WO1992014137A1

WO1992014137A1 - Procedure and apparatus for determining the fluorescence of a liquid sample

Info

Publication number: WO1992014137A1
Application number: PCT/FI1992/000029
Authority: WO
Inventors: Hannu Harjunmaa; Pertti Ekholm; Osmo Suovaniemi
Original assignee: Biohit Oy
Priority date: 1991-02-05
Filing date: 1992-02-04
Publication date: 1992-08-20
Also published as: FI910549A; FI910549A0

Abstract

Procedure and apparatus for determining the fluorescence of a liquid sample. The apparatus comprises a cuvette (1) in which the liquid sample is placed; a light source (2); optical means (3) by which is separated the desired wavelength from the light of the light source for forming an excitation light beam (4) and for directing it into the cuvette for producing fluorescence in the liquid sample; a fluorescent light detector (5) for observing the light emitted in a predetermined measuring direction. The fluorescent light detector (5) is arranged from below, relative to the cuvette, to measure from an optically delimited area substantially exclusively internal fluorescence of the liquid sample.

Description

PROCEDURE AND APPARATUS FOR DETERMINING THE FLUORES¬ CENCE OF A LIQUID SAMPLE

The present invention concerns a procedure as defined in the preamble to Claim 1. Furthermore, the invention concerns an apparatus as defined in the pre¬ amble to Claim 5.

In prior art, a number of devices is known which serve the determination of fluorescence from sam- pies presented in a cuvette, such as a microtitre tray. A number of devices is likewise known which serve the determination of the absorbance of samples presented in a cuvette, such as a microtitre tray. Such devices are, for instance Multiskan and Fluoroskan of the company Eflab Oy, the apparatus of the Dynatech Corporation, etc., which are based in a technology presented e.g. in the paper: Suovaniemi, Jarnefelt: "Discrete multichan¬ nel analysis systems", Internat. Laboratory 4, 1082, and in the paper; Suovaniemi: "The vertical measurement principle in photometry", Internat. Biotech. Lab. 5/6, 1984. All these only measure either fluorescence or absorbance because heretofore no attempts to combine said two measuring methods in a simple way have suc¬ ceeded. The matter has certainly been studied and in- novative solutions can be found in the literature (e.g. Harjumaa: "Theory, Design and Immunological Applica¬ tions of a Vertically Measuring Fluorometer", Labsys- tems Research Laboratories Report, 1986; and US 4,678,326). Methods and apparatus of prior art are also embarrassed by the major problem that the background fluorescence of the plastic material of the microtitre tray interferes with the measurement of the fluores¬ cence from the liquid sample. For this reason, known fluorescence measuring devices measure the fluorescence from the topside if the cuvette and an opaque cuvette is employed, which has low or no fluorescence. The use of a non-transparent cuvette further excludes the pos¬ sibility of measuring absorbance from the same micro¬ titre tray because in absorbance assays light is meas¬ ured which has gone through the sample. Moreover, the free surface of the liquid sample is within the field of vision of the fluorescence measurement, a circum¬ stance which detracts from the reproducibility of meas¬ urements.

The object of the invention is to eliminate the drawbacks mentioned.

It is particularly an object of the invention to provide a simple procedure and apparatus by which on the side of fluorescence measurement also absorbance can be measured, without transferring the liquid sample from one apparatus or cuvette to another and without need concessions as regards the performance capacity in either type of measurement.

It is furthermore an object of the invention to provide a procedure and apparatus in the use of which the background fluorescence from the structural material of the cuvette cannot affect the results of measurement.

The procedure of the invention is character¬ ized by that which has been stated in Claim 1. The apparatus of the invention is characterized by that which has been stated in Claim 5.

As taught by the invention, in the procedure the liquid sample is placed in a cuvette; an excitation light beam is directed on the cuvette; and the fluores- cence excited by said beam in the liquid sample is measured. As taught by the invention, the fluorescence is measured from below, relative to the cuvette, on an optically delimited measuring area, substantially ex¬ clusively as internal fluorescence of the liquid sam- pie. When the fluorescence is measured from below rela¬ tive to the cuvette, the measurement will be well re¬ producible because the free surface of the liquid sam- pie is not within the field of vision of the fluores¬ cence measurement.

In an embodiment of the procedure, the emis¬ sion light from fluorescence produced by action of the excitation light beam on the structural components of the cuvette is delimited to lie outside the measuring area. In other words, when the measuring area is delim¬ ited to lie entirely within the cuvette, that part of the cuvette's structure, e.g. of its bottom, where the excitation light beam enters the cuvette is not visible to the fluorescent light detector and therefore cannot interfere with the measurement of the fluorescence from the liquid sample.

In an embodiment of the procedure, the excita- tion light beam is directed into the cuvette from below substantially in vertical direction, and the quantity of light that has passed through the sample is measured in order to determine the absorbance.

In an embodiment of the procedure, in absorb- ance measurements the excitation light beam is directed from below along the central axis of the cuvette; in fluorescence measurements the excitation light beam is displaced, parallelling the central axis of the cu¬ vette, laterally to a distance from the central axis of the cuvette; and the central axis of the fluorescence measuring direction lies on the plane defined by the central axis of the cuvette and the central axis of the excitation light beam, the central axis of the measur¬ ing direction enclosing with the central axis of the cuvette an angle which is in the range of 5 to 50°.

The apparatus of the invention comprises a cuvette in which the liquid sample is placed; a light source; first means for separating from the light of the light source the desired wavelength for producing an excitation light beam and for directing same into the cuvette to cause fluorescence in the liquid sample; and a fluorescent light detector for observing the light caused by fluorescence and emitted in a predeter¬ mined measuring direction. As taught by the invention, the fluorescent light detector is arranged from below, relative to the cuvette, to measure in an optically delimited measuring area substantially only the inter¬ nal fluorescence of the liquid sample.

In an embodiment of the apparatus, the appara¬ tus comprises second optic means for delimiting the emitted light due to fluorescence elicited in the structures of the cuvette, e.g. in its bottom, to be outside the measuring area.

In an embodiment of the apparatus, the appara¬ tus comprises an absorbance light detector disposed to measure the absorbance of the liquid sample on the basis of the light passing through the liquid sample. The absorbance light detector and the fluorescent light detector may be separate light detectors. The detectors may equally consist of one and the same light detector, in which case the apparatus may comprise an optical conductor by which the light signal of absorbance meas¬ urement or that of fluorescence measurement is option¬ ally conducted to said one light detector.

In an embodiment of the apparatus, the cuvette is a cuvette with planar bottom. In an embodiment of the apparatus, the appara¬ tus comprises a transport means for moving the cuvette and/or the excitation light beam and to position the central axis of the excitation light beam, with a di¬ rection parallelling the central axis of the cuvette, at a distance from the latter; and second optic means are arranged to direct the central axis of the fluores¬ cence measuring direction in the plane defined by the central axis of the cuvette and the central axis of the excitation light beam in such manner that the angle between the central axis of the measuring direction and the central axis of the cuvette is in the range of 5 to 50°. In an embodiment of the apparatus, the appara¬ tus comprises s reference light^*detector for monitoring the intensity of the light directed on the sample, and a divider means, such as a beam divider, for separating a part from the excitation light beam directed on the liquid sample and directing this part to said reference light detector.

In an embodiment of the apparatus, the first optical means comprise a first lens for focussing the light beam on the cuvette, a first stop for delimiting the solid angle of the light beam bundle conducted into the cuvette, a first wavelength selector, such as a filter or a grating monochromizer, for determining the wavelength of the light beam, and a mirror for refract- ing the light beam to enter the cuvette from below and substantially vertically; and the apparatus comprises a second lens for directing the light passing through the sample to go to the absorbance light detector, and a first measuring electronics circuit arranged to trans- form the signal from the absorbance light detector into am absorbance reading, using the signal from the refer¬ ence light detector as aid.

In an embodiment of the apparatus, the second optical means comprise an aperture limiter disposed before the fluorescent light detector to delimit the measuring area; a third lens for focussing an image of the aperture limiter into the sample; a second stop for limiting that solid angle in which fluorescent light generated in the measuring area will be included in the measurement; a second mirror for refracting the light beam emitted from the liquid sample in the measuring direction and ^cor determining the measuring direction; a econd wavelength selector, such as a filter or a grating monochromizer, for determining the wavelength of the fluorescent light that will be measured; and the apparatus comprises a second measuring electronics cir¬ cuit disposed to transform the signal from the fluores- cent light detector into a fluorescence reading, using as aid the signal from the reference light detector.

The paths both of the excitation light beam and of the fluorescent emission light can be refracted with the aid of prisms and mirrors in ways known in themselves in the art and which shall not be more closely described here, without affecting the measuring event in the cuvette, taught by the invention.

The invention affords the advantage that when fluorescence is being measured the absorbance can also be measured with the same apparatus, without transfer¬ ring the liquid sample from one apparatus or cuvette to another and without need of concessions relating to performance in respect of either measuring method. Further, thanks to the invention, the advan¬ tages of fluorescence measurement taking place from below relative to the cuvette can be utilized and pho¬ tometry and fluorometry can be combined, at the same time avoiding the detriments caused by background fluo- rescence.

It is moreover an advantage of the invention that it is applicable with cuvettes arranged in a matrix array and in particularly with the microtitre tray format, while it may equally be applied with cu- vettes of other shape.

In the following, the invention is described in detail, referring to the attached drawing, wherein

Fig. 1 presents schematically and sectioned, an embodiment of the apparatus of the invention; Fig. 2 shows the section II-II of Fig. 1; and

Fig. 3 displays, in an embodiment of the pro¬ cedure of the invention, the path of the light beams in the region of a cuvette belonging to a microtitre tray. In Fig. 1 is depicted an apparatus for deter- mining the fluorescence of a liquid sample, in which the liquid sample has been placed in a flat-bottomed cuvette 1, there being a plurality of such cuvettes. assembled in matrix configuration to form a microtitre tray 26. The apparatus comprises a light source 2. The light source is here a xenon flash lamp receiving its requisite voltage pulses from a voltage source 27. The anode and cathode ends of the arc of the lamp 2 are covered with a mask in the interest of better reprodu- cibility. The apparatus comprises first optical means 3 for separating the desired wavelength from the light of the light source to form the excitation light beam 4 and for directing this beam into the cuvette in order to produce fluorescence in the liquid sample; and a fluorescent light detector 5 for observing the light produced by fluorescence and emitted in a predetermined measuring direction. The fluorescent light detector 5 is disposed to measure, from below relative to the cu¬ vette 1 and from an optically delimited measuring area, substantially nothing but internal fluorescence of the liquid sample. The apparatus further comprises second optical means 6 for delimiting any fluorescent emission light due to the effect of the excitation light beam 4 on the bottom of the cuvette 1, to be outside the meas¬ uring area.

The apparatus is also used to measure the ab¬ sorbance of the liquid sample, and therefore the appa- ratus comprises an absorbance light detector 7, dis¬ posed to measure the absorbance of the liquid sample on the side above the cuvette, on the basis of the light that passes through the liquid sample. The light source 3 used in absorbance measurement is the same as the light source 2 used in measuring fluorescence.

The apparatus further comprises a reference light detector 12 for monitoring the intensity of the light directed on the sample, and a divider means 14, such as a beam divider, for separating a part from the excitation light beam directed on the liquid sample and for directing this part to the reference light detector 12. The first optical means 3 comprise a first lens 14, disposed to focus an image of the arc of the flash lamp 2 into the cuvette 1. Furthermore, the first optical means 3_. comprise a first stop 15, with which the solid angle (relative aperture) of the light beam bundle conducted into the cuvette 1 is delimited, a first wavelength selector 16, or a filter, with which the wavelength of the light beam is fixed, and a mirror 17 for refracting the light beam to ho into the cuvette from below and substantially in vertical direction. As can be seen in Fig. 2, there may be several filters 16 and among these a suitable filter may be selected for each application. The filter 16 is employed to select the wavelength for absorbance measurement or for fluo- rescence excitation. Since the solid angle of the beam bundle is small, it is possible to use interference filters for the filters 16 without need of separate collineation of the beam bundle. The mirror 17 may be produced ny aluminium-coating the surface of the prism. By refracting the beam bundle at right angles, as shown in the figure, the bulk of the apparatus can be re¬ duced.

For absorbance measurement, the apparatus com¬ prises a second lens 18, by which the light that has gone through the sample is directed to the absorbance light detector 7, and a first measuring electronics circuit 19, in which the signal from the absorbance light detector 7 is transformed into an absorbance reading with the aid of the signal from the reference light detector 12.

The second optical means 6 provided in the apparatus for fluorescence measurement comprise an aperture limiter 20, disposed before the fluorescent light detector 5 and serving the purpose of delimiting the measuring area. The fluorescent light radiates, in principle, isotropically from the sample, i.e., uni¬ formly in all directions from the path where the exci- tation light beam passes. The measuring area is that region (hatched area in Fig. 3) where excitation light is present and which is in the field of vision of the fluorescent light detector 5. It is thus understood that the aperture limiter 20 is used to delimit the size of the measuring area, the image of said aperture limiter 29 being produced in the liquid sample by means of a third lens 21. The second optical means further comprise a second stop 22, by which that solid angle is delimited in which the fluorescence produced in the measuring area 29 will get to be measured. A second mirror 23 refracts the light beam emitted in the meas¬ uring direction from the liquid sample, and it fixes the measuring direction. The mirror 23 may be the aluminized surface of a prism. The mirrors 17 and 23 may, as shown in the figure, be established on adjacent facets of one and the same prism. The mirror 23 is used to refract the fluorescent emission light bundle to be horizontal, which enables the bulk of the apparatus to be restricted.

The second optical means 6 further comprise a second wavelength selector, or filter, 24 for selecting the wavelength of the fluorescent light that is being measured. There may be several filters 24, of which a suitable filter can be chosen for each particular application.

In the exemplary embodiment, the wavelength of the excitation light and of the emitted light is selec¬ ted with the filters 16 and 24. It is equally possible to employ, for instance, a grating monochromizer for selecting the wavelength of one of said lights, or both, in which case alterations known in themselves in the art have to be made in the optical system, which are not discussed her in greater detail. The apparatus further comprises a second meas¬ uring electronics circuit 25, disposed to transform the signal from the fluorescent light detector 5 into a fluorescence reading, using as aid the signal from the reference light detector 12.

The fluorescent light detector 5 is a photo- multiplier tube, e.g. of the well-known type 1P21. In the exemplary embodiment the channels of the excitation light and of the emission light are as closely similar as possible. This implies that the lenses 14 and 21 are similar, preferably quartz lenses. If interference filters are used for filters 16 and 24 in both channels, it should be taken into account that for oblique beams the transmission graph of the filter shifts toward shorter wavelengths. However, this effect is minimal in the system here described.

When using the apparatus, one may for instance first measure the absorbance, the light beam being con¬ ducted through the liquid sample along the central axis 10 of the cuvette (see Fig. 3).In principle, the fluo¬ rescence could be simultaneously measured, but it is advantageous to move the cuvette 1 laterally so that the emission light beam, due to fluorescence, emitted in the measuring direction need not pass too close to the lower margin of the cuvette 1. Therefore the appa¬ ratus may include a transport means (not depicted) by means of which the cuvette can be moved relative to the excitation light beam 4 and the central axis 9 of the excitation light beam displaced to a distance from the central axis 10 of the cuvette, still parallel to it, as shall be described next.

In Fig. 3 is seen the situation in the cuvette 1 during fluorescence measurement. The central axis 10 of the cuvette is indicated with a dot-and-dash line. The central axis 9 of the excitation light beam bundle, presented with a three-dot-and-dash line, parallels the central axis 10 of the cuvette but instead of coincid- ing with it, runs at a distance from it. The excitation light 4 excites on the bottom 30 of the cuvette 1, fluorescence on an area 31, which has been indicated with oblique hatching in the figure. Since the surface is downwardly concave, the reflection is directed away from the fluorescence measuring direction, of which the central axis is presented with a two-dot-and-dash line 11, and the reflection does not interfere with the fluorescence measurement. The central axis 11 of the measuring direction encloses in the interior of the sample with the central axis 9 an angle of about 22°, corresponding to an angle about 30° in the air space outside the cuvette 1. The angle is so selected that the emitted light from the sample, which is to be meas¬ ured, does not pass through that area 31 of the bottom of the cuvette where the excitation light 4 strikes, and also so that the downward extension 32 provided on the lower margin of the cuvette does not intersect the light to be measured which is emitted in the measuring direction of light emitted in the liquid sample sue to fluorescence.

The apparatus can also be used to make turbi- dimetric and nephelometric measurements. A turbidimet- ric measurement is carried out substantially like an absorption measurement (photometric measurement) and a nephelometric measurement, substantially like a fluoro- etric measurement. The invention is not delimited exclusively to concern the embodiment examples presented in the fore¬ going; numerous modifications are feasible while stay¬ ing within the scope of the inventive idea defined by the claims.

Claims

1. A procedure for determining the fluores¬ cence of a liquid sample, in which the liquid sample is placed in a cuvette; on the cuvette is directed an ex¬ citation light beam; and the fluorescence produced in the liquid sample by the excitation light beam is meas¬ ured, characterized in that the fluorescence is meas¬ ured from below relative to the cuvette, from an opti- cally delimited measuring area, substantially exclu¬ sively as internal fluorescence of the liquid sample.

2. Procedure according to claim 1, character¬ ized in that the emission light of the fluorescence produced by effect of the excitation light beam on structural components of the cuvette is delimited to be outside the measuring area.

3. Procedure according to claim 1 or 2, char¬ acterized in that the excitation light beam is directed into the cuvette from below, substantially vertically, and the quantity of light that has passed through the sample is measured for determination of absorbance.

4. Procedure according to claim 3, character¬ ized in that in absorbance measurement the excitation light beam is directed from below along the central axis of the cuvette; that in fluorescence measurement the excitation light beam is displaced, retaining its direction parallel to the central axis of the cuvette, to one side to a distance from the central axis of the cuvette; and that the central axis of the fluorescence measuring direction lies in the plane defined by the central axis of the cuvette and the central axis of the excitation light beam, the central axis of the measur¬ ing direction forming with the central axis of the cu¬ vette an angle which is in the range of 5 to 50°.

5. Apparatus for determining the fluorescence of a liquid sample, comprising a cuvette (1) in which the liquid sample has been placed; a light source (2); first optical means (3) for separating the desired wavelength from the light of the light source for form¬ ing the excitation light beam (4) and for directing it into the cuvette for producing fluorescence in the liquid sample; a fluorescent light detector (5) for observing the light emitted in a predetermined measur¬ ing direction, characterized in that the fluorescent light detector (5) is arranged from below, relative to the cuvette, to measure from an optically delimited area substantially exclusively internal fluorescence of the liquid sample.

6. Apparatus according to claim 5, character¬ ized in that the apparatus comprises second optical means (6) for delimiting the fluorescent emission light produced by effect of the excitation light beam (4) on structural components, e.g. the bottom, of the cuvette (1) to be outside the measuring area.

7. Apparatus according to claim 5 or 6, char¬ acterized in that the apparatus comprises an absorbance light detector (7) disposed to measure the absorbance of the liquid sample on the basis of the light passing through the liquid sample.

8. Apparatus according to any one of claims 5-7, characterized in that the cuvette (1) is a cuvette with even bottom.

9. Apparatus according to claim 7 or 8, char¬ acterized in that the light source employed in absorb¬ ance measurement is the same as the light source (2) employed in fluorescence measurement.

10. Apparatus according to any one of claims

5-9, characterized in that the apparatus comprises a transport means (8) for moving the cuvette and/or the excitation light beam (4) and positioning the central axis (9) of the excitation light beam (4), retaining its parallellity with the central axis (10) of the cu¬ vette, at a distance from the latter axis; and that second optical means (6) are disposed to direct the central axis (11) of the fluorescence measuring direc¬ tion to lie in the plane defined by the central axis of the cuvette and the central axis of the excitation light beam so that the angle between the central axis of the measuring direction and the central axis of the cuvette is in the range of 5 to 50°.

11. Apparatus according to any one of claims 5-10, characterized in that the apparatus comprises a reference light detector (12) for monitoring the inten- sity of the light directed into the sample, and a di¬ vider means (13, such as a beam divider, for separating a part from the excitation light beam directed on the liquid sample and directing said part to the reference light detector.

12. Apparatus according to any one of claims

5-11, characterized in that the first optical means (3) comprise a first lens (14) for focussing the light beam into the cuvette, a first stop (15) for delimiting the solid angle of the light beam bundle conducted into the cuvette, a first wavelength selector (16), such as a filter or grating monochromizer, for fixing the wave¬ length of the light beam, and a mirror (17) for re¬ fracting the light beam to go into the cuvette from below, substantially vertically; and that the apparatus comprises a second lens (18 for directing the light that has passed through the sample, to the absorbance light detector (7), and a first measuring electronics circuit (19) disposed to transform the signal from the absorbance light detector onto an absorbance reading, using as aid the signal from the reference light detec¬ tor (12) .

13. Apparatus according to any one of claims 5-12, characterized in that the second optical means (6) comprise an aperture limiter (20) disposed before the fluorescent light detector (6) for delimiting the measuring area; a third lens (21) for focussing the image of the aperture limiter into the sample; a second stop (22) for delimiting that solid angle in which the fluorescent light produced in the measuring area gets to be measured; a second mirror (23) for refracting the light beam emitted from the liquid sample in the meas- uring direction and for determining the measuring di¬ rection; a second wavelength selector (24), such as a filter or grating monochromizer, for fixing the wave¬ length of the fluorescent light to be measured; and that the apparatus comprises a second measuring elec- tronics circuit (25) disposed to transform the signal from the fluorescent light detector into a fluorescence reading, using as aid the signal from the reference light detector (12).