FI70086C - ANCILLATION OF FLUORESCENT TURBIDITY LUMINESCENS ELLER ABSORPTION - Google Patents

Description

70086

Apparatus and method for measuring fluorescence, turbidity, luminescence or absorption

The invention relates to an apparatus for riveting both fluorescence, turbidity, luminescence and absorption from liquid samples.

Several different devices are known for separately measuring the fluorescence, turbidity, luminescence or absorption of a liquid sample. Some devices are also known in which two or more of the above-mentioned functions are combined. In this case, however, it has not been possible to achieve as good performance in any of the measurements as with the device designed for one measurement.

In this connection, it is also known to take advantage of the total internal reflection of the cuvette. For example, DE 3 122 895 discloses a device in which a light beam passes through a chamber filled with a horizontally flowing sample and in which the absorbance is measured using total reflection. What is special about this device is that? mi the refractive index of the wall of the sampling vessel is smaller than that of the dimensions-20 of the liquid, whereby the total reflection occurs from the inner surface of the vessel.

Photometric measurement through a sample using a vertical light beam is known, for example, from patent publications FI 57665 and FI 55093.

The object of the present invention is to provide a device whose performance in all measurements is state-of-the-art and in particular in fluorometry substantially better than conventional devices.

The object of the invention is achieved by the means set out in claims 30. In the device according to the invention, the sample to be examined is located in a cuvette whose wall material has a refractive index greater than that of both the liquid to be measured and the surrounding medium. The cuvette is cylindrical and of its two 70086 '). . * · · - ‘· · c. * - ««.

one end perpendicular to the axis of the cylinder is open and facing upwards. The bottom of the cylinder is transparent. Most of the optics of the device are located under the cuvette. Immediately below the cuvette is a lens system to which an optical fiber bundle is rigidly attached. The central axis of the cuvette, the optical axis of the lens system, and the central axis of the fiber bundle head converge. The fiber bundle has four zones: a circular central zone, two annular outer zones, and an annular intermediate zone between the central zone and the outer zones. The intermediate zone does not contain optical fibers. The fiber zone in the middle directs light from the light source to the cuvette. The outer fiber zones are used to conduct light from the cuvette to the light detector.

The purpose of the lens system is to collimate the light emanating from the central zone so that substantially the entire sample becomes illuminated by light rays parallel to the walls of the cuvette. The purpose of the lens system is also to collect radiation from the region of the bottom of the cuvette to the outer zones at an angle less than a certain boundary angle to the central axis of the cuvette.

20 The abovementioned cut-off angle must be less than 6 ° from the axis of the cuvette; the maximum light collection efficiency is achieved at an angle of 6 °.

The two most important advantages of the device according to the invention are that the sample becomes evenly illuminated and that an equal proportion of the light emitted by the 25 samples is passed to the detector from each point of the sample. In fluorometry and nephelometry, both advantages are essential to the operation of the device. In luminometry, where the sample is not illuminated, the uniformity of illumination is irrelevant. Thus, the known advantages of vertical measurement are valid in these three measurement methods, and in the absorption measurement as is known in the art: The measurement result depends only on the total amount of substance to be measured in the cuvette and not on the amount of solvent or possibly uniform deviation of the substance to be measured in the cuvette; the sensitivity of the measurement can be adjusted by changing the amount of liquid in the cuvette; see, e.g., Suovaniemi, O., "Performing n 70086 ance and properties of the Finnpipette Analyzer System", Proceedings of the Second National Meeting on Biophysics and Biotechnology in Finland, Kairento, AL, Riihimäki, E. and Tarkka, P. , Eds., Pp. 1δ3 ~ ΐ87 (1976), and Suovaniemi, 0., 5 Järnefelt, J., "Discrete Multichannel Analysis Systems", International Laboratory, April 1982 (and American Laboratory, June 1982).

In addition, the invented apparatus is also well suited for measuring a fluorescently labeled antibody attached to microspheres.

The operation of an exemplary device implemented according to Figures 1 and 2 in fluorometry, luminometry, nephelometry and absorption geometry is shown below.

This limit value is justified as follows (see

Figure 15 2): The radiation emitted from any part of the liquid sample at an angle of more than Al ° to the axis of the cuvette 1 exits the cuvette through its side wall 2. The radiation emitted at an angle of less than 41 ° to the axis of the cuvette experiences total reflection and continues to propagate within the cuvette while the absolute value of the directional angle remains unchanged. If the barely just reflected beam of light now moving at an angle of Jil ° reaches the bottom 3 of the cuvette, then according to Snellius' law it changes its directional angle so that it is 6l ° in the air space below the flat-bottomed cuvette. The given kul-25 ma values are true when the refractive index of the sample is 1.33. It should be noted that if the wall and bottom of the cuvette are of equal thickness, they do not affect said orientation angles, even if their refractive index is greater than 1) 33 · Each orientation angle between 0 ° ... 6 ° corresponds to a certain transverse distance from the fiber system in the focal plane 4 of the lens system. from the center of the head.

In fluorometry, the excitation light passing along the central fiber zone A passes through the lenses 5 and 6 and then forms a bundle of parallel light rays 35 which illuminates the contents of the cuvette uniformly and is thus limited so that the vertical walls of the cuvette do not become illuminated. Some of the excitation light is reflected back from the surface of the lenses 70086 and from the inner and outer surfaces of the bottom of the cuvette and the free surface of the liquid. From those surfaces which are cut perpendicularly by the excitation light beam, it is reflected back to its starting point. Since zone A is not a point light source, some of the reflected light 5 falls outside it. The intermediate zone B is a protection zone whose outer radius is, for example, three times larger than that of zone A. The rays reflected from the perpendicular surfaces which do not fall in zone A fall into zone B and do not enter the part of the fiber bundle 7 from which the emission is measured. If the sample fluoresces, it emits light in all directions with a wavelength greater than that of the excitation light. The total reflection from the vertical wall of the cuvette causes any light beam whose direction angle inside the sample, measured from the downward direction of the axis of the cuvette, is less than 1 °, exits the sample through the bottom 3 of the cuvette. As the light beam moves from the sample to the air, its directional angle increases so that the directional angle of the beam with a directional angle in the sample of ^ 1 ° in air is 61 °. The lenses 20 refract all incident light rays having an orientation angle of less than 61 ° into the focal plane at the end of the fiber bundle. In the combustion plane, the emission light passes to the fibers in the outer belt zones C and D and down to the light detector.

25 In luminometry, the sample emits light due to a chemical reaction. The device collects the emitted light and directs it to the light detector in exactly the same way as in fluorometry. The only difference, then, is that in luminometry, the light source is not in operation.

30 Nephelometry measures the light scattered by solid particles in a sample, ie the light that deviates from its direction. The light source used in nephelometry is either the same light source as in fluorometry or a light beam from above was cured along its central axis, such as e.g. In the latter case, the light beam strikes the fiber zone A after passing through the cuvette and the lens. The refracted light is measured only using the fiber zone D. Sii-

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• ·. ::: '. ·: · ·:; · - 5 ·; Only those rays of light whose scattering angle is between a ... H10, where a is determined by the width of zone D, shall be blocked. 70086 shall be prevented from entering the light detector along the fibers of zone C.

5 The absorption measurement can be performed in two ways: the light passing through the sample can be measured either with a light detector located above the cuvette or with the same light detector as in other measurement methods. In the latter case, a light-reflecting surface 10 should be placed above the cuvette, which should preferably be opaque white.

The device may have a detachable cuvette and separate optics for each cuvette. The side walls of the cuvette cylinders are transparent or reflective. The light can also be directed to the detector completely without lenses, because the optical fiber also has its own boundary angle, at which the light rays coming at a larger angle do not propagate in the fiber. The bottom of the cuvette, if properly shaped, can also act as a lens.

20 The device may also have a second, reference light detector to which the light is conducted directly from the light source. In this case, the device may have a light exchanger which switches the light of the light source to pass alternately along the light path leading to the cuvette and alternately directly to the reference light detector. The light-25 exchanger can be in three positions, which in its first position causes the light source to pass along the light path leading to the cuvette and in its second position directly to the reference light detector, and in its third position prevents light from entering both light paths. The alternation ta-30 takes place in particular asymmetrically and in such a way that the light path from the light source to the cuvette is open most of the time.

Claims (6)

Apparatus for measuring the fluorescence, luminescence, turbidity and absorption of a liquid sample in a cylindrical cuvette (1) with a transparent base, the wall material (2) having a refractive index higher than both the liquid to be measured and the surrounding medium and adapted to direct light from an optical A) along the central axis of the cuvette, the apparatus comprising an optical system (5; 6) whose optical axis coincides with the central axis of the 10 cuvettes to be measured, and a light detector, characterized in that the optical system (5; 6) is adapted to collect a substantially equal proportion of each from the light emanating from the volume element of the sample by total reflection from the outer walls of the cuvette 10 and that said optical system is adapted to also collimate the light of the light source being introduced into the cuvette. Apparatus according to claim 1, characterized in that the optical system (5; 6) is located below the cuvette or part of it is arranged at the bottom of the cuvette 20 itself and the end of the fiber bundle (A) and the fiber bundle leading from the optical system to the light detector are attached to the optical system. C; D) head. Apparatus according to claim 1 or 2, characterized in that the fiber bundle (C; D) leading to the light detector is arranged concentric around the fiber bundle (A) from the light source so that in the common end plane of the fiber bundles on the optical system they are spaced apart, that there is a ring zone between them. 30 Apparatus according to claim 2 or 3, characterized in that the optical system (5; 6) between the end of the fiber bundles and the bottom of the cuvette is two planar convex lenses, the plane surface of the first (6) being fixed in the common end plane (4) and the second ( 5) The 35 planar surfaces are turned towards the cuvette, the center of curvature of the convex surface of the first lens being at the end of the fiber bundle (A) to which any light from the light source passes. 70086 Z>. Apparatus according to one of the preceding claims, characterized in that on the side opposite to the light source of the hardener there is a second light detector which is used to measure the absorption of the sample. 5 A method for measuring the fluorescence, luminescence, turbidity and absorption of a liquid sample in a cylindrical cuvette (1) with a transparent base, the refractive index of the wall material (2) of which is greater than that of both the liquid to be measured and the surrounding medium. fibers along the central axis of the cuvette, in an apparatus comprising an optical system (5; 6) whose optical axis coincides with the central axis of the cuvette to be measured, and a light detector, characterized in that a substantially equal proportion of each sample volume is collected from the outer walls of the cuvette via an optical system (5; 6) to the light detector and that the light of the light source to be introduced into the cuvette is also collimated by means of said optical system. 8 70086