FR2502783A1 - Measurement appts. for oxidation-reduction state of living organism - uses UV and IR radiation to produce fluorescent and reflected beams - Google Patents

Abstract

The apparatus for measuring the oxidation-reduction state of an organism in a living body comprises an ultraviolet pulsed laser generator (1), a second pulsed laser generator (3) for a different light, esp. infrared light, receivers (25,20) for the fluorescence generated by the UV light and for the reflected other, esp. IR light, and devices (7,14, 15,16) for concentrating or focussing the two source lights to a common point (10). This point (10) is at one end of a photoconducting fibre (11), the other end (12) of which is located in the organ investigated. The photoconducting fibre thus transmits the UV and IR light to the organ, and transmits the fluorescent and reflected radiation in the opposite direction. The appts. is used for measuring the oxidation-reduction state of a living organism, particularly within a very short cycle such as a single heartbeat, in a perfectly natural physiological situation, where access to the organ is obtained by simple puncturing, intravascular catheter or endoscopic investigation.

Description

Device for measuring the oxidation-reduction state of a living organ in situ
The present invention relates to a device for measuring the redox state of a living organ in situ.

 We know of a device of this type described in the article entitled "Optical consequences of blood substitution on tissue oxidation-reduction state microfluorometry" (Shigeki Kobayashi et al: extract from the journal of the United States of America "Journal of applied physiologyn vol 31 , no. 1, June 1971. This device comprises a radiation source comprising a mercury vapor lamp and filters. This source is capable of illuminating an organ with two radiations of different wavelengths, ultraviolet radiation (366 nm ) and red radiation (720 nm). Illumination of the organ by ultraviolet radiation leads to the formation of fluorescence (## O - 480 nm) picked up by a photoelectric receiver.

 The organ returns part of the energy of the red radiation in a reflected beam picked up by another photoelectric receiver. This device also includes a system for recording the output signals of the two receivers.

 The intensity of the fluorescence measured is representative of the state of redox of the organ.

 However, variations in intratissular red blood cell concentration disturb photometric measurements, especially when experiments are performed on a living organ in situ. Indeed, a decrease in the intratissular concentration or red cells leads to an increase in the recorded fluorescence.

 The variations in intensity of the reflected red radiation are representative of the intratissular concentration in red blood cells.

 By operating on an organ maintained in a constant oxidation-reduction state, Kobayashi was able, using the device described in this article, to draw a graph and establish an equation relating the values of intensity of fluorescence and red reflection obtained on the one hand with the organ completely emptied of its blood and on the other hand with the organ containing various levels of red blood cells. Kobayashi believes that the results obtained in his study show that it is possible to electronically compensate for variations in fluorescence as a function of the intratissular concentration in red blood cells.

The device described in this article allows, on an isolated organ artificially perfused, therefore on a model far removed from the physiological state, outside the organism, the study of the report
NADH / NAD in its wide, slow and provoked variations, during experimentation which can only be held in the laboratory.

 The object of the present invention is to provide a device allowing the study of rapid variations (in particular during a single cardiac cycle), spontaneous and / or caused, of small amplitude, of the NADH / NAD ratio, at the level of a organ in place in his body, normally perfused with blood, in physiological situation, in particular by simple puncture, intravascular catheterization or endoscopic examination, this device being susceptible of clinical applications, in particular during surgical interventions.

 The subject of the present invention is a device for measuring the redox state of a living organ in situ, comprising - means for illuminating the organ by ultraviolet radiation and by other radiation, - a first photoelectric receiver arranged to detect the fluorescence emitted by the organ in response to illumination by ultraviolet radiation - and a second photoelectric receiver arranged to detect the beam returned by the organ in response to illumination by the other radiation, characterized in what - the means for illuminating the organ comprise a first laser generator capable of emitting pulses of ultraviolet radiation, a second laser generator capable of emitting pulses of infrared radiation, these pulses constituting the other radiation,. means for concentrating the ultraviolet and infrared pulses at a single point and an optical fiber capable of transmitting the ultraviolet and infrared pulses from one of its end faces to the other, a first end face of this fiber being disposed at said same point , the second end face being disposed in said member, - and in that it further comprises a processing circuit receiving the electrical signals delivered by the first and second receivers, this circuit being capable of developing, from these signals, a electrical output signal independent of the rate of red blood cell concentration in the organ, this signal being representative of the redox state of the organ.

 Particular embodiments of the object of the present invention are described below, by way of example, with reference to the accompanying drawings in which - Figure 1 schematically shows an embodiment of the device according to the invention - and FIG. 2 is a more detailed diagram of an electronic system forming part of the device illustrated in FIG. 1.

 In FIG. 1, a nitrogen laser generator 1 emits a pulse of ultraviolet radiation 2 of wavelength 337 nm towards the tank of a dye laser 3 along an axis 4. The nitrogen laser 1 is for example excited by a current wave propagating along a flat line of excitation, this wave being created by the electric discharge of a spark gap. The dye laser 3 comprises a tank filled with a liquid constituted for example by a mixture of two dyes in a solvent, the two dyes being diethyloxatricarbocyanine iodide (DOTC) and hexamethylindotricarbocyanine iodide (HITC), and the solvent being dimethyl sulfoxide (DMSO). This tank is provided with a resonant optical cavity comprising two mirrors, so as to be able to emit a pulse of infrared radiation of wavelength 805 nm along an axis 6 perpendicular to the axis 4.

 An optical plate 7 is arranged at the output of the laser 1 and inclined at 450 on the axis 4. This plate reflects at 900, along an axis 8, ten percent of the energy of the pulse 2 and lets 90 pass through. cent of the energy of this pulse along the axis 4 towards a converging cylindrical lens 5 placed between the blade 7 and the laser 3.

 Along an axis 9 parallel to axis 4 are successively arranged - one end face 10 of an optical fiber 11, the other end face 12 of which is disposed in a living organ 13, the axis 9 being perpendicular to the face 10, this face possibly having a rounded shape, for example hemispherical, - a converging lens 14 centered on the axis 9 and of which a focal point is located on the face 10, - an optical plate 15 disposed substantially at the intersection of the axes 8 and 9 and inclined at 450 on the axis 9 perpendicularly to the blade 7, - an optical blade 16 disposed substantially at the intersection of the axes 6 and 9, parallel to the blade 16, - an optical blade 17 arranged parallel to the blade 16, substantially at the intersection of a return axis 18 with the axis 9, - an optical filter 19 arranged perpendicular to the axis 9 - and a photoelectric receiver 20.

 On the axis 8, an optical attenuator 21 can be placed between the blades 7 and 15, and a photoelectric receiver 22 can be located beyond the blade 15.

 Likewise, on the axis 6 an optical attenuator 23 can be placed between the laser 3 and the blade 16, # and a photoelectric receiver 24 can be located beyond the blade 16.

 On the axis 18, is arranged a photoelectric receiver 25 and a filter 26 situated between the plate 17 and the photodetector 25.

 The electrical outputs of the four photodetectors 20, 22, 24 and 25 are connected respectively to four inputs 27, 28, 29 and 30 of a processing circuit 31.

 FIG. 2 is a diagram of the processing circuit 31. In this diagram, one end of a branch 32 is connected to the input 30 of the processing circuit. The branch 32 comprises in series, from the input 30, an amplifier 33, an integrator 34 and a sampling circuit 35. The other end of the branch 32 is connected to an input of a divider circuit 36. At the other input of circuit 36 is connected one end of a branch 37 comprising in series, from circuit 36, a sampling circuit 38, an integrator 39 and an amplifier 40. The other end of branch 37 is connected to input 28 of the processing circuit.

 A pulse generator 41 is connected to the branch 37 between the input 28 and the amplifier 40.

 One end of a branch 42 is connected to the input 27 of the processing circuit. The branch 42 comprises in series, from the input 27, an amplifier 43, an integrator 44 and a sampling circuit 45. The other end of the branch 42 is connected to an input of a divider circuit 46. At the other input of circuit 46 is connected one end of a branch 47 comprising in series, from circuit 46, a sampling circuit 48, an integrator 49 and an amplifier 50. The other end of branch 47 is connected to input 29 of the processing circuit.

 The outputs of the two divider circuits 36 and 46 are respectively connected to the two inputs of a multiplex circuit 51, the output of which is connected to the input of an analog-digital converter 52. The output of the converter 52 is connected to the input a computer 53 whose outputs are connected to the inputs of a recorder 54.

 The device described above and illustrated by Figures 1 and 2 operates in the following manner.

 First, an ultraviolet pulse 2 of the nitrogen laser 1, of wavelength 337 nm, is triggered.

 The plate 7 reflects 10% of the energy of this pulse along the axis 8 towards the plate 15. The latter reflects part of the energy of the radiation of wavelength 337 nm and lets the other part pass. this energy towards the photoelectric receiver 22. The ultraviolet pulse coming from the blade 7 is therefore returned by the blade 15 parallel to the axis 9 to be concentrated on the face 10 of the fiber 11 by means of the lens 14.

The blade 7 transmits 90% of the energy of the pulse 2 along the axis 4. The energy thus transmitted is concentrated by the cylindrical lens 5 in the tank of the dye laser 3 so as to excite it
The laser 3 then emits an infrared pulse of wavelength 805 nm along the axis 6. The plate 16 is partially reflective at the wavelength of 805 nm; along the axis 9 it returns half the energy of the pulse coming from the laser 3 and lets the other half of this energy pass to the photoelectric receiver 24. The plate 15 is transparent to light of wavelength 805 nm. It therefore lets the impulse returned by the blade 16 pass to the lens 14. The lens 14 concentrates this impulse on the face 10 of the fiber 11.

 In practice, it can be considered that the two ultraviolet and infrared pulses arrive on the face 10 at substantially the same time.

 As an indication, the optical fiber may include a silica core with a diameter of 400 microns surrounded by an optical cladding also made of silica but with a lower refractive index. The rounded end of the fiber in contact with the member makes it possible to reduce parasitic Fresnel reflection. The optical sheath can have an outside diameter of 500 microns and itself be surrounded by a plastic protective sheath with an outside diameter of 700 microns. This plastic material is sterilizable and chosen from that which does not have a coagulating effect in the presence of blood.

Of course, such an optical fiber can be introduced, if necessary, into a catheter or a hypodermic needle.

 The optical fiber 11 transmits the two ultraviolet and infrared pulses from the face 10 to the face 12 so as to illuminate the organ 13 which can for example be the heart of a patient during operation.

 The ultraviolet pulse causes in the organ 13 a blue fluorescence of average wavelength 480 nm which is transmitted by the fiber 11 from the face 12 to the face 10. Then this pulse is directed by the lens 14 along the axis 9 by successively crossing the plates 15 and 16 which are transparent to radiation of wavelength 480 nm.

The plate 17 reflects this radiation along the axis 18 towards the receiver 25 through the filter 26 which is a bandpass filter transmitting only the fluorescence
Part of the energy of the infrared pulse transmitted to the member 13 by the fiber 11 is reflected by this member and transmitted in the opposite direction by the fiber 11 from the face 12 to the face 10.

The lens 14 then directs along the axis 9 the reflected infrared pulse. This crosses the plate 15 transparent to light with a wavelength of 805 nm. Half of the energy of this pulse passes through the blade 16 towards the blade 17 which is transparent to this radiation. The pulse is finally received on the receiver 20, after passing through the filter 19 which is a bandpass filter transmitting only the infrared radiation emitted by the laser 3.

 The attenuators 21 and 23 optionally make it possible to adjust the respective intensities of the ultraviolet and infrared pulses so as to adapt them to the different types of organs to be examined. These attenuators are therefore not essential in all cases. It is also possible to eliminate the photoelectric receivers 22 and 24, insofar as the lasers 1 and 3 deliver pulses of perfectly stable power.

 It is particularly advantageous to use an infrared laser emitting at the wavelength of 805 nm. Indeed for this wavelength called "isosbestic", the reflection coefficient of the organ does not depend on its oxidation-reduction state, nor on the state of oxygenation of the blood which circulates there.

 In the information processing circuit, the divider 36 makes the relationship between the fluorescence signal and the emission signal of the ultraviolet laser 1, these signals being shaped in the respective branches 32 and 37. This thus gives the output of the circuit 36 a fluorescence signal F independent of the intensity of the laser signal delivered by the receiver 22.

 Similarly, the divider circuit 46 performs the relationship between the infrared reflection signal and the emission signal from the laser 3, these signals being shaped in the respective branches 42 and 47. Thus, at the output of the circuit 46, a signal I independent of the laser signal delivered by the receiver 24.

After crossing circuits 51 and 52, the signals F and I enter the computer 53. The latter is capable of deducing from the values F and I a value Fo corresponding to the equation
Io / I = 1 + K ln (Fo / F) in which lo and Fo denote the values of I and F which it is possible to obtain when the organ is completely drained of its blood, K and Io being constants which can be determined by previous tests.

 The value of Fo thus obtained is independent of the intratissular concentration of red blood cells; it is representative of the redox state of the organ considered.

 The pulse generator 41 makes it possible, from each laser pulse emitted by the laser 1, to open predetermined time windows in all the elements of the branches 32, 37, 42 and 47, as well as in the elements 51 and 52 of the circuit 31. To this end, the pulse generator 41 has fourteen outputs represented by arrows, and each element concerned has a control input also illustrated by an arrow. Each output of generator 31 is connected to a control input. Thus the output 55 of the generator 41 is connected by a link 56 to the control input 57 of the amplifier 40. These time windows authorize the operation of each element only for a short time interval. whose limits frame the moment of arrival of the pulse. This makes it possible to avoid recording spurious signals and in particular the reflection signals of the laser pulses on the face 10 of the optical fiber 11.

 As indicated in FIG. 2, the recorder 54 makes it possible to record not only the signal Fo, but also the signals I and F.

 The device described above has many advantages.

 The high light power delivered by the different types of ultraviolet laser operating in pulses, at a repetition rate which, for information, can be between 20 and 120 Hz, makes it possible to obtain a high level of fluorescence while maintaining at a low value the average illumination power of the organ examined. Under these conditions, this illumination does not in itself bring any disturbance of the value to be measured (NADH / NAD ratio, for example) in particular by thermal effect, and, a fortiori, does not alter the tissue studied.

 The repetition rates indicated above make it possible to deliver to the tissue to be studied a sufficient number of pulses per cardiac cycle to obtain an accurate measurement in the case of measurements on humans (heart rate at rest 1.2 Hz) or on the animal (rat heart rate: 5 Hz).

 Although it is also possible to use excimer or exciplex lasers to obtain fluorescence, the nitrogen laser is preferably used, the emission wavelength (337 mm) of which is very close to the peak of absorption of NADH, which increases the signal / noise ratio.

 The use of a dye laser as a source of infrared radiation has two advantages: on the one hand this laser can be excited by part of the energy of the pulses emitted by the ultraviolet laser so as to obtain two measurement pulses, ultraviolet and infrared, almost simultaneous, and on the other hand the dye laser can be tuned on the wavelength of 805 nm isosbestic for hemoglobin.

 The use of a single optical fiber, of relatively large diameter to capture as much fluorescence as possible, nevertheless makes it possible to obtain sufficient flexibility to follow, for example, the vascular path of a catheter. The single fiber also has the advantage of having a single fiber-fabric interface, which is preferable to a transmission interface distinct from a reception interface, so as to increase the energy ratio of the radiation received to the radiation emitted.

 Finally, it is possible to currently produce an electronic processing circuit having a very short response time, so as to carry out all of the operations during a time interval significantly less than the period separating two laser pulses.

 The device according to the present invention can be used to carry out continuous measurements in situ of the instantaneous NADH / NAD ratio (having regard to the time scale of the phenomena considered), which is of capital biological importance.

 This device can be applied for example to the study of cardiac metabolism and in particular of its variations in pathology and during cardiac surgical interventions. The study of cardiac metabolism can then be done at the endocardium, that is to say inside the cardiac cavities, by simple catheterization, the peripheral approach can be venous or arterial.

 This device can also be applied to the study of other organs (brain, liver, kidney) in the most diverse circumstances (pharmacological interventions, tumor transformations for example).

Claims (10)

1 / Device for measuring the oxidation-reduction state of a living organ in situ, comprising - means for illuminating the organ by ultraviolet radiation and by other radiation, - a first photoelectric receiver arranged to detect fluorescence emitted by the organ in response to illumination by ultraviolet radiation - and a second photoelectric receiver arranged to detect the beam returned by the organ in response to illumination by other radiation, characterized in that - the means to illuminate the organ comprise a first laser generator (1) capable of emitting pulses of ultraviolet radiation,. a second laser generator (3) capable of emitting pulses of infrared radiation, these pulses constituting the other radiation,. means (7, 14, 15, 16) for concentrating at the same point the ultraviolet and infrared pulses. and an optical fiber (11) capable of transmitting the ultraviolet and infrared pulses from one of its end faces to the other, a first end face (10) of this fiber being disposed at the same point, the second end face (12) being arranged in said member, - and in that it further comprises a processing circuit (31) receiving the electrical signals delivered by the first (25) and second (20) receivers, this circuit being capable of developing, at from these signals, an electrical output signal independent of the rate of red blood cell concentration in the organ, this signal being representative of the state of redox of the organ. 2 / Device according to claim 1, characterized in that the first laser generator (1) is a nitrogen laser generator. 3 / Device according to claim 1, characterized in that the second laser generator (3) comprises an active medium consisting of a dye, this active medium being capable of being excited by the pulses (4) emitted by the first laser generator ( 1). 4 / Device according to claim 3, characterized in that the excited dye is capable of emitting pulses of wavelength 805 nanometers. 5 / Device according to claim 3, characterized in that it further comprises - a third photoelectric receiver (22) arranged to detect the pulses emitted by the first laser generator - and a fourth photoelectric receiver (24) arranged to detect the pulses emitted by the second laser generator. 6 / Device according to claim 5, characterized in that said means for concentrating at one point the pulses of ultraviolet radiation and infrared radiation comprise - an optical system (14) centered along a first axis (9), said same point being a focal point of the optical system, the second receiver (20) being disposed on the first axis on the side opposite the focal point of the optical system, - a first optical blade (16) disposed on the first axis (9) between the optical system (14) and the second receiver (20), this strip partially reflecting the infrared radiation emitted by the second laser generator (3), this strip being inclined on the first axis to reflect this radiation towards the optical system along the first axis, this strip being transparent to fluorescence radiation and being disposed on a second axis intersecting the first axis between the second laser generator (3) and the fourth receiver (24), - a second optical plate (7) disposed on a third axis parallel to the first axis, the first (1) and the second (3) laser generator being located on this third axis, this second optical blade being arranged perpendicular to the first optical blade (16) to return along a fourth axis part of the energy of the pulses emitted by the first laser generator (1), the other part of this energy passing through the second blade (7) to excite the dye of the second laser generator (3) - and a third optical blade ( 15) arranged parallel to the first optical blade (16) on the one hand on the first axis (9) between the optical system (14) and the first optical blade (16) and on the other hand on the fourth axis between the second optical plate (7) and the third photoelectric receiver (22), this plate reflecting the radiation emitted by the first laser generator (1) and being transparent to the fluorescence and to the radiation emitted by the second laser generator (3). 7 / Device according to claim 6, characterized in that it further comprises - a first optical attenuator (21) of ultraviolet radiation disposed on the fourth axis between the second (7) and third (15) optical blades - and a second optical attenuator (23) disposed on the second axis between the second laser generator (3) and the first optical blade (16). 8 / Device according to claim 7, characterized in that it comprises a fourth optical blade (17) arranged parallel to the first optical blade (16) on the one hand on the first axis (9) between the first optical blade (16 ) and the second receiver (20) and on the other hand on a fifth axis intersecting the first axis, the fourth optical plate partially reflecting the infrared radiation emitted by the second laser generator, this fourth optical plate being transparent to the radiation emitted by the second laser generator.  9 / Device according to claim 1, characterized in that the second end face (12) of the optical fiber (11) has a rounded shape. 10 / Device according to claim 5, characterized in that the processing circuit (31) comprises - first dividing means (36) capable of measuring the ratio between the amplitude of the signals originating respectively from the first (25) and third receivers ( 22), - second dividing means (46) capable of measuring the ratio between the amplitude of the signals coming respectively from the second (20) and fourth receivers (24), - a computer (53) capable of correcting, according to a predetermined law the signals delivered by the first dividing means (36) as a function of the signals delivered by the second dividing means (46), so as to obtain a compensated signal - and a recording system (54) of said signals.