RU2182329C2 - Fluorometry detector - Google Patents

Abstract

FIELD: optical instrumentation. SUBSTANCE: fluorometric detector incorporates source of luminous flux, focusing optics, capillary with specimen, parabolic mirror, light filter and photodetector. Detected volume of specimen is so placed in focus of mirror that fluorescent radiation collected by mirror forms quasi-parallel beam. Detector is fitted with spherical mirror whose focus is conjugate to focus of parabolic mirror, both mirrors being located on one axis perpendicular to plane in which axis of excitation radiation and capillary are positioned. Detector has CCD matrix of additional photodetector installed in direction of specular ray reflected from capillary with specimen. In this case radiation source and/or capillary are mounted for movement in direction perpendicular to their axes. Apart from this axis of radiation source is located at Brewster angle with reference to capillary. EFFECT: enhanced sensitivity of fluorometry measurements, simplified adjustment and improved operational characteristics of detector. 1 cl, 3 dwg

Description

 The invention relates to the field of optical instruments for measuring concentrations and identification of substances and can be used in highly sensitive analytical instruments and systems for monitoring product quality, in pharmacology, biotechnology, in medical and biological research.

 A known fluorimetric detector for a flow system [1], consisting of a closed ellipsoidal mirror cell, a radiation source and a photodetector with a light filter. The capillary is in one focus of the ellipsoidal mirror, and the photodetector is in the second focus. The fluorescence light from a substance located in the capillary is redistributed to a photodetector, and the collection efficiency of light radiation approaches 75%.

 The disadvantage of this scheme is that, in addition to fluorescence, the ellipsoidal mirror collects all the light reflected and scattered in the capillary from the radiation source and directs it to the photodetector. As practice shows, the proportion of reflected and scattered light can be very significant, which significantly reduces the sensitivity of measurements and leads to nonlinearity of the calibration characteristic.

 Also known is a device for collecting fluorescence [2], consisting of an ellipsoidal mirror, a laser with focusing optics, a capillary, a light filter and a photodetector. The luminous flux from the radiation source falls on the capillary at an angle of 90 degrees. An ellipsoidal mirror located on the same axis as a radiation source, a light filter and a photodetector collects about 50% of the fluorescence flux.

 The disadvantage of this scheme is also that the mirror, together with the fluorescence flux, focuses the reflected and scattered radiation from the light flux source to the photodetector. Moreover, the higher the fluorescence light gathering efficiency for the described devices, the more re-reflected and scattered radiation hits the photodetector, which reduces the sensitivity and distorts the measurement results. The use of light filters does not allow sufficiently effective to eliminate spurious illumination from a powerful source of exciting radiation - a laser. In addition, in these devices there is no convenient and simple way to align, which is very important when measuring fluorescence in capillaries having an internal diameter of tens to several microns.

 Closest to the technical nature of the claimed device is a device for fluorimetric detection, containing a radiation source, focusing optics, collecting a parabolic mirror, a capillary with a sample, a light filter and a photodetector, the light flux from a radiation source, a capillary with a sample and the optical axis of a parabolic mirror in three mutually perpendicular planes, and the photodetector is located on the same optical axis as the mirror [3]. The luminous flux of the radiation source excites the fluorescence of the sample in the capillary located at the focus of the parabolic mirror, which directs the collected fluorescence through a light filter to the photodetector with a quasi-parallel beam. Excess light radiation that passes through the capillary is discharged along the beam through the hole in the mirror.

 A disadvantage of the known device is that, in addition to the fluorescence signal, the photodetector receives radiation reflected and scattered from the walls of the capillary, which reduces the sensitivity of the measurements. In addition, the alignment of the optical system in the known detector is carried out according to the maximum Raman signal scattering of the contents of the capillary using rotating filters, which is a laborious, complex and expensive operation, leading to a complication of the detector design.

 The proposed optical device solves the problem of increasing the sensitivity of fluorimetric measurements, simplifying the alignment of the device, makes it possible to accurately align the device when operating in spectral ranges falling out of the visible part of the spectrum, which ultimately improves the operational properties of the fluorimeter.

 The problem is solved due to the fact that a fluorometric detector containing a light source, focusing optics, a capillary with a sample, a parabolic mirror, a light filter and a photodetector, while the detected sample volume is located in the focus of the parabolic mirror so that the fluorescence radiation collected by the mirror forms a quasiparallel beam, equipped with a spherical mirror, the focus of which is conjugated with the focus of a parabolic mirror, and the mirrors are located on the same axis, perpendicular plane, in which the axis of the exciting radiation and the capillary are located, and contains the CCD matrix of an additional photodetector installed in the direction of the mirror reflected from the capillary with a beam breakdown, while the radiation source and capillary are movable with the possibility of movement in the direction perpendicular to their axes. In addition, the axis of the radiation source is located at a Brewster angle relative to the capillary.

 When the exciting radiation interacts with the sample, fluorescence of the substance in the capillary is caused. The fluorescence intensity depends on the amount of fluorescent agent in the sample. The luminous fluorescence flux with the help of mirrors is sent by a quasi-parallel beam through a filter to a photodetector, forming a signal proportional to the concentration of the desired substance.

 A system of two mirrors with conjugated foci makes it possible to solve the problem - to increase the sensitivity of measurements, since it allows to increase the solid angle of fluorescence flux collection. In this case, an increase in the solid angle to approximately 2π radians is achieved. In addition, it becomes possible to use replaceable capillaries installed in a separate construct (cartridge), which allows for quick replacement of the capillary and thereby improves the operational properties of the device.

 In addition, to reduce or completely eliminate the effect of the reflected and scattered light flux on the measured signal contributes to the fact that the exciting radiation falls on the capillary at a Brewster angle. In this case, it is possible to significantly reduce the reflection of radiation from the walls of the capillary, since polarized exciting radiation in a plane parallel to the axis of the capillary will not undergo reflections at the capillary-air interface and will completely penetrate the capillary, and also allows directing reflected radiation in such a way that it didn’t get on the mirrors and then on to the photodetector.

 The use of capillaries with a small inner diameter increases the detection sensitivity and resolution, but there is a need for precise alignment of the fluorimeter. In this case, it is necessary to achieve an accurate passage of the laser beam through the inner diameter of the capillary, since if the beam passes between the inner and outer walls of the capillary, then due to the very small radius of curvature of the capillary, the fraction of rereflected radiation from the walls of the capillary increases significantly. When the exciting beam passes through the center of the capillary, the reflected radiation is of the least importance, which in turn leads to an increase in sensitivity and an improvement in the calibration characteristic.

 The invention is illustrated by drawings, where figure 1 (a) shows a diagram of the inventive device, which depicts a top view, and figure 1 (b) shows a front view. In FIG. 2 is a diagram of the formation of two rays reflected from the first and last walls of the capillary. Figure 3 shows the course of the rays in the cross section of the capillary.

 Fluorimetric detector [Fig. 1 (a)] contains a radiation source 1 located at a Brewster angle to the axis of the capillary with sample 2, a parabolic 3 and a spherical 4 mirror, in the focus of which is a capillary, a filter 5, a photodetector 6. A CCD array is installed in the beam reflected from the capillary 7. Parabolic 3 and spherical 4 mirrors are located on the same optical axis with a light filter 5 and a photodetector 6.

 The proposed device works as follows: the light flux from the radiation source 1 at a Brewster angle falls on the capillary and causes fluorescence of the sample in the capillary. The fluorescence flux in a solid angle of 22π radians is collected by parabolic 3 and spherical 4 mirrors having a conjugate focus in which a capillary with a sample is mounted. The collected fluorescence flux by a quasi-parallel beam is directed to a light filter 5 and then to a photodetector 6. The beam passing through the capillary goes out between two mirrors. Part of the radiation incident on the capillary will be reflected, and a CCD array 7 is installed in the direction of the reflected beam.

 Precise alignment improves the sensitivity of measurements and provides linear calibration characteristics.

Precision adjustment of the fluorimeter can be performed in the following ways:
a) the movement of the exciting beam or
b) displacement of the capillary perpendicular to the plane in which they are located.

 The maximum displacement can be limited by the size of the outer diameter of the capillary, that is, it is enough to move the exciting beam or capillary by the value of the outer radius of the capillary left or right relative to its center.

 The adjustment can be controlled by the laser beam reflected from the capillary in the visible region of the spectrum by the eye and in the invisible region of the spectrum using the CCD matrix 7, since part of the exciting beam will be reflected.

 In this case, two rays reflected from the capillary are observed, running parallel to each other. Figure 2 illustrates how two reflected beams (2 and 3) are obtained when the exciting radiation passes through the capillary. They are formed at the interface between two media: air and the material of which the capillary consists. When incident on the capillary, the incident ray 1 is refracted and passes inward, but a small part of it will be reflected at the air – material interface — ray number 2. When passing through the capillary, the ray undergoes refraction and ray 3 is reflected from the last material – air interface. The rest, most of the radiation will come out (beam 4). Reflection at the material-sample interface (the sample fills the inner diameter of the capillary and has a refractive index close to that of the material of the capillary) is much smaller and slightly affects the formation of the reflected light flux. Both beams are recorded by a CCD matrix 7.

 These two reflected beams make it possible to precisely align the excited radiation to the center of the capillary. Figure 3 shows that if the excited beam 1 does not coincide with the center of the capillary, the CCD will register two reflected beams, 2 and 3, which are located at a certain distance from each other. The greater the deviation of the incident laser beam from the center of the capillary, the greater the angle of reflected beams relative to each other, and the correspondingly, the greater the distance between the light spots on the CCD.

 Moving the laser or capillary perpendicular to the optical axis on the CCD matrix, one can observe the divergence or fusion of the two reflected beams, depending on whether the incident beam 1 will be further or closer to the center of the capillary. The passage of the exciting beam 5 exactly through the center of the capillary corresponds to the almost complete coincidence of the light spots on the CCD.

 Aligning optical systems in fluorimetric detectors with respect to the maximum fluorescence signal is laborious, complex, and not always optimal. In addition, with such methods, the capillary must be filled with some kind of fluorescent substance, and in the proposed method, the capillary can be empty inside, which facilitates and speeds up the alignment process.

 The alignment method of the proposed detector with a moving radiation source and / or capillary can be used when tuning detectors with lasers operating in different spectral ranges, especially it is indispensable for radiation sources whose spectral region lies outside the visible region of the spectrum.

 With this alignment method, you can move either the laser or the capillary depending on the design capabilities of the device, which gives flexibility in the design of the device.

Sources of information
1. Pat. 3946239, G 01 N 21/38, US, 03.23.76. "Ellipsoidal cell flow system".

 2. Pat. 4088407, G 01 N 1/10; G 01 N 21/52, US 9.07.76. "High pressure fluorescence flow-through cuvette."

 3. Pat. 5614726, G 01 N 27/00; G 01 N 21/31, US 03.23.95. "Automated optical alignment system and method using Raman scattering of capillary tube contents".

Claims (2)

 1. A fluorimetric detector containing a light source, focusing optics, a capillary with a sample, a parabolic mirror, a light filter, a photodetector, while the detected volume of the sample is located in the focus of the mirror so that the fluorescence radiation collected by the mirror forms a quasi-parallel beam, characterized in that that it is equipped with a spherical mirror, the focus of which is conjugated with the focus of a parabolic mirror, and the mirrors are located on one axis perpendicular to the plane in which the axis in of excitation radiation and a capillary, and contains a CCD matrix of an additional photodetector installed in the direction specularly reflected from the capillary with a beam breakdown, while the radiation source and / or capillary are movable with the possibility of movement in a direction perpendicular to their axes.  2. The fluorimetric detector according to claim 1, characterized in that the axis of the radiation source is located relative to the capillary at an angle of Brewster.

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