GB2218511A

GB2218511A - Apparatus for the automatic photometric analysis of small specimens

Info

Publication number: GB2218511A
Application number: GB8909010A
Authority: GB
Inventors: Karl-Ernst Suesse; Matthias Schmidt; Bernhard Knull
Original assignee: Carl Zeiss Jena GmbH
Current assignee: Jenoptik AG
Priority date: 1988-05-09
Filing date: 1989-04-20
Publication date: 1989-11-15
Anticipated expiration: 2009-04-20
Also published as: SE8901623D0; GB2218511B; DE3902609A1; DD271953A1; SE8901623L; FI890566A; FI890566A0; GB8909010D0

Description

2218S l 1

DESCRIPTION APPARATUS FOR THE AUTOMATIC PHOTOMETRIC ANALYSIS OF SMALL QUANTITIES OF A SPECIMEN

The invention relates to an apparatus for the automatic photometric analysis of small specimens, whereby the transmission (absorption) and the fluorescence of predominantly liquid specimens can be determined, particularly in the case of specimens less than the order of 10)jl. The fields of application lie in medicine, veterinary medicine, agriculture, biology, biotechnology and chemistry.

Known photometer arrangements usually comprise a light source, a specimen receptacle with a holder, a photoelectric receiver and electronics for the operation, the control and the evaluation of data. Furthermore, a spectral selecting system is frequently incorporated. Image-forming systems serve to guide electromagnetic waves between their source and the receiver. In order to perform analysis, the photometer unit is connected to peripheral devices, such as devices for preparing the specimens; devices for feeding the specimen into the measuring vessel (metering technology); devices for feeding the vessel, or a plurality of vessels one after the other, into the path of the electromagnetic radiation; means for tempering; and devices for removing the specimens and cleaning the measuring vessels.

1 1 It Co-operation of the photometer unit with the peripheral devices, and the formation and evaluation of the measured value, may be controlled by microprocessors', hence permitting the automatic measurement of a plurality of specimens.

One disadvantage of the above-mentioned technical solution resides in the fact that a considerable expenditure on apparatus is necessary for the simultaneous detection of a plurality of identical or different specimens, and for automating the analysis. Limits are set to the miniaturisation of the photometer unit for its use "in situ".

Further to this, photometric arrangements are known which utilise the interaction of evanescent decaying waves with the specimen.

In EP 181 291, it is proposed to guide electromagnetic waves through a liquid specimen located in a special vessel by means of fibre optics.

In EP 206 433, the electromagnetic wave is guided in a prism by means of total internal reflection. The prism is immersed in the specimen to be investigated, and the decaying component of the guided wave interacts with the specimen.

The interaction of evanescent components of guided waves with specimens to be investigated is highly suitable if components of the specimen absorbed on the waveguide are to be detected (EP 170 376).

i i x 1 4_ However, the known technical solutions utilising the interaction of evanescent components of guided waves with the specimen do not offer the possibility of extensive miniaturisation of the photometer, process control, simultaneous detection of a plurality of specimens or extensive automation.

The aim of the present invention is to achieve a high rate of processing of specimens coupled with low expenditure on apparatus, thereby increasing the universality of use.

In accordance with the present invention, there is provided an apparatus for the automatic photometric analysis of small quantities of specimen, in which electromagnetic radiation spectrally selected in a photometer unit is directed onto a receiver by wave-guiding means after interaction with a specimen, and in which the photometer unit is coupled to means for specimen prepartion and after-treatment in that at least one waveguide path forming part of the wave guiding means is incorporated in a carrier and at least sections of its surface are adapted to be in optical contact with at least one specimen when the apparatus is in use. Means, incorporated in or on the carrier, for coupling electromagnetic radiation into the waveguide path, are provided for each waveguide path. The carrier is secured to a conveying means j -4which establishes contact between the carrier and the means for specimen preparation and after-treatment.

Since the wave guide paths have a higher refractive index than the carrier material, the electromagnetic radiation is guided to the specimen to be investigated and, after interaction with the latter, to the receiver or another optical element.

In order to avoid losses during guidance of the radiation, the carrier and the waveguide paths are made from a material which is transparent to the guided radiation.

Either specimen vessels mounted on the specimen carrier, or a passage guided through the carrier, can be provided for receiving the specimens to be investigated.

If specimen vessels are mounted on the carrier, the implemented waveguide paths extend in directions parallel to the surface of the carrier and terminate at the surface. The bottom or a wall of each specimen vessel is then formed by the waveguide carrier having at least one implemented waveguide path.

The evanescent tails of the waves guided in the waveguide paths are absorbed by reason of their interaction with the specimen. The absorption of the specimen and magnitudes (for example concentration) derived therefrom can be determined by measuring the t -5intensity of the guided wave by a receiver at the end of the waveguide path, before and after introducing the specimen into a specimen vessel.

Since an effective interaction of the evanescent tails of a guided wave with the specimen is only effected in one layer with a thickness in the order of magnitude of the wavelength of the guided radiation mode, very small quantities of specimen are sufficient for a measurement.

In order to distribute these small quantities of specimen uniformly over the waveguide path after pipetting, means for distributing the specimen in the cell are advantageous, and, in the simplest case, reside in pressing a cover onto the specimen, analogously to the cover glass known in microscopy. The rate of analysis can be increased by disposing a plurality of specimen vessels on each waveguide path. For this purpose, the specimens are pipetted successively into the specimen vessels, the following measuring cycle being observed:

1. Measuring the intensity of radiation at the end of the waveguide path; 2. Pipetting into the first specimen vessel and measuring at the end of the waveguide path; and 3. Pipetting into the second specimen vessel and measuring at the end of the waveguide path, etc.

8 1, It is advantageous to coat the boundary of the specimen vessels which is formed by the waveguide path or which carries the waveguide path. Thus, the sensitivity of detection is increased by solvent-rejecting and specimen-absorbing substances whose coating thickness is smaller than the wavelength of the guided mode and which does not absorb the latter.

In addition to taking measurements of transmission and absorption, it is also possible to use the described device to measure the fluorescence of the specimen stimulated by evanescent waves. For this purpose, a further boundary wall of the specimen vessels is in the form of a radiation receiver. or a radiation receiver disposed adjacent to a transparent boundary wall of the specimen vessel.

Surface regions of the specimen in the immediate vicinity of the waveguide are measured by the described interaction with the evanescent tails of the guided wave. If volumetric ranges are to be included in the measurement, that wall of the specimen vessel which is at right angles to the longitudinal direction of the waveguide path is in the form of a coupling-out prism for electromagnetic radiation from the waveguide path. The refractive index of this coupling-out prism is equal to, or greater than, that of the waveguide path.

1 1 In order to measure the absorption, that wall of the specimen vessel which is opposite the coupling-out prism is in the form of a radiation receiver, or that wall of the specimen vessel which is opposite the coupling-out prism is of transparent construction, and a radiation detector is disposed downstream thereof.

In order to measure the fluorescence, the wall in the form of a radiation detector, or the receiver disposed downstream thereof, is positioned at right angles to the coupling-out prism.

Alternatively, a coupling-out grid which is located on the waveguide path and forms a wall of the specimen vessel, may be used instead of the coupling-out prism.

If the specimen is not pipetted into mounted vessels, and is pumped through a passage in the carrier,, the waveguide paths are formed in the carrier in such a way that they form a wall of the passage, at least in sections.

The photometric measurement is effected by the interaction of the decaying components of the guided waves with the specimen.

Measurements of fluorescence may also be performed by constructing that wall of the passage which is located opposite the waveguide path as a radiation receiver, or by making the wall transparent and disposing a radiation receiver downstream thereof.

0 Simultaneous measurements of a pluality of samples, as well as identical samples at various wavelengths, are made possible by having a plurality of waveguide paths formed in or on the carrier and in which polychromatic or monochromatic radiation is used. In the use of polychromatic radiation, that end of the waveguide at which the radiation emerges is imaged by way of an imaging holographic grid onto a receiver arrangement such as, for example, a diode array or a CCD-line or-matrix. Thus, by virtue of the interaction of the evanescent waves with the specimen, it is possible simultaneously to measure the spectral dependenpe of the specimen.

Either a coupling-in grid, a coupling-in prism, or a monochromatic source of laser light integrated into the carrier at the beginning of each waveguide path, is provided for coupling monochromatic radiation into the waveguide paths.

The effect wherein a waveguide leads, at a predetermined wavelength, to radiation which is coupled into the waveguide at a predetermined angle, the so-called "zig-zag angle", is utilised for the coupling-in of monochromatic radiation by means of a grid or prism. Thus, it is possible to couple monochromatic radiation into the waveguide paths from a polychromatic parallel beam by prescribing the angle -9of incidence onto the grid or onto the prism. Therefore, the polychromatic beam is directed onto the coupling-in prism or grid by way of a reflector which is independently variable for each waveguide path.

If convergent polychromatic radiation impinges on the coupling-in grid or prism, polychromatic radiation is coupled into the waveguide path.

Advantageously, the sources of laser light at the beginning of a waveguide path are lasers with distributed feedback which are pumped either optically or electrically.

Also, electrical conduction paths can be integrated in the carrier for tempering the specimen and to regulate the tempering of the specimen in a defined manner.

An automatic cyclic process for the sequence of measurement can be achieved in that the carrier is brought successively and cyclically into contact with the means for metering the specimen and distributing the specimen as means for preparing the specimen, and with the means for after treatment which resides in the cleaning of the measuring surfaces, so that-an interaction can take place.

An advantage of the present invention therefore lies in the possibility of providing a compact photometer unit for measuring even the smallest _10quantities of specimens'and which, in various embodiments, enables a plurality of discrete specimens, as well as continuously fed specimens, to be analysed by the same or different wavelengths simultaneously and automatically and which can be integrated in peripheral technology with low expenditure on apparatus.

The invention is described further hereinafter, by way of example only, with reference to the accompanying diagrammatic drawings, in which:

Fig.1 is a perspective view of one embodiment of a carrier in accordance with the present invention; Fig.2 is a view of a further embodiment of a carrier in accordance with the invention; Fig.3 shows a first specimen vessel; Fig.4 shows a second specimen vessel; Fig.5 is a view of a further embodiment of a carrier which is suitable for the measurement of a flow; and Fig.6 shows an arrangement of carriers, in accordance with the invention, on a rotating drum serving as a conveying means.

Referring to Fig.1, a coupling-in grid 2 and two waveguide paths 3 and 4 are provided in or on a carrier 1. Specimen containers in the form of cells 6, 7, 8 and 9 are mounted onto the carrier 1 in such i 5:

1 way that their bottoms are formed by the carrier 1 having the waveguide paths 3, 4 extending on its surface, the side.walls of these cells being made from material whose refractive index is less than the refractive index of the waveguides. Polychromatic beams 10,11 emanating from sources of radiation (not illustrated) are directed onto the coupling-in grid 2 by way of reflectors 12,13 which are adjustable independently of one another. Detectors 14,15 are disposed beyond the waveguide paths 3, 4 at a short distance of approximately 0.1 mm.

The beams 10,11 directed onto the coupling-in grid 2 by way of the reflectors 12,13 excite radiation modes of differing wavelengths X in the waveguide paths 3, 4 dependant on the angle of incidence. The wavelength is determined by the equation:

X = d (sin c, + sin 'I') for the diffraction on the grid (1st order) in the case of oblique incidence of radiation., in which d = grid constant C. = angle of incidence V,,= llzig-zag" angle of the guided radiation in the waveguide.

The effectiveness of coupling-in is increased by a blazed grid.

1 It will be appreciated that the coupling-in grid 2 may be replaced by a prism which acts in an equivalent manner. The photometric measurement of predominantly liquid specimens located in the specimen containers is effected by the absorption of the evanescent tails of the radiation modes, guided in the waveguide paths 3, 4, by the specimens, a layer thickness of a fewpm being adequate.

In order to increase the sensitivity of measurement-, it is possible to apply to the surface of the carrier 1 a solvent-repelling and specimenabsorbing substance as a coating having a thickness of from 1 nm to 10 nm. By way of example, phenyl silane is suitable in the case of water-soluble specimens.

In a further embodiment as illustrated in Fig.2, radiation sources 16,17 can be incorporated in or on the carrier 1 in addition to the waveguide paths 3,4. These radiation sources are in the form of distributedfeedback-lasers (DFB-1asers) which are optically pumped by the beams 10. 11. The wavelength coupled into the waveguide is determined according to the period of modulation of the refractive index of the M laser.

Alternatively, it is possible to use specimen vessels 18 (Fig.3) for the described solutions, one cell wall of each vessel being in the form of a W 511, 1 -13coupling-out prism 19 having a refractive index greater than, or equal to, the refractive index of the waveguide paths 3 or 4. The cell wall located opposite the coupling-out prism 19 then constitutes a radiation receiver 20. Photometric measuring with these cells is not effected by the absorption of. evanescent waves, but by the absorption of the radiation which is coupled out of the waveguide paths 3,4 by the coupling- out prism 19 and which is recorded by the detector 20 after passing through the specimen in the specimen vessel 18.

When measuring fluorescence, a wall directed at right angles to the coupling-out prism 19 can be in the form of a rdiation receiver. It will be appreciated that, in both types of measurement described, it is also possible to dispose the radiation receiver beyond the specimen vessel wall which must then be transparent to the radiation.

The specimen vessel 21 shown in Fig.4, and whose opening is directed upwardly, is optically connected to the waveguide path 3 (or 4) by way of a coupling-out grid 22 which is disposed directly on the waveguide path 3 (or 4). A photodiode 23 serving as a receiver is mounted on the specimen vessel wall located opposite the coupling-out grid 22.

Analogously to the example shown in Fig.3, the specimen vessel wall, beyond which the photodiode 23 i is disposed, may itself also be in the form of a photodiode.

In the same way as in the example of Fig.2, radiation sources 16,17 are integrated in the carrier 1, shown in Fig.5. Specimen fluid for continuous analysis may be pumped through a passage 24 extending through the carrier 1. A portion of one wall of the passage 24 is formed by sections of the waveguide paths 3,4.

Furthermore, in the embodiment of Fig.5, one wall of the passage may be in the form of a radiation receiver, or a radiation receiver may be disposed beyond the wall, in order to be able to take measurements of fluorescence.

If polychromatic radiaiton is to be guided in the waveguide paths 3,4 for the purpose of investigating specimens, the ends of the waveguide paths 3, 4 in all examples form entry gaps of a polychromator in which the radiation entering is directed onto a receiver arrangement by means of an imaging holographic grid. Sicne an adequate number of examples of a polychromator arrangement of this kind exist and are well known in the art, it is not shown in the present drawings.

Furthermore, electrical conduction paths may be provided for tempering purposes in all the variants of the carrier.

1 -is- Referring to Fig.6, three carriers 1, V, V' are secured to a carrier frame 26 which is rotatable about an axis 25, so that the carriers can pass cyclically through three different positions in which metering, measuring, cleaning and drying are effected.

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Claims

-16CLAIMS

1. An apparatus for the automatic photometric analysis of small quantities of specimens, in which electromagnetic radiation spectrally selected in a photometer unit is directed onto a receiver by wave-guiding means.after interaction with a specimen, wherein at least one waveguide path forming part of the wave-guiding means is provided in or on a carrier, at least sections of whose surface are adapted, in use, to be in optical contact with at least one specimen, and means in or on the carrier for coupling electromagnetic radiation into the waveguide path being provided for the or each waveguide path.

2. An apparatus as claimed in claim 1, wherein the waveguide paths extend parallel to and terminate with a surface of the carrier, and at least one specimen vessel is mounted on each waveguide path, so that a spatial boundary is formed by the carrier and optical contact is established.

3. An apparatus as claimed in claim 1, wherein the waveguide paths form, at least in sections, a wall of a passage which is provided as a specimen vessel in the carrier and through which the specimen to be investigated can be pumped.

4. An apparatus as claimed in claim 2 or 3, wherein electrical conduction paths pass through the carrier for heating the carrier.

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5. An apparatus as claimed in claim 1, wherein a first grid is provided between the waveguide path and the specimen vessel for establishing optical contact.

6. An apparatus as claimed in any of claims 2 to 4, wherein at least one second grid or prism is provided 4s a means for coupling converging polychromatic radiation into the wayeguide paths.

7. An apparatus as claimed in any of claims 2 to 4, wherein at least one second grid integrated in the carrier is provided for coupling spectrally selected radiation into the waveguide paths, parallel polychromatic radiation being directed onto the said second grid at a variable angle of incidence.

8. An apparatus as claimed in any of claims 2 to 4, wherein at least one prism is provided as means for ccupling spectrally sel-ected radiation into the waveguide paths, parallel polychromatic radiation being directed onto the said prism at a variable angle of incidence.

9. An apparatus as claimed in any of claims 2 to 4, wherein an optically or electrically pumped source of laser light for each waveguide path is integrated in the carrier as a means for coupling spectrally selected radiation into the waveguide paths.

10. An apparatus as claimed in claim 9, wherein the wavelength of the laser light is variable.

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11. An apparatus as claimed in any of claims 2 to 10, wherein the boundaries of the specimen vessel which are in optical contact with a waveguide path are covered with a solvent repelling and specimen absorbing coating which is from 1 nm to 10 nm thick.

12. An apparatus as claimed in claim 2, wherein the wall of the specimen vessel which faces the means for coupling-in the electromagnetic radiation is in the form of a coupling-out prism.

13. An apparatus as claimed in claim 2, 3, 5 or 12, wherein a further specimen vessel wall is in the form of a radiation receiver.

14. An apparatus as claimed in claim 2, 3, 5 or 12, wherein a radiation receiver is disposed beyond a further specimen vessel wall which is transparent to radiation.

15. An apparatus as claimed in claim 5, wherein the radiation-emitting end of at least one waveguide path serves as the entry gap of a polychromator, and the radiation emitted is directed onto a receiver arrangement by way of an image-forming holographic grid.

15. An apparatus for the automatic photometric analysis of small quantities of specimens, in which electromagnetic radiation spectrally selected in a photometer unit is directed onto a receiver by j 41 4 1 -19wave-guiding means after interaction with a specimen, wherein at least one waveguide path forming part of the wave-guiding means is provided in or on a carrier, at least sections of whose surface are adapted, in use, to be in optical contact with at least one specimen, and means in or on the carrier for coupling electromagnetic radiation into the waveguide path are provided for the or each waveguide path, the carrier being secured to a conveying means which establishes contact between the carrier and a means for specimen preparation and after-treatment.

16. An apparatus for the automatic photometric analysis of small quantities of specimens, substantially as hereabove described, with reference to and as illustrated in the accompanying drawings.

Published 1989 at The Patent Office. State House. 66"71 High Holborn. London WC1R 4TP. Further copies maybe obtamedfrom The Patent Office.

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