The present invention relates to the field of intraretinal mapping.
Intraretinal mapping is usually carried out by angiography or by taking photographs of the fundus of the eye. However, these techniques provide results that are qualitative and sometimes difficult to interpret. Moreover, document WO92/12412 discloses a device and a method of measuring the pH of a target, which can be used endoscopically, using fluorescent markers. The method disclosed in that document overcomes measurement condition problems by working with ratios of fluorescence levels (and not with fluorescence levels themselves). However, it is not possible to map the pH, in two dimensions, of a target area.
The object of the present invention is to provide a noninvasive method and device for the fluorescence mapping of the intraretinal pH which employ only well-controlled techniques and means and which provide a faithful representation that can be used directly for subsequent diagnostic purposes.
According to a first aspect, the invention proposes a method of mapping the intraretinal pH, whereby, after the retina has been marked by a fluorescent marker, the fluorescence emission spectrum of which has a peak whose amplitude depends on the pH:
a) the retina is illuminated with first excitation radiation having a narrow spectrum centered on a wavelength falling within the excitation spectrum of the fluorescent marker, and a first image of the fluorescence emitted by the retina is formed, in the form of a two-directional matrix of pixels, at the wavelength of the peak of the fluorescence emission spectrum;
b) the retina is illuminated with second excitation radiation having a narrow spectrum centered on a wavelength of the excitation spectrum of the fluorescent marker that is different from that of the first radiation, and a second image of the fluorescence emitted by the retina is formed, in the form of a two-dimensional matrix of pixels, at the wavelength of the peak of the fluorescence emission spectrum; and
c) the ratio of the fluorescence levels emitted in response to the two respective excitation wavelengths at each pixel is calculated, this ratio being representative of the pH of the retina, and a pH map of the retina is deduced therefrom.
According to a second aspect, the invention proposes a method of mapping the intraretinal pH, after the retina has been marked with a fluorescent marker, the fluorescence emission spectrum of which has a peak whose amplitude depends on the pH. This method involves only a single excitation radiation and comprises the following steps:
a) the retina is illuminated with excitation radiation having a narrow spectrum centered on a wavelength falling within the excitation spectrum of the fluorescent marker;
b) a first image of the fluorescence emitted by the retina is formed, in the form of a two-dimensional matrix of pixels, at the wavelength of the peak of the fluorescence emission spectrum;
c) a second image of the fluorescence emitted by the retina is formed, in the form of a two-dimensional matrix of pixels, at a wavelength corresponding to an isosbestic point in the fluorescence emission spectrum; and
d) the ratio of the fluorescence levels emitted at the two respective wavelengths falling within the emission spectrum at each pixel is calculated, this ratio being representative of the pH of the retina, and a pH map of the retina is deduced therefrom.
According to a third aspect, the invention relates to a device for mapping the intraretinal pH of a retina marked with a fluorescent marker, the fluorescence emission spectrum of which has an intensity peak whose height depends on the pH.
It is known that retinal ischemia, which is characterized among other things by acidification of the internal neuroretina, may lead to the formation of retinal neovessels. These retinal vessels may bleed, fibrose and cause problems of significant reduction in visual acuity. The use of a method and a device according to the invention allows better quantification of the ischemia than the methods and procedures of the prior art, and facilitates diagnosis by a practitioner and makes it easier to determine indications for subsequent treatment.
According to a fourth aspect, the invention relates to a device for photocoagulating the peripheral areas of a retina, which device may incorporate the elements of the mapping device. This photocoagulation device comprises the same elements as the intraretinal pH mapping device and a laser provided with means for focusing and orienting an output beam onto the point or points on the retina that are represented, on the pH mapping image provided by the device, by pixels corresponding to pH values below a given threshold value.
Other features and advantages of the invention will become apparent over the course of the following description of embodiments, these being given by way of nonlimiting example and with reference to the appended drawings.
In the drawings:
In the various figures, the same references denote identical or similar elements.
The source 2 is for example a xenon lamp. The power is for example 150 W. The band of the filters 3a and 3b nm has for example a width at mid-height of 10 nm. It would also be possible to use, instead of the source 2 associated with the filter system 3, laser diodes of respective wavelength λexc1 and λexc2.
The device 1 furthermore includes a focusing optic 5 for focusing the radiation onto the human retina.
Along the return path of the light, the device includes a filter 6 designed to let through only a narrow band containing the wavelength λ0 of the peak of the fluorescence emission spectrum, which corresponds to the peak in pH sensitivity. This filter thus receives the fluorescence from the retina illuminated by the light source.
In one advantageous method of implementation, the device is incorporated into an angioretinography device and furthermore includes two mirrors 7, 8 for folding the return path. An aperture 9 in the mirror 8 allows the emission fluorescence to pass through it, allowing the incident channel to be separated from the return channel of the retina.
The device 1 furthermore includes a matrix camera 10 for forming a 2D image. This camera provides an image with, at each pixel, a value representative of the fluorescence level.
The device also includes means 11 for calculating the ratio of the values at each pixel for two images provided. It is also provided with means 12 for storing these values. In general, the device 1 also includes means 13 for displaying an image.
The camera may for example be a CCD camera, sensitive for wavelengths of the emission spectrum of BCECF. It may comprise 512×512 pixels. The calculating means 11, storage means 12 and display means 13 are for example the microprocessor, the hard disk and the monitor of a computer.
The first mapping method according to the invention is carried out in one implementation as follows. A fluorescent marker, whose fluorescence depends on the pH, is injected beforehand into a vein. Many pH-dependent fluorescent markers can be used (SNAFL, SNARF, HPTS, fluoresceins and carbofluoresceins, etc.). The method of implementation shown below uses BCECF(2′,7′-bis(2-carboxyethyl) 5(and 6)carboxyfluorescein), the sensitivity range of which lies within physiological pH values. BCPCF (2′,7′-bis(2-carboxypropyl)-5(and 6)carboxyfluorescein) may also be used. BCPCF is a derivative of BCECF in which the carboxyethyl group has been replaced with a carboxypropyl group. BCPCF has a wavelength at the isosbestic point of the excitation spectrum of 454 nm and a wavelength at the isosbestic point of the emission spectrum of 504 nm.
The amount of BCECF injected lies for example within the range from 0.1 to 10 mg per kg of the patient's body. In the method of implementation shown below, this amount is 1 mg/kg.
Once sufficient time has elapsed after injection for the retina to have received the marker, the retina is illuminated with first excitation radiation having a narrow spectrum (mid-height width of 10 nm) centered on a wavelength λexc1 of the excitation spectrum of the fluorescent marker. This radiation corresponds to the radiation of the light source 2 filtered by the first filter of wavelength λexc1 of the filter system 3. A first image of the fluorescence emitted by the retina is formed simultaneously on the sensitive member of the camera 10. This first image, in the form of a two-dimensional matrix of pixels, represents the fluorescence from the retina filtered by the filter system 6 at the wavelength λ0. This image is stored in digital form in the storage means 12.
Advantageously, λexc1 is equal to the wavelength of the peak of the excitation spectrum of the fluorescent marker. In the case of BCECF, the excitation spectrum of which is shown in
The first filter is then replaced with the second filter, centered on λexc2, by moving the arm 4. The retina is illuminated with second excitation radiation having a narrow spectrum centered on the wavelength λexc2 of the excitation spectrum of the fluorescent marker that is different from that of the first radiation. A second image of the fluorescence emitted by the retina is formed, in the form of a two-dimensional matrix of pixels, again at the wavelength λ0.
For example, λexc2 may be taken to be equal to 470 nm.
Advantageously, in another method of implementing the invention, λexc2 may also be chosen to be equal to the 450 nm wavelength of the isosbestic point of the excitation spectrum (the point having the same level of absorption whatever the pH) shown in
The microprocessor 11 calculates, at each pixel, the ratio of the fluorescence levels at the wavelength λ0, after possible storage, emitted in response to the two respective excitation wavelengths λexc1 and λexc2.
Finally, a pH map of the retina is determined from this ratio, which is representative of the pH of the retina. This image may be displayed on the screen 13.
The pH measurement is reliable and not dependent on the measurement conditions, as it uses a fluorescence intensity ratio for two different excitation wavelengths. The pH value is obtained from the calculated fluorescence ratio, using a predetermined ratio/pH correspondence curve.
In another method of implementing the invention, a device according to an alternative embodiment is used in which the filter system 3 includes at least one narrow-band filter (mid-height width of 10 nm) centered on a wavelength λexc of the excitation spectrum of the fluorescent marker. The device includes a filter system, instead of the filter 6, suitable for filtering in succession, at two different wavelengths, λ0 and λIbEm respectively, the fluorescence from the retina illuminated by the light source. λ0 and λIbEm correspond to the wavelength of the peak and the wavelength of the isosbestic point, respectively, of the fluorescence emission spectrum.
The method employing this latter device is as follows.
A pH-dependent fluorescent marker is injected. The retina is then illuminated with excitation radiation having a narrow spectrum (typically a mid-height width of 10 nm) centered on the wavelength λexc of the excitation spectrum of the fluorescent marker, using the light source 2, the emission from which is filtered by the filter 3 centered on the wavelength λexc. The camera 10 forms a first image of the fluorescence emitted by the retina, in the form of a two-dimensional matrix of pixels, at the wavelength λ0 of the peak of the fluorescence emission spectrum. The corresponding data is stored by the storage means 12.
The first filter of the filter system is then replaced with the second filter centered on λIbEm and a second image of the fluorescence emitted by the retina is formed, in the form of a second two-dimensional matrix of pixels, at the wavelength λIbEm corresponding to the isosbestic point of the emission spectrum.
Advantageously, an optical system may be used that allows the CCD camera to form both images of the fluorescence.
Finally, a pH map of the retina is determined from the calculated ratio, at each pixel, of the fluorescence levels emitted at the two wavelengths of the fluorescence emission spectrum, this ratio being representative of the pH of the retina.
In one advantageous method of implementing the invention, the wavelength corresponding to the excitation spectrum peak may be chosen as the excitation wavelength λexc.
For example, if the fluorescent marker C-SNAFL-1 is used, the method described above may be implemented using a single excitation at 514 nm and by forming two images corresponding to the fluorescence at the respective wavelengths of 540 nm and 635 nm.
In a method according to the invention, the measurements are carried out after injection within a period lying between a minimum of three minutes and a maximum of three hours, usually a few minutes.
A look-up table showing the correspondence between fluorescence ratios and pH may for example be established by means of a prior measurement, reproducing the planned conditions of use, by comparison with the data provided by a pH electrode on similar tissue and under illumination conditions identical to those of the measurements that will be carried out using the method.
The device may be supplemented with means for photocoagulating areas of the retina based on the intraretinal pH mapping according to the invention, by adding a treatment laser to the mapping device.
By photocoagulating areas of the retina, especially the peripheral areas, it is possible to destroy the isochemic areas which lead to the formation of neovessels.
The procedure, using the device shown in
The pixels corresponding to those points on the retina whose pH is below a given threshold are then identified and a laser pulse is directed onto only those points.
The pH threshold in question is for example equal to 7.2. These points on the retina, which correspond to the isochemic areas, are treated with the laser.
To do this, in the case shown in
The laser may also be coupled directly to the retinograph used in the embodiment of the device 1 described above.
The laser may for example be a green monochromatic argon laser or a 532 nm frequency-doubled Nd:YAG laser.
In the case of retina opacity, a krypton laser may be used. The impacts on points identified on the map are defined by the focusing means between 200 μm and 500 μm in diameter, generally with an exposure time of 0.1 to 0.2 s.
The power of the laser used is for example 100 to 500 mW and it is adjusted according to the effect and also according to any possible cataract that attenuates the laser beam. Four to six sessions, each consisting of 500 impacts, may be carried out, seeking to obtain a retina of clean white appearance.
Number | Date | Country | Kind |
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0303174 | Mar 2003 | FR | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/FR04/00534 | 3/5/2004 | WO | 6/21/2006 |