GB2062889A - Fluorimetry - Google Patents

Abstract

An apparatus for the fluorometric evaluation of samples with a radiation source, having a source (8) of primary, exciting radiation directed onto a sample vessel (1) by means of an arrangement of lenses, mirrors and filters (10, 14, 6, 7, 9) to cause the sample in the vessel (1) to produce fluoroscence, which can be evaluated by an optical and electronic receiving arrangement (11, 12, 15, 4). The mirror (6) located between the radiation source (8) and the sample vessel (1) is permeable to the exciting radiation but reflects fluorescent radiation, whereas the mirror (7) located between the evaluation element (4) is permeable to fluorescent radiation but reflects the exciting radiation, this arrangement maximising the efficiency of the use of the exciting radiation whilst minimising background radiation at the evaluation element (4) <IMAGE>

Description

SPECIFICATION Apparatus for the fluorometric evaluation of samples The present invention relates to an apparatus for the fluorometric evaluation of samples with a radiation source, the primary exciting radiation causing fluorescence in a sample, and having optical elements for the alignment of the primary radiation with the sample, a container for the sample and receiving and evaluating elements for the fluorescence radiation, whereby the optical elements are fixedly arranged filters, lense and mirrors.

In a fluorometer a sample is irradiated by exciting light and hence emits fluorescent light. Both are subjected within the sample to absorption according to the Lambert-Beer's Law, resulting in irregular comprehension of the measurement volume. This irregular comprehension of the measurement volume has hitherto been reduced by extensive dilution, to negligible values, but this causes the detection limit to suffer therefrom (Christoffers, D., Fluorometerische Lysinbestimmung in Getreidemuehlen Diss. Hannover 1976, S. 1 ff). An almost complete utilisation of exciting and fluorescence light has hitherto been impossible, since, in order to keep the background dark, the exciting and measurement beams of rays assume a right angle position to one another.Transmitted light beams have the disadvantage that residues of the exciting light, which also penetrate the suppression filter, produce a considerable background interference at the receiver.

Fluorometers are used more especially for the reproducible determination of the chlorophyl content of plants (e.g. algae). By evaluating the fluorescence signal an indication of the photosynthetic activity and the effect of substances (e.g. herbicides) on the timed course of the photosynthesis reaction can be produced, if it is possible to carry out the stimulation of the fluorescence pulsewise with sufficient intensity and evaluate the individual pulses separately.

Chlorophyl previously was extracted and photometrically measured with chemical aids.

This required numerous operations which can be carried out only in a laboratory, and thus field measurements are not possible. Alternatively, the chlorophyl content was determined from the fluorescence of live plants, but with the disadvantage that the quantity yield of fluorescence varied in an unforeseeable manner upon the physiological state (Govindjee, The Absorption of Light in Photosynthesis, Scientific American, Dec. 1 974; Samuelson G. et al, The Variable Chlorophyl; a fluorescence as a measure of photosynthetic capacity in algae, Mitt. Internat. Verin Limnol. 21, 1978). This dependence is based on the competitive behaviour of photosynthesis and fluorescence.

The photosynthetic activity has hitherto been determined by a chemical determination of the oxygen production or the carbon diox-* ide consumption (i.e. by marking with radioactive 14C). These methods require long exposure periods (some hours) and numerous manipulations which cannot be carried out in the field. In (Samuelson, G. et al, The Variable Chlorophyl; a fluorescence as a measure of photosynthesis in algae, Mitt. Intern. Verein Limnol. 21, 1978) a conclusion is drawn from the difference of the fluorescence before and after poisoning with DCMU on the photosynthetic activity. What is disregarded is that the measurement value before posioning is dependent neither upon preliminary exposure nor preliminary exposure and other physiological factors, which makes measurement unreliable.

Herbicide analyses are carried out after a plurality of completely different chemical methods, mainly extraction with subsequent gas or liquid chromatographic separation. An alternate method is described in German PS 2626915.

The aim of the invention is to present an apparatus producing a uniform irradiation with exciting light and identical evaluation of all component with the evaluation of fluorescent light (independent of sedimentation and homogeneity), whereby exciting and fluorescent light are exploited almost without loss and operating with low exciting energy, so that no bleaching-out effects occur and the chlorophyl content, the photosynthetic activity and/or the course of the photosynthesis reaction is also ascertainable in natural, unprepared populations.

According to the present invention there is provided an apparatus for the fluorometric evaluation of samples with a radiation source of primary exciting radiation to produce fluorescence in a sample, optical elements for alignment of the primary radiation on the sample, a container for the sample, and receiving and evaluating elements for the fluorescent radiation, in which the optical elements including fixedly arranged lenses and/or mirrors, and in front of the sample container, seen in direction of the path of the primary radiation, a first mirror, permeable to the primary radiation and reflecting the fluorescence radiation, is located, and behind the sample container a second mirror, permeable to the fluorescent radiation and reflecting the primary radiation, is arranged, and the fluorescence light passing through the second mirror is directed against the receiving and evaluating elements.

The particular advantages of the invention reside in that the exciting radiation penetrates through the measuring volume and is reflected by the selective mirror coating of the sample. Due to this, the sample, after being irradiated from one side, is again illuminated from the other side, even if only with a somewhat lesser intensity. The same applies to the fluorescence light. The light escaping in the direction of the exciting radiation is conducted directly to the receiver. Due to this: 1.

the absorption conditioned irregularities in evaluating the measurement volume are substantially compensated; 2. exciting and fluorescent light is utilised to a much improved extent; and 3. with a simultaneous sensitivity increase, the proportion of exciting radiation is substantially reduced at the emission signal.

The overall fluorescence yield increases thereby. Further filters in the exciting and fluorescent beam paths reduce the residual light which interferes in conventional arrangements, down to an unmeasureable level.

These filters may also be replaced by selective mirrors.

The production of exciting radiation occurs by means of a flash tube similar to those in electronic flash devices for photography. The flash technique has hitherto not been used, however, for energy conservation or to maintain a definite period of illumination after preceding dark intervals. Energy conservation is essential in field devices (weight of batteries/accumulators). Maintaining a definite dark interval and producing definite light flashes are essential in numerous fluorescent dyestuffs which can decompose subject to the influence of the exciting light, more especially in the chlorophyl fluoresence described below.

A lower weight and lesser energy consumption (field device) are also promoted by using a semiconductor photoelement as a light receiver. This renders a stabilised high tension supply unnecessary for the secondary electron multiplier otherwise used in such cases because of low fluorescent light yield. The special selection of an element of smaller time constant thus permits a connection with the flash excitation.

In accordance with the invention therefore a higher linearity with otherwise equal expenditure or use of greater sample sizes and hence maximum sensitivity with equal linearity are enabled. The good utilisation of exciting and fluorescent light permit smaller light sources, lower energy requirement, less damage to the sample. The pulse operation lowers the energy consumption of the light source and hence the weight of the battery-accumulator.

It makes possible definite dark and illuminating phases with small technical expenditure.

This is important especially for decomposable fluorescent dyestuffs. The semi-conductor detector saves costs and weight. The excitation of the sample downwardly and the evaluation from two sides of the fluorescent radiation cause an improved investigation of sedi mented particles compared with the normal vertical beam paths. The optical elements are mounted in a compact, vibration proof and readily interchangeable form. All transmitting and reflecting components (filters and mirrors), and the sample holder are built into a common block which-should a change of wavelength be necessary-may be interchanged as a whole. Instead of arcuate surfaces selectively treated with vapour deposited films, flat surfaces may also be used.

The invention will be described further, by way of example, with reference to the accompanying drawings, in which: Figure 1 shows a first embodiment of an apparatus according to the present invention; Figure 2 shows an alternative embodiment; and Figure 3 illustrates graphically the timed course of fluorescence after a long dark interval.

Fig. 1 shows schematically the structure of a compact path of beams. The excitation radiation 3 (direction of diffusion or optical axis 2), transmitted by a pulse source 8 (Xehigh pressure flash, Heimann Type 7701), is reflected by the mirror 9, selectively treated with vapour for the excitation frequency (glass mirror with vapour treatment of thorium oxide, such that for A = 682 nm, A = 435 nm transmission or reflection maxima occur), and then passes through an excitation filter 10 (Schott-lnterference filter Type Al 435) to a mirror 14. This mirror 14, of glass treated with thorium oxide evaporation, reflects the excitation radiation incident at 45 (A = 435), selectively.A filter 6, following in the path of beams 2, located in front of a sample vessel 1, is treated with thorium oxide evaporation so that it is transparent for the excitation or primary radiation 3, but which reflects the fluorescene radiation 5 (A = 682) produced in the sample. The sample vessel 1 contains the test substances homogeneously or inhomogeneously mixed, regardless as to whether there is any sediment, and its diameter is 25 mm, its height 30 mm.

In the path of beams 2 located after the sample bush 1, there is a further optical element or filter 7 which reflects the excitatior.

radiation 3 which passed through the sample vessel 1, but transmits the fluorescent radiation 5, by suitable treatment with thorium oxide vapour. The fluorescent light 5 is now reflected at an angle of 45 via a further deflecting mirror 1 5 (corresponds to the deflecting mirror 14). A concave lens 12 having f= 25 cm images the bottom of the flask 1 on a photo-diode 4 (Hamamatsu HTVS 874-8K), and for further suppression of spu rious radiation a further filter 11 is located in front of the photo-diode 4 (Schott Interferen zfilter Type Al 682), which is transparent to the fluorescence radiation. The twice deflected radiation path 2 permits a compact mechani cal structure of the apparatus.

Fig. 2 shows a second embodiment, in the path of beams 2 is right. The same refer ence numerals Have been used for corre sponding parts to those of the device shown in Fig. 1. With a straight path for beam 2 the deflecting mirrors 14 and 1 5 may be dispensed with (it would, of course, also be possible for selective filters 14' and 15' corresponding to the diverting mirrors 14 and 15, having a vertical transparency for excitation light 3 or fluorescent light 5, to be contained in the beam path). Contrary to the arrangement of Fig. 1, the mirror 9 is formed as a focussing mirror whereby the light yield from the source 8 of excitation radiation 3 is increased. A lens 1 3 forms a parallel ray path 2, which, via the excitation filter 10 and filter 6, impacts the sample flask 1.To reinforce the reflection effects of the filters 7 and 6 for excitation of fluorescent radiation 3 or 5, these are formed as spherical mirrors which are each focussed on the sample flask 1. The fluorescent light 5 in turn impacts the detecting element or photo-diode 4 and is evaluated.

Fig. 3 shows the timed course of the fluorescence after a long dark interval, and after poisoning with CMU or DCMU. The varying fluorescence yield with or without this poisoning is a measure for the photosynthetic activity before poisoning. This poisoning (blocking of the electron transport chain of the photosystem II) is to be attained by the addition of CMU.

The fluorescence value before poisoning after a dark interval is in good reproducible relation to the maximum of the graph in Fig.

3 (A). The difference of the fluorescent values before and after poisoning shows a good correlation to the value (B). By measuring the timed course and extraction of the data A and B, the same figures may be obtained, in principle, as by measurement of the fluorescence before and after poisoning.

The measure of the photosynthetic activity is derived as already described from the difference of the fluorescence value before and after poisoning or from the magnitude of the fluorescence values A and B. The use of a dark interval, the use of CMU instead of DCMU or the extraction of the fluorescence value from the graph are essential.

Numerous herbicides contain CMU, DCMU or other substances which block the electron transport chain of the photosystem II and show the effect on the fluorescence. If in an intact algae suspension suspected water is added, then the timed fluorescence effect shows the possible presence of a herbicide, which has the same mechanism.

Chlorophyl determination is hence possible without extraction steps and without the errors of a simple fluorescence measurement under specific conditions (dark interval), a measurement of the photosynthetic activity under the same advantages succeeds and presence of a number of herbicides may be proved substantially instantaneously without extraction steps and chromatographic determinations and similar chemical manipulations.

Claims (5)

1. An apparatus for the fluorometric evaluation of samples with a radiation source of primary exciting radiation to produce fluorescence in a sample, optical elements for alignment of the primary radiation on the sample, a container for the sample, and receiving and evaluating elements for the fluorescent radiation, in which the optical elements including fixedly arranged lenses and/or mirrors and in front of the sample container, seen in direction of path of the primary radiation, a first mirror, permeable to the primary radiation and reflecting the fluorescence radiation is located, and behind the sample container a second mirror, permeable to the fluorescent radiation and reflecting the primary radiation, is arranged, the fluorescence light passing through the second mirror is directed against the receiving and evaluating elements.

2. An apparatus according to claim 1 in which in front of the radiation source seen in the direction of the path of the primary radiation an additional mirror is arranged, which reflects primary radiation from the radiation source and also primary radiation reflected from the second mirror in the direction of the path of the primary radiation.

3. An apparatus according to claim 1 and 2, in which the mirrors are of flat or spherical structure.

4. An apparatus according to any preceding claim in which between the radiation source and the first mirror, an excitation filter is provided for preselection of the primary radiation, and, between the second mirror and receiving elements, a further fluorescence filter is arranged.

5. An apparatus for the fluorometric evaluation of samples substantially as hereinbefore described with reference to and as illustrated in the accompanying drawings.