm to 25 μm and the lateral resolution from 2 to 8 μm. This allows visualization of nuclei located in the first two to three cell layers. The processing of the fluorescence signal to reconstruct the image can be carried out in the same way as that carried out from a signal coming from a confocal plane as explained in the

  detailed description given below.

  The present invention also provides an apparatus for carrying out the method mentioned above.

  It offers high resolution fluorescence imaging equipment comprising: - an image guide made up of thousands of optical fibers; a source emitting continuously at the excitation wavelength of at least one targeted fluorophore, - scanning and fiber after fiber injection means of the excitation beam produced by the source in an XY plane corresponding to the input section of the image guide; means for separating the excitation wavelength and the fluorescence wavelengths; - means for detecting the fluorescence signal; and - means for processing the detected signal allowing the reconstruction of an image; characterized in that: the end of each fiber is adapted to produce an excitation beam which is divergent and is intended to be placed directly bare in contact with the surface of the tissue to be observed.

  According to the second object of the invention, the method and the apparatus described above are more particularly suitable for in situ in vivo imaging in real time.

  Such a method is characterized in that the excitation signal is deflected at a speed corresponding to the acquisition of a number of frames per second sufficient for use in real time and in that the fluorescence signal at a detection frequency corresponding to a minimum fiber sampling frequency one by one.

  Respecting the sampling of the fibers (according to Shannon's criterion) makes it possible to obtain a point-to-point image corresponding well to each fiber. This allows not to lose information by sampling all of the fibers 1 5 one by one while respecting a minimum average number of images per second, namely in practice at least 12 images per second for a maximum mode of 640 x 640 pixels. The choice of the detection frequency (bandwidth of the detector) as a function of this minimum sampling then makes it possible for each fiber to detect the greatest possible number of fluorescence photons.

  Thus, according to a possible embodiment, using an image guide of approximately 30,000 flexible optical fibers, the sampling frequency and the bandwidth of the detection system (an avalanche photodiode or equivalent) are substantially fixed. at 1.5 MHz, corresponding to approximately 12 pixels 25 per fiber, thus making it possible to obtain a minimum of 12 images / s in maximum mode 640 × 640 pixels.

  In practice, according to an advantageous embodiment, allowing suitable rapid scanning of the fibers to obtain a real-time image, the deflection of the beam is adjusted by determining a rapid resonant frequency of a "line" resonant mirror and a frequency of slow resonance of a galvanometric "frame" mirror.

  According to the invention, the duration of detection of a stream coming from a fiber is limited in time and very short to respect the acquisition of an image in real time. According to the invention, there are therefore further provided optimization means - 6 for collecting and detecting the maximum flow coming from the sample during this limited detection period, in particular: - optical deflection means are used, injection, focusing and detection with a degree of achromaticity depending on the difference in wavelength between the excitation wavelength and the maximum fluorescence wavelength, as well as the width spectral of the fluorescence signal emitted; this advantageously makes it possible to optimize the fluorescence flux collected over the entire spectral band of fluorescence emitted; and - a detector having a quantum efficiency at the wavelengths of fluorescence to be detected of at least 50% is chosen.

  According to the invention, the image processing then carried out on the detected flux is also optimized, in order to obtain a very good quality image from this limited flux of detected photons. This optimization is carried out as follows.

  1 5 A series of steps is carried out prior to the acquisition of images in real time: - a step of detecting the location of each fiber of a chosen set of fibers intended to be used (ie the whole of the image guide or a selected subset); this step is to be carried out at least on each change of image guide; a step for calibrating the injection rate in each fiber, that is to say defining an injection rate specific to each fiber; and a step for detecting the background image (without sample).

  In operation, the optimization of the image processing comprises in particular the steps consisting, after digitization of the detected signal: - in defining the real flux collected by each fiber, that is to say coming only from the sample, after correction as a function of the own injection rate of the fiber and of the subtraction of the background image, so as to obtain a corrected signal; - Then to carry out a reconstruction of the image from this corrected signal, with the aim in particular of transforming an image having a mosaic of fibers into an image without apparent fibers.

  According to the invention, these last two steps must be possible in real time. With regard to signal correction, this can be done in real time thanks to processing adapted to the structure of the signal observed and an optimized algorithm. As regards the reconstruction of the image, this can be done by the choice of a number of operations per pixel achievable in real time making it possible to obtain the desired result in terms of image quality. Gaussian low-pass filtering represents a good compromise between the complexity of the processing, the quality of the result and the calculation time.

  To save time and increase the complexity of the processing corresponding to the reconstruction of the image, it is also possible to increase the processing capacity of the hardware, for example by using a specific processing card and / or a parallel architecture such as a multiprocessor.

  The present invention will be better understood and other advantages will appear in the light of the description which follows of embodiments, 1 description made with reference to the drawings in which: - Figure 1 is a schematic view of an apparatus according to the invention; and - Figure 2 is a detail view of the end of an optical fiber in an apparatus according to the invention; and According to the example chosen and shown in Figures 1 and 2, the apparatus 20 comprises: - a light source 1 - means 2 for shaping the excitation beam; - Wavelength separation means 3; - scanning means 4; - means 5 for beam injection; - an image guide 6 consisting of flexible optical fibers; - Means 8 for rejection of the excitation beam; - means 9 for focusing the fluorescence signal; means 10 for spatial filtering of the fluorescence signal; Means 11 for detecting the fluorescence signal; and means 12 for electronic and computer processing of the detected fluorescence signal and for display.

  These various elements are detailed below. - 8

  The light source 1 is a laser emitting at an excitation wavelength making it possible to excite a wide range of fluorophores, for example 488 nm. To optimize the injection into one of the fibers of the image guide 6, the excitation beam is circular in order to be able to inject a fiber of section 5 also circular and, to optimize the injection rate, the laser is preferably longitudinal singlemode to present the best possible wavefront for injection into a weakly multimode optical fiber. The laser emits continuously and steadily (lowest possible noise, <1%). The available output power is around 20mW. As examples, one can use a quantum well laser (VCSEL), a diode pumped solid laser, a laser diode or even a gas laser such as Argon.

  At the output of the source 1, the means 2 for shaping the excitation laser beam are placed. They consist of an afocal optical system of magnification different from 1, composed of two lenses L1 and L2 which make it possible to modify the diameter of the laser beam. The magnification is calculated so that the diameter of the beam is adapted to the means of injection 5 into a fiber.

  The reshaped excitation laser beam is then directed towards the means 3 provided for separating the excitation and fluorescence wavelengths. It is for example a dichroic filter having a transmission efficiency of 98 to 99% of the excitation wavelength and which therefore substantially reflects the other wavelengths. The fluorescence signal, taking the same optical path as the excitation signal on return, will thus be sent almost completely to the detection channel (8-11). The rejection means 8 placed on the detection channel serve to completely eliminate the 1 to 2 25% of parasitic reflections at the excitation wavelength 488 nm which pass to the detection channel (for example a rejection filter at 488 nm or a bandpass filter allowing for example only a transmission between 500 and 600 nm).

  The scanning means 4 then resume the excitation beam. According to the example chosen and represented in FIG. 1, these means comprise a mirror M1 resonating at 4 KHz serving to deflect the beam horizontally and therefore to produce the lines of the image, of a galvanometric mirror M2 at 15 Hz serving deflecting the beam vertically and therefore producing the frame of the image; and two afocal systems of unit magnification, AF1 located between the two mirrors and AF2 located after the mirror M2, these afocal systems being used to combine the planes of rotation of the two mirrors Ml and M2 with the injection plane in one fibers. According to the second object of the invention, the scanning speed is fixed to allow observation of the tissues in vivo in situ in real time. 5 For this, the scanning must be fast enough so that there are at least 12 images I s displayed on the screen for a display mode of 640 × 640 pixels corresponding to the slowest mode. For display modes with fewer pixels, the number of images acquired per second will thus always be greater than 12 images / s. As a variant, the scanning means can comprise in particular a rotary mirror, integrated components of the MEMs type (X and Y scanning mirrors), or an acousto-optical system.

  The excitation beam deflected at the output of the scanning means is directed towards the optical means 5 in order to be injected into one of the fibers of the image guide 6. These means 5 consist here of two optical assemblies El and 1i5 E2. The first optical assembly El makes it possible to partially correct the optical aberrations at the edge of the field of the scanning means 4, the injection being thus optimized over the entire optical field (at the center as well as at the edge). The second optical assembly E2 is intended to carry out the injection proper. Its focal length and its digital aperture have been chosen to optimize the rate of injection into the optical fibers of the guide 6. According to one embodiment making it possible to obtain the achromaticity criterion, the first set El consists of a doublet of lenses, and the second set E2 of two pairs of lenses followed by a lens located near the image guide. As a variant, this injection optic could consist of any other type of standard optics, such as for example two triplets, or lenses with an index gradient or else a microscope objective (however more expensive).

  The image guide 6 can be composed of approximately 5,000 to 100,000 flexible optical fibers, depending on the outside diameter of the guide that is desired, which itself depends on the intended application (endoscopic, field size 30 sought , etc.). They may, for example, be image guides marketed by the company FUJIKURA. The core diameter of the fibers is preferably between 1 and 4 μm. This leads to a beam divergence at the exit of the fiber having an angle of about 180 for a numerical aperture of 0.42 in water. A core diameter of 1 µm can be obtained by a method of stretching the end of the entire image guide. The end of the guide 6 is polished and does not include optical means. The purpose of polishing is to give the same surface condition to the face of the guide and therefore to the bare fibers in contact with the fabric so that, on the one hand, the background of the image obtained is as homogeneous as possible and, on the other hand, to eliminate the possible problems of adhesion of the fibers with the fabric which would risk damaging it. The polishing can be flat or rounded to match the shape of the fabric. The digital aperture of each optical fiber is preferably chosen to be as large as possible in order to collect the maximum of fluorescence photons, that is to say for example 10 0.42. The inter-core distance gives the lateral resolution of the image obtained (distance between two points of the image). The smaller this distance, the better the resolution, however it will be at the expense of the size of the field to be imaged. It is therefore necessary to find a good compromise between the number of guide fibers and the inter-core distance between the fibers to obtain good lateral resolution (between 2 and 8 μm) with an appropriate field size for observing the desired tissue elements. .

  Two examples of guides that may be suitable according to the invention are given below: Example 1 Example 2 Number of fibers 6,000 30,000 Image field diameter 300 iim 650 Flm Outside diameter of the guide 330 Uim 750 jlm Core diameter of a fiber 3 Ftm 1.9 pm Inter-core distance 4 jm 3.3 jlm Maximum signal integration depth from 15-20 im 10-15 jlm the surface (shown schematically by the plane P in Figure 1) or axial resolution Lateral resolution 4 Flm 3.3 am - 11 In operation, the distal end of the image guide is brought into contact with the sample 13, the end face of the fibers thus being directly in contact with the surface of the tissue. The latter is biological tissue or cell culture. The expression of fluorescence is carried out either by a fluorophore which is injected (systemic fluorescence), or by a fluorophore produced by the cell itself by modification of a gene (transgenic fluorescence). In these two cases, the fluorophore present in the excited micro-volume according to the invention re-emits photons over a spectral band more or less large which can range from ten nanometers to more than one hundred nanometers.

  On the detection channel, the fluorescence signal, at the output of the rejection filter 8, is then focused by the means 9, consisting for example of a detection lens, in a filtering hole of the spatial filtering means 10. The focal length of the detection lens is calculated so that the fluorescence signal 1 5 from a fiber is the size or slightly smaller than that of the filtering hole. The latter makes it possible to conserve the fluorescence light coming only from the fiber illuminated by the incident beam. It allows to reject the light which could have been coupled in the fibers adjacent to that which is illuminated. The size of the hole is calculated so that the image of a fiber fits perfectly into it. Furthermore, in order to optimize the quantity of photons passing through the filtering hole, and therefore the detected flux, the scanning means 4, the injection means 5 and the detection means 8, 9 and 10 are suitable. to the detected fluorophore: these means are chosen to be sufficiently achromatic to collect photons on the broadest emission band of the fluorophore.

  The detection means 11 have maximum sensitivity to the fluorescence wavelengths studied. One can use for example an avalanche photodiode (APD) or a photomultiplier. Furthermore, to obtain an image in real time, the bandwidth is preferably chosen to optimize the integration time of the fluorescence signal. It is 1.5 MHz, which corresponds to the minimum sampling frequency of the image guide with an optimized integration time on each pixel. - 12

  The electronic and computer means 12 for controlling, analyzing and digitally processing the detected signal and for displaying includes the following cards: - a synchronization card 20 which has the following functions: - to synchronously control the scanning, this is ie the movement of line mirrors Ml and frame M2; - to know at all times the position of the laser spot thus scanned; and to manage all the other cards by means of a microcontroller itself which can be controlled; a detector card 21 which includes an analog circuit which in particular performs an impedance matching, an analog digital converter then a programmable logic component (for example an FPGA circuit) which formats the signal; a digital acquisition card 22 which makes it possible to process a stream of digital data 15 at variable frequency and to display it on a screen 23; - a graphics card 24.

  Alternatively, one can use a single card combining the functionality of these different cards.

  Image processing is done as follows.

  When an image guide is placed in the apparatus, a first operation is carried out to recognize the pattern of the fibers in the image guide, and therefore to know the real location of each fiber intended to be used.

  The following operations are also carried out prior to the use of the apparatus: the determination of the injection rate specific to each fiber, using a homogeneous sample, this injection rate possibly varying from one fiber to another and - the measurement of the background image, carried out without sample.

  These two operations can be performed regularly depending on the frequency of use of the equipment. The results obtained will be used to correct in operation the digital signal at the output of the detector card. - 13

  In operation, according to the invention, 2 processing groups are carried out on the digital signal at the output of the detector card: The first group consists firstly of correcting the digital signal in particular to take account of the actual natural rate of injection of the fiber. 5 from which said signal comes and to subtract from it the part of the flux corresponding to the background image. This makes it possible to process only a signal actually corresponding to the sample observed. For this processing group, a conventional calculation algorithm is used which can be optimized to respect, if necessary, the constraint of real time.

  The second group then consists, from the corrected signal, of reconstructing the digital image which will be displayed by the practitioner. The aim of the processing carried out is to offer the display a reconstructed digital image which is not simply a mosaic of image elements each corresponding to a digital signal corrected by a fiber placed side by side, 1 5 but of offer a reconstructed digital image that no longer reveals the fibers. We use for this an algorithm intended to perform a certain number of operations on each pixel, the algorithm being chosen to respect the real time constraint, that is to say that it must represent a good compromise between the complexity of the operations. requested, the quality of the result that can be obtained and the calculation time. As an example, a Gaussian low-pass filtering algorithm can be used.

  The operation of the apparatus is as follows. The source 1 produces a circular parallel excitation beam at λ = 488 nm, which is then reshaped in the afocal system 2 in order to give it the adequate size for the best possible injection into the core of a fiber. This beam is then sent to the dichroic separation system 3 which reflects the excitation wavelength. The incident beam is then deflected angularly in time in both directions of space by the optomechanical scanning system of mirrors 4, and injected by means of the optical injection means 5 30 into one of the fibers of the image guide. 6. The electronic means 12 serve to control the injection at a given instant of one of the optical fibers of the image guide by angularly deflecting the beam by means of the mirrors, and this point by point for a given line, and line after line, to constitute the image. At the outlet of the guide, the diverging light emerging from the injected fiber is scattered - 14 in a microvolume of the sample located between the surface and a maximum depth of 25 µm (depending on the core diameter of the fibers and their ON). Thanks to scanning, the sample is illuminated micro-volume by micro-volume. At each instant, the micro-volume excited in the tissue then emits a fluorescence signal 5 which has the particularity of being shifted towards longer wavelengths. This fluorescence signal is picked up by the same optical fiber as that used for the excitation, then follows the reverse path of the excitation beam to the dichroic filter 3 which will transmit the fluorescence signal to the detection channel. The parasitic reflections occurring at the excitation wavelength 10 will then be rejected by the rejection filter 8. Finally, the fluorescence signal is focused in the filtering hole 10 to select only the light coming from the fiber excited and the photons are detected by the avalanche photodiode 11. The detected signal is then digitized and corrected.

  The detected signals, one after the other, are processed in real time by virtue of the image processing described above to allow reconstruction of a real-time image viewed on the screen. -

Claims (27)

claims   1. Method for producing a high resolution fluorescence image using an image guide made of several thousand optical fibers, an excitation signal being emitted by a continuous source, deflected and injected in turn into one of the optical fibers of said image guide and a fluorescence signal emitted in reaction being collected by the same optical fiber as that used for the excitation, then detected and digitized to form an image element, characterized in that the end of the fibers is intended to be placed bare directly in contact with the surface of the tissue to be imaged, each fiber being adapted to produce a divergent bundle capable of exciting a micro-volume of the tissue situated from the surface to a maximum depth depending in particular on the core diameter of the optical fibers.   2. Method according to claim 1, characterized in that the image guide 1 5 preferably comprises substantially between 5,000 and 100,000 fibers of 0 of core preferably preferably comprised between 1 and 4, tm and inter-core distance of preferably substantially between 2 and 8 pm.   3. Method according to claim 1 or 2, characterized in that the end of the fibers is polished.   4. Method according to one of claims 1 to 3 for producing an in vivo image in situ, characterized in that the excitation signal is deflected at a speed corresponding to the acquisition of a number d Images per second sufficient for use in real time and in that the fluorescence signal is detected at a detection frequency corresponding to a minimum sampling frequency of the fibers one by one.   5. Method according to claim 5, characterized in that one adjusts a deflection speed of the excitation beam by determining a fast resonant frequency of a line resonant mirror and a slow frequency of a raster galvanometric mirror.   6. Method according to one of the preceding claims, characterized in that optical deflection, injection, focusing and detection means are used having a degree of chromaticity making it possible to collect photons over the entire width of the emission band of the excited fluorophore. - 16   7. Method according to one of the preceding claims, characterized by a quantum efficiency of the detection at the fluorescence wavelengths to be detected of at least 50%.   8. Method according to one of the preceding claims, characterized by a preliminary step of detecting the location of the fibers of the image guide intended to be used.   9. Method according to one of the preceding claims, characterized by a prior step of determining the actual injection rate specific to each fiber.   10. Method according to one of the preceding claims, characterized by a prior step of determining the collected flow corresponding to the background image.   11. Method according to claims 9 and 10, characterized by a step of correcting the digitized signal coming from a fiber by subtracting the flux corresponding to the background image and adapting to the actual own injection rate of said fiber.   12. The method of claim 11, characterized by a step of reconstructing the image from the corrected signal.   13. The method of claim 12, characterized in that the image reconstruction step comprises a Gaussian low-pass filtering.   14. High resolution fibered fluorescence imaging apparatus for implementing the method according to one of the preceding claims, comprising: - the image guide (6); - the source (1) continuously emitting at the excitation wavelength of at least one targeted fluorophore, - scanning (4) and injection (5) fiber after fiber of the excitation beam produced by the source (1) in an XY plane corresponding to the input section of the image guide (6); - means (3) for separating the excitation wavelength and the fluorescence wavelengths; - means for detecting (11) the fluorescence signal; and - means (12) for processing the detected signal allowing the production of an image; - 17 characterized in that the end of each fiber is adapted to produce a beam which is divergent and is intended to be placed naked directly in contact with the surface of the tissue to be observed.   15. Apparatus according to claim 14, characterized in that the image guide 5 preferably comprises substantially between 5,000 and 100,000 fibers of 0 of core preferably preferably substantially between 1 and 4 μm and of inter-core distance preferably between 2 and 8 pm.   16. Apparatus according to claim 14 or 15, characterized in that the end of the fibers is polished.   17. Apparatus according to one of claims 14 to 16, characterized in that the scanning means are adapted to move the excitation beam at a speed corresponding to obtaining an image in real time and the detection means have a bandwidth whose frequency is fixed as a function of the minimum sampling frequency of the fibers one by one.   1 5 18. Apparatus according to claim 17, characterized in that the excitation beam produced by the source (1) is longitudinal single mode having an optimal wavefront quality for injection into a weakly multimode optical fiber.   19. Apparatus according to claim 17 or 18, characterized in that the section of a fiber being circular, the excitation beam produced by the source is circular so as to optimize the injection into a fiber.   20. Apparatus according to one of claims 14 to 19, characterized by means (2) for shaping the beam used at the output of the source (1) to shape the excitation beam in order to adapt it to injection means 25 (5) in the image guide (6).   21. Apparatus according to one of claims 14 to 20, characterized in that the means for separating the excitation and fluorescence wavelengths comprise a dichroic filter (3) having maximum efficiency at the length of excitation wave.   22. Apparatus according to one of claims 14 to 21, characterized by rejection means (8) placed upstream of the detection means (11) and adapted to eliminate the excitation wavelength. - 18   23. Apparatus according to one of claims 14 to 22, characterized in that the scanning means (4) comprise a resonant line mirror (Ml), a weft galvanometric mirror (M2), a first afocal optical system of unit magnification ( AF1) making it possible to combine the two mirrors and a second afocal system (AF2) of unitary magnification making it possible to conjugate the plane of rotation of the second mirror with the plane of injection into one of the fibers.   24. Apparatus according to one of claims 14 to 23, characterized in that the injection means (5) comprise two optical assemblies (El, E2), the first assembly (El) being adapted to correct the optical aberrations at 10 edge of the field of the scanning means (4) and the second assembly (E2) being adapted to carry out the actual injection into one of the fibers of the image guide (6).   25. Apparatus according to claim 24, characterized in that the first optical assembly (El) comprises a doublet and the second optical assembly (E2) 1 5 comprises two doublets followed by a lens.   26. Apparatus according to one of claims 14 to 25, characterized by a filtering hole (10) placed before the detection means (11) whose diameter is chosen so that the image of a fiber is registered there.   27. Apparatus according to claim 26, characterized by means for focusing (9) the fluorescence signal on the filtering hole (10).