EP1192431A1

EP1192431A1 - Fluorescence measuring device

Info

Publication number: EP1192431A1
Application number: EP99966998A
Authority: EP
Inventors: Stig Tormod
Original assignee: Amersham Bioscience AB
Current assignee: Cytiva Sweden AB
Priority date: 1998-12-28
Filing date: 1999-12-22
Publication date: 2002-04-03
Also published as: SE9804551D0; JP2003522323A; WO2000039545A1

Abstract

A fluorescence measuring unit comprising a monochromatic light source (13), a detector unit (23a, 23b), an excitation beam (13a) going from the light source (13) to the spot (18), an emission beam going from the spot (18) to the detector unit (23a, 23b) and an acousto optic tuneable filter (AOTF) (12), arranged in a non-collinear mode, and such that the excitation beam path and the emission beam path coincide but propagate in the AOTF in opposite directions. An arrangement comprising two laser sources (27) defining two beam paths leading to a common beam path (31) with the features that: a) said laser sources have polarisation planes perpendicular to each other and b) a non-collinear AOTF (29) is placed in the conjunction of said two beam paths and oriented such that, when tuned to the respective wavelength of the laser light from the respective laser source, the laser light from the two sources will pass and exit the second AOTF (29) in one and the same beam path (31).

Description

FLUORESCENCE MEASURING DEVICE

The field of invention

The present invention is related to a unit and method for measuring fluorescence.

Background to the invention

Previous methods for the measurement of fluorescence: A molecule that is fluorescent will always emit light at a longer wavelength than the wavelength of the light that it is excited with. This is valid for non-resonant absorption processes. Interference filters of type cut-off, cut on, or bandpass are commonly used to separate the emission from the excitation light. A common set-up of the system layout is where the filtered excitation beam is perpendicular to the detection system. In some cases it is not possible to have 90° difference in direction. If the sample is situated in a narrow and deep well or on a non-transparent or transparent microscope slide it is an advantage to excite and measure the emitted florescence from the same side. Previously this has been done with, e.g. a dichroic mirror that reflects the excitation light and lets the emitted light pass. The exciting and emitting light are therefore coaxial at the sample side. This is the case in some micro-titre plate readers and in confocal microscopy. There are several drawbacks with interference filters. They are fixed in wavelength and if there are many fluorophores in the sample with different excitation and emission spectra many different filters are needed. To change between these takes considerable time. It is not possible to obtain continuous spectra with discrete filters.

The objective of the invention is to render these problems simpler.

Brief Description of the drawings

Figure 1 Illustrates what happens when white light passes an

AOTF that is tuned to one wavelength of the passing light. Figure 2 Illustrates the wavelength dependency of light intensity of light that has passed an AOTF tuned to a certain wavelength, λ_emission. (λ_em) Figure 3 Illustrates a preferred set up of a measuring unit according to the invention. Figure 4 Shows a monochromatic light source utilising two different lasers.

Acousto-Optic Tuneable Filters, hereafter abbreviated AOTF, are electronically controllable, solid-state, narrow band optical filters. They are tuneable from ultraviolet to near- infrared, have multi-wavelength possibilities with bandpass variability, high throughput, and wavelength switching times in the tens of microseconds .

AOTFs have previously been used for the measurement of fluorescence. See WO-A- 9730331. However, the AOTF has then only been passed by light in one direction (emitted light from the sample) .

Function of AOTFs (figure 1) : An AOTF is made of a birefringent anisotropic crystal (1) , e.g. paratellurite (Te0₂) that is used for visible and infrared light or crystalline quartz for ultraviolet and visible light. The crystal (1) is oriented and cut and polished to special face angles. To one face of the crystal a piezoelectric transducer (2) is firmly attached and to the opposite side an acoustic absorber (3) . By applying a radio frequency (RF) signal (4) in the MHz range to the transducer (2) an ultrasonic wave is created in the crystal (1) . This propagating acoustic wave (5) creates a sinusoidal pattern of compression and rarefaction and therefore a periodic modulation of the index of refraction. The crystal can now act as a Bragg grating. An incident beam of unpolarised white light (6) at an angle to the acoustic wave can enter the crystal at an input end (7) and interact with the acoustic wave. Only a limited band of wavelengths can satisfy the phase-matching condition and be diffracted to the first order at the output end (8) of the crystal. The diffracted light is orthogonally polarised and due to birefringence the ordinary and extraordinary beams (9 and 10, respectively) are separated by a certain angle. This angle is the same for all wavelengths that means that the detectors can be fixed. The wavelengths that are not diffracted pass the crystal unaffected in the zero order (11) . When the crystal is run under these conditions the device is called a noncollinear AOTF. By changing the RF frequency the centre wavelength of the diffracted beams changes accordingly. This can be done in microseconds. Several different RF frequencies can be run simultaneously giving multiple diffracted wavelengths as output.

The zero order beam (11) has its full intensity except for the wavelength the AOTF is tuned to where the AOTF acts as a notch filter. See Fig.2. These features are utilised in this application of an AOTF. In the context of the invention an AOTF comprises the control circuit for tuning, i.e. as described in figure 1, the piezoelectric transducer (2), the acoustic absorber (3) and the radio frequency (RF) transmitter (4) .

Object of the invention

An object of the invention is to provide an improved device and method for measuring fluorescence.

The invention

The object of the invention is achieved by devices having the features of claims 1 & 13 and methods in accordance with claim 11 & 16.

The inventive measuring unit thus is intended for measuring fluorescence in a spot (18) and comprises a monochromatic light source (13), a detector unit represented in the figure 3 by two detector subunits (23a, 23b) , an excitation light beam path (13a) with wavelength λ_ex going from the light source (13) to the spot (18) , an emission light beam path with wavelength λ_em going from the spot (18) to the detector unit and an acousto optic tuneable filter (AOTF) (12) in the beam path between the spot (18) and the detector unit of the fluorescence measuring unit. The spot (18) may be a sample.

The unit has the features that : a) the AOTF (12) is arranged in a non-collinear mode; b) the excitation light beam path and the emission beam path coincide but pass through the crystal of the AOTF in opposite directions.

The AOTF of the unit is thus placed both between the excitation light source (13) and the spot (18) and between the spot (18) and the detector unit in figure 3 represented by two subunits (23a, 23b) .

In the inventive unit, the AOTF (12) receives excitation light, typically monochromatic from a monochromatic light source (13) , at its input end (14) . Between the light source (13) and the AOTF (12) , there may be means for collimating the light, preferably a lens system (15) . A polarising filter or the like (15a) may be inserted in the excitation beam path in front of the AOTF (12) if fluorescence depolarisation or anisotropy is studied. When the measuring unit is in use, the control circuit of the AOTF (12) is tuned to the emission wavelength λ_em of the fluorophore to be measured, which means that the collimated excitation light passes unaffected straight through the AOTF (12) without being diffracted. In the excitation beam path after the AOTF (12) , there preferably are means to focus the light on the spot (18) . When the excitation light (λ_era) hits the appropriate fluorophore in the spot (18) the fluorophore will emit light (λ_em) which is guided into the AOTF (12) through its output end (16) . Thereafter the light propagates in the AOTF (12) which, as said above, is tuned by a suitable RF signal to the emitted wavelength (λ_e . In the crystal the light is now dispersed into two orthogonally polarised beams (19,20) which after exiting the AOTF may be focused by a respective lens system (21,22) or the like to a detector subunit (23a, 23b) . The means for focusing the excitation light to the spot (18) and the means for directing emitted light into the output end

(16) of the AOTF (12) may coincide as represented by lens system (17) . This lens system functions both to focus the light on the spot (18) and to collect and collimate the emitted light to a parallelity that is acceptable for the AOTF

(12) used.

The detector unit may comprise two more or less equal subunits (23a, 23b), i.e. one for each of the orthogonally polarised beams (19, 20), or one common unit for both beams or only a single unit for one of the beams. In the case that there is a common detector unit for both beams there are light guiding means (32) for guiding the light from the AOTF (12) to the detector unit (23b) (now acting as a common detector as subunit 23a shown in figure 3 would not be present) . In the case that a common detector is used the arrangement may equivalently comprise light guiding means, similar to (32) , for each emission light path for guiding light to the common detector unit. The detector unit/subunits (23a, 23b) may comprise a light guide (24) for guiding the light to a detector (25) , typically a photomultiplier tube or a photo diode. Light guides may be in form of reflection means such as an optical fibre and/or one or more mirrors.

In each light beam path between the end (14) of the AOTF (12) and the detector (25) there may be placed a filter (26) for decreasing stray light from the AOTF (12) . This type of filter may be a so called "liquid crystal tuneable filter" (LCTF) (for instance from Cambridge Research Instrumentation, Boston, MA., U.S.A.). The location of the filter is preferably within the detector unit/subunits (23a, 23b) and as close as possible to the detector (25) .

The monochromatic light source (13) may comprise a broadband light source and a monochromator utilizing another AOTF or a grating or an interference filter. As a further alternative the light source (13) may comprise a laser source. In the first variant the lens system (17) may be a set of anamorphic cylindrical lenses in order to accomplish an optimal throughput of emitted light in the AOTF (12) . In the alternative variant the lens system (17) may comprise a beam expanding lens system, preferably followed by a positive lens system focusing the excitation light on the spot (18) . The beam expanding lens system may be composed of a first negative lens followed by a positive lens . Positive lenses in lens system (17) should preferably have a large diameter giving an efficient high numerical aperture fluorescence light collection.

Two different laser sources (27, 28) may also be used as the monochromatic light source (13). See Figure 4. Each laser source then shall provide polarised light of one or more wavelengths. The polarisation plane of light from one source shall be perpendicular to the polarisation plane of the light from the other source. In this alternative a second non- collinear AOTF (29) may be placed in the beam path of the excitation light in front of the first AOTF (12) . The laser sources should then be oriented such that the direction and the polarisation planes of the beams coincide with the first order diffraction direction and polarisation planes of the AOTF (29) . This second AOTF can be tuned to the respective wavelengths of the laser light from the laser sources, which means that the respective excitation light will exit the second AOTF (29) in one and the same beam path (31) . A pair of beam stoppers (30) are placed after the AOTF (29) to block unwanted zero order laser light.

In fluorescent life time studies one utilises laser light. Typically the laser beam is intensity modulated by a fast modulator, e.g. an acousto-optic modulator that is situated in the light source (13) just in front of the laser.

The inventive unit can be used in most fluorescent assays techniques. Examples are steady state fluorescence, delayed florescence, fluorescence depolarisation or anisotropy, energy transfer fluorescence, and fluorescence lifetime provided that the unit is equipped/modified in the appropriate way as required in the art for these techniques . Fluorescence depolarisation and anisotropy, for instance, will require separate measurement of the ordinary and extraordinary beams, i.e. separate detectors. Spectral information of the fluorescent light can be obtained by rapidly scanning the

AOTF. It is then possible to detect multiple fluorophores in the spot (18) .

The unit can be used for the measurement of fluorescence in the context of various type of assays that are based on the reaction between two reactants I and II and detection of the reaction by a reactant that is labelled with a fluorescent- causing substance. The labelled reactant -may be either reactant I, reactant II or any other reactant that may bind to either of these two reactants or to a reaction product formed, either directly or indirectly, as a consequence of the reaction between these two reactants. The unit may also be used for the measurement of fluorescent-causing substances as such.

The spot may correspond to a sample, for instance derived from any of the assay method discussed above. One or more spots may be present on a surface or the like, for instance a microtiter plate or a spotted surface. The inventive measuring unit may be arranged so that it is possible to move the surface and unit relative each other thereby scanning the surface for fluorescence in the spots one by one. By utilising a suitable data processing unit it will be possible to create an image of the surface represented by the fluorescence of the spots (imaging) .

One separate aspect of the invention is a generalised arrangement of the arrangement described in figure 4 for linking laser lights of perpendicular polarisation planes into a common beam path. This aspect is thus an arrangement comprising a first laser source (27) and a first beam path leading from the first laser source to a common beam path (31) and a second laser source (28) and a second beam path leading from the second laser source to said common beam path (31) .

The arrangement has the features that : a) said laser sources are provided such that the laser light entering the first beam path has a polarisation plane that is perpendicular to the laser light entering the second beam path and b) a non-collinear AOTF (29) is placed in the conjunction of said first and second beam paths and oriented such that, when tuned to the respective wavelength of the laser light from the respective laser source, the laser light from the two sources will pass and exit the second AOTF (29) in one and the same beam path (31) . There may be beam stoppers (30) placed after the AOTF .(29). to block unwanted zero order laser light. Either or both of the laser sources may be of the type enabling switch between laser light of different wavelength. The AOTF (29) can be tuned to the wavelengths provided by the laser sources (27,28) .

Claims

1. A measuring unit, for measuring fluorescence in a spot (18) , comprising a monochromatic light source (13) , a detector unit (23a, 23b) , an excitation light beam path (13a) going from the light source (13) to the spot (18) , an emission light beam path going from the spot (18) to the detector unit (23a, 23b) and an acousto optic tuneable filter (AOTF) (12) in the beam path between the spot (18) and the detector unit (23a, 23b) , characterised in that a) the AOTF (12) is arranged in a non-collinear mode, and b) the excitation light beam path and the emission light beam path coincide but propagate in the AOTF in opposite directions .

2. The fluorescence measuring unit of claim 1, characterised in that there is one detector unit (23a) for the ordinary ray and/or one detector unit (23b) for the extraordinary ray derived from the emitted light .

3. The fluorescence measuring unit of any of claims 1-2, characterised in that there is a common detector unit

(23b) for the extra-ordinary and ordinary ray and that there are light guiding means (32) for guiding the two rays to the common detector unit .

The fluorescence measuring unit of any of claims 1-3, characterised in that there are means, preferably a lens system (17) , in the emission beam path before the AOTF for collimating the emission light.

5. The fluorescence measuring unit of any of claims 1-4, characterised in that the unit has means permitting the measurement of emitted light (fluorescence) as steady state fluorescence and/or delayed fluorescence and/or fluorescence depolarisation or anisotropy and/or energy transfer fluorescence and/or fluorescence life time.

6. The fluorescence measuring unit of any of claims 1-5, characterised in that the light source (13) comprises a laser providing the excitation light.

7. The fluorescence-measuring unit of any of claims 1-5, characterised in that the light source (13) comprises a broadband light source.

8. The fluorescence measuring unit of any of claims 1-7, characterised in that a) the light source (3) comprises one or two different laser sources (27,28), i) each of which sources provide polarised laser light of one or more wavelengths and ii) the polarisation plane for light from one source being perpendicular to the polarisation plane of the light from the other source, and b) a second non-collinear AOTF (29) placed in the beam path of the excitation light between the first AOTF (12) and the laser sources (27,28) and oriented such that, when tuned to the respective wavelength of the laser light from the respective laser source, the excitation light from the two sources will pass and exit the second AOTF (29) in one and the same beam path (31) .

9. The fluorescence measuring unit according to any of claims 1-8, characterised in that, between the first AOTF (12) and the spot (18) , there is a lens system (17) focusing the excitation light on the spot (18) and collecting and collimating emitted light to enter the AOTF (12) through the end (16) .

10. The fluorescence measuring unit of claim 8, characterised in that a) the monochromatic light source (13) comprises a broad band light source and the lens system (17) comprises a set of anamorphic cylindrical lenses, or b) the monochromatic light source (13) comprises a laser source and the lens system (17) comprises a beam expanding lens system combined with a positive lense 5 focusing the expanded excitation beam on the spot (18) and collecting emitted light from spot (18) .

11. The use of the measuring unit of any of claims 1-10 for measuring fluorescence from a spot (18) .

10

12. The use according to claim 11, characterised in that the spot (18) is on a surface containing several spots and that the fluorescence of each spot is measured one by one.

15 13. An arrangement comprising a first laser source (27) and a first beam path leading therefrom to a common beam path (31) and a second laser source (28) and a second beam path leading therefrom to said common beam path (31) , characterised in that

20 a) said laser sources are provided such that the laser light entering the first beam path has a polarisation plane that is perpendicular to the laser light entering the second beam path and b) a non-collinear AOTF (29) is placed in the conjunction

25 of said first and second beam paths and oriented such that, when tuned to the respective wavelength of the laser light from the respective laser source, the laser light from the two sources will pass and exit the s AOTF (29) in one and the same beam path (31) .

30

14. The arrangement of claim 13, characterised in that a pair of beam stoppers (30) is placed after the AOTF (29) .

15. The arrangement of any of claims 13-14, characterised in

35 that either or both of the laser sources may provide switch from one wavelength to the other.

16. The use of the arrangement of any of claims 13-15 for linking two laser lights of perpendicular polarisation planes deriving from a respective laser source into one common beam path.

17. The use according to claim 16 characterised in that the common beam path (31) is linked into an AOTF of a fluorescence measuring unit as defined in claims 1-10.