|5108179||System and method for determining changes in fluorescence of stained nucleic acid in electrophoretically separated bands||1992-04-28||Myers||356/344|
|5062942||Fluorescence detection type electrophoresis apparatus||1991-11-05||Kambara et al.||204/299|
|4997275||Process for the production of a device for the optical analysis of a microparticle flux and application to the production of a cytofluorimeter||1991-03-05||Gaucher et al.||356/246|
|4929561||Absorption-emission optrode and methods of use thereof||1990-05-29||Hirschfeld||436/116|
|4927265||Detector for fluorescence and absorption spectroscopy||1990-05-22||Brownlee||356/73|
|4801552||Method and a spectrometer for measuring absorbance difference spectra of radical reactions||1989-01-31||Hoff||436/173|
|4786171||Spectral analysis apparatus and method||1988-11-22||Le Febre et al.||356/246|
|4675300||Laser-excitation fluorescence detection electrokinetic separation||1987-06-23||Zare et al.||436/172|
|4657397||Light collector and its use for spectroscopic purposes||1987-04-14||Oehler et al.||356/414|
|4565448||Particle counting apparatus||1986-01-21||Abbott et al.||356/246|
|4367040||Multi-channel optical sensing system||1983-01-04||Goto||356/44|
|4273443||Method and apparatus for measurement of reradiation in particle flow cell systems||1981-06-16||Hogg||356/343|
|4199686||Dark field illuminator and collector apparatus and method||1980-04-22||Brunsting et al.||356/342|
|4172227||Flow microfluorometer||1979-10-23||Tyrer et al.||250/461.1|
|4088407||High pressure fluorescence flow-through cuvette||1978-05-09||Schoeffel et al.||356/246|
|3795450||DUAL BEAM OPTICAL ABSORPTION PHOTOMETRY DETECTOR ASSEMBLY FOR HIGH PRESSURE APPLICATIONS||1974-03-05||Munk||356/246|
|EP0404646||1990-12-27||Electrophoretic capillary with incorporated optical system.|
This application is a continuation-in-part of the copending application Ser. No. 07/772,823 filed Oct. 8, 1991 and entitled "Enhanced Fluorescence Detection of Samples in Capillary Column." The contents thereof are incorporated by reference.
This invention relates to the efficient collection and detection of low levels of radiation arising within microliter volumes of sample.
The radiation may be in the form of luminescence from a chemical reaction or result from the interaction of an intense light source and the sample. Alternatively, the processes of light scattering, Raman scattering, fluorescence and phosphorescence may be used.
The technique of light scattering is particularly useful in the detection of particles, and two broad classifications of instruments can be identified. Particles of diameters significantly smaller than the wavelength of probe light scatter isotropically, i.e. equally in all directions. This type of scattering is usually called Rayleigh scattering. Particles of diameters significantly larger than the wavelength of probe light scatter primarily in the forward direction and, often, the intensity of scatter is a complicated function of scattering angle. This type of scattering is usually called Mie scattering. The intensity of forward scattered light varies as the sixth power of the particle diameter; hence, special precautions must be taken when attempting to measure the light scattered from very small particles.
The present invention permits for the determination of relative particle size and/or concentration of very small particles, particularly in a flowing system.
Intense light sources such as lasers are used for studying small particles. The use of such sources introduces problems associated with discrimination of light scattered by instrumental components from that scattered by the sample. The intensity of light scattered from small particles may be less than one millionth the intensity of laser radiation. To measure such low intensities, it is necessary to collect as much of the light scattered by the sample as possible, yet reject that scattered by the instrument. In general, it becomes increasingly difficult to eliminate instrumental light scattering as the angle of detected scattering decreases. Consequently, when measuring the light scattered by particles significantly smaller than the wavelength of probe light, it is best to avoid detection of the small angle scattering, while collecting as large a solid angle of scattered light as possible.
When measuring the light scattered by small particles, considerable attention must be paid to the overwhelming level of light scattered from unwanted foreign particles of large size. For this reason, it is advantageous to keep the size of the scattering volume (volume of sample simultaneously illuminated and detected) as small as possible. In a flowing sample, these particles will be evidenced as signal "spikes" abruptly rising from a base signal. The base signal represents the signal from the more numerous small particles.
Large angle scattered light instruments described in the prior art have used large scattering volumes and require extensive sample treatment to minimize the detection of large foreign particles. Furthermore, sample volume has to be large to avoid detection of light scattered from the instrument. These prior art instruments have usually used lenses and apertures to define the scattering angle and, of necessity, capture a small solid angle of scattered light.
Moreover, most of the prior art has addressed the detection of relative large biological cells (cytometry). The signals from these large particles are much greater than those provided by the small particles of particular interest in the present invention.
Raman scattering is used for the chemical characterization of the samples rather than particle size. Like Rayleigh scattering, the signal intensities are very low and it is important to capture as large a solid angle of Raman scattered light as possible while also using very intense sources of incident radiation. The Raman scattered signals are of different wavelength from the incident light. Chemical characterization of the sample is determined by the difference between the frequency of the Raman signal and the frequency of the exciting radiation. It is customary to use a blocking filter to reject the incident radiation followed by a monochromator to determine the frequency of the Raman signal. While this frequency difference helps in the discrimination between exciting and Raman signals, the Raman bands are often narrow; hence, the signals are very weak. It is important to use whatever geometric means are available to minimize the detection of the incident radiation and maximize the detection of the weak Raman radiation from the sample.
Fluorescence techniques are used when the sample or some component(s) of the sample can be tagged with a fluorescing dye. The sample is then excited by an intense beam of exciting radiation, usually from a laser. The wavelength of the exciting radiation is chosen to correspond to the wavelength of maximum sample excitation. The wavelength of the fluorescing radiation will usually be greater than the exciting wavelength, and a filter or monochromator will be chosen to pass only that radiation corresponding to the wavelength of maximum fluorescence emission. When the sample concentration is low, great care is needed in the choice of filters or monochromators in order to discriminate between exciting and fluorescing wavelengths. No filters provide a perfect discrimination, and it is desirable to use whatever geometric means are available to minimize the detection of exciting rays scattering from the instrument. Under ideal conditions, tagged molecules can be detected at extremely low concentration.
Phosphorescence techniques are employed where the sample, itself, or the tagged sample continues to emit radiation a significant time after excitation. Time as well as wavelength can then be used to discriminate between exciting and emitting radiation.
Luminescence techniques are used when the sample or some component of the sample emits radiation as a result of a chemical reaction of the sample with a reagent. No external exciting radiation is needed; hence, all of the radiated light may be detected without recourse to filtering. However, the luminescence signals are usually very weak and depend on the number of reacting molecules within the detected sample volume. A compromise must usually be made between detected sample volume and concentration of reacting molecules. The efficient collection and detection of the radiation emitted within the detected sample volume is of great importance.
There is a need to provide a system for radiation collection and detection which provides significant advances over the prior art.
By this invention, there is provided a system for detecting a radiation signal in a manner which minimizes disadvantages of the prior art.
According to the invention, a generated radiation signal from an excited sample is collected over a large solid angle by means of a reflecting curvilinear surface of revolution and is directed to a detector. The angle should preferably be between about 35° and 145°.
The sample enters a detection volume in a flowing stream and the axis of sample flow is collinear with the axis of revolution of the reflecting curvilinear surface of revolution. The scattering volume, which is part of the detection volume, is retained relatively small.
In one preferred form, the radiation signal after the above interaction has the same wavelength as the radiation signal incident on the sample. This type of interaction is called "light scattering." The incident radiation is of high intensity and is usually produced by a laser. There are means for directing this laser beam along an axis of the curvilinear reflection means.
In particular, small volumes of sample flow past the point of interaction. The axis of sample flow is coaxial with the axis of a reflective light collector. There are also means for directing a sample flow in a tube along an axis collinear with the laser beam in such a way that the beam impacts the sample without impacting the sample tube wall. In this manner, the three axes are collinear.
The scattered light ray output is the generated signal at the focus of the reflecting means.
The output reflection signal from the reflective means can be a collimated beam which is directed to a detector directly. The reflecting curvilinear surface of revolution producing a collimated beam is selectively a paraboloid. The collimated beam may also be focused onto a detector by a focusing lens. Alternatively, the curvilinear means can reflect the generated signal directly to a remote focal point. The curvilinear means for doing this is preferably an ellipsoid.
In different forms of the invention, the generated radiation signal may arise selectively from light scattering, Raman scattering, fluorescence, phosphorescence or luminescence.
The invention is further described with reference to the accompanying drawings.
FIG. 1 shows the invention used to monitor some property of a sample as it changes in time as a result of passage through a separation means. The sample may be mixed with a reagent, and the geometric axes in the vicinity of the point of radiation generation are illustrated.
FIGS. 2A and 2B are respectively a side diagrammatic view and end diagrammatic view of the system for detection of scattered light.
FIG. 3 shows the details of the containment vessel insert fitting into the curvilinear reflecting surface.
FIG. 4 shows an alternate design of the insert in a configuration optimized for the detection of sample luminescence.
FIG. 5 is a diagrammatic view of an embodiment wherein an annular aperture determines the detected scattering angle of a scattering signal.
FIG. 6 is a diagrammatic view of a monochromator suitable for addition to any of the above light collection systems. A Fery prism is used to disperse the radiation onto one or more detectors. An aperture plate in the focal surface and in front of the detector(s) determines the portion of the spectrum detected. Alternatively, an array detector may be located at the focal surface of the prism and the spectrum measured.
FIG. 7 is an embodiment illustrating an ellipsoidal version of the reflective surface, and is similar to FIGS. 2A and 2B.
FIG. 1 identifies the major components of the system. A sample processed by some type of separation system 1, such as a chromatograph column, mixes with a reagent from a delivery system 2 at point 3. The mixture enters a sample containment vessel 4. An intense light source 5 such as a laser produces a beam 102 which is filtered by a filter 6 and focused by a lens 7 onto a point of radiation generation 8. This generation point 8 corresponds to a focal point of a curvilinear surface of revolution 9 milled into a block 10.
In this illustration, the curvilinear surface 9 is a paraboloid. The surface 9 of this paraboloid is reflecting and collimates the radiation generated at the point 8. From the point 8, there is generated a beam 111 of incident radiation which is directed through a large solid angle to reflective surface 9. The collimated beam 100 of reflected radiation is filtered by a filter 11 and focused by a lens 12 onto an aperture 13 in a plate 14. Radiation passing through the aperture 13 is detected by a detector 15.
An imaginary straight line 101 from the intense light source 5 to the aperture 13 defines the axis of the curvilinear surface 9 and also the axis of sample flow in the vicinity of the point of generation 8. Where pertinent, this imaginary line 101 defines the axis of the exciting light beam 102 in the vicinity of the point 8 of radiation generation.
FIG. 2A shows an embodiment of the invention specifically for the detection of scattered light. The light beam 102 from a laser 5 is focused by the lens 7 and directed by means of a two-axis adjusting device 16 down the axis of a channel 17 in the sample containment vessel 4 which screws into the block 10 of the paraboloid. The sample from point 3 enters the containment vessel 4 at 18 and exits at 19.
The laser beam 102 must not strike the walls of the channel 17 in the sample containment vessel 4. Alignment is accomplished by viewing the generation point 8 by means of viewing lens 20 and a hole 21 in block 10 into which the curvilinear surface 9 is formed.
The lens 12 and the aperture in plate 14 serve as a spatial filter and allow only the radiation generated at 8 from passing to the detector 15. In some applications, this spatial filter is unnecessary, but the photosensitive area of the detector 15 must then be as large as the mouth of the paraboloid.
FIG. 2B is an end view of the apparatus looking down the axis of revolution 101 of the curvilinear surface 9. Looking into the open of the paraboloid, one "sees" the end of the end cap 27 of the containment vessel. The inner edge 103 and outer edge 104 of the paraboloid mouth as well as the outer edge of the lens 12 are visible.
FIG. 3 shows the details of the sample containment vessel 4 and the end cap 27. The parts are made of a material such as black "Delrin." The containment vessel 4 is threaded at 105 to fit into the paraboloid block 10 which has mating threads and position the generation point 8 at the focal point of the paraboloid surface 9.
The sample enters at 18 through a stainless steel tube 22 tightly inserted in a hole 23 drilled into the black plastic. At a point below the stainless steel tube 22, a second hole 24, at 90° to the first hole 23, is drilled into the black plastic and conveys the sample mixture to channel 107 running down the axis of the containment vessel. The second hole 24 is closed by plug 25 to keep the sample mixture from escaping from the desired channel 24. A glass tube 26 fits tightly into bore 107 in the sample containment vessel 4 and extends into the second black plastic end cap 27.
A glass plate 28 serves as a window to allow laser radiation 102 to pass into the containment vessel while keeping the sample in its channel 24. The glass plate 28 is held against an "O ring" 29 by means of retainer 30. The glass tube 26 does not come in optical contact with the glass plate 28. Thereby, the light scattered at the sur-face of the glass plate 28 does not channel into the glass tube 26. Were a significant fraction of the light scattered by glass plate 28 to enter tube 26, some unwanted radiation could escape from tube 26 in the vicinity of the generation point 8.
The end cap 27 allows the sample to exit the system through hole 108 and also acts as a light trap for the excess incident radiation from laser beam 102. A plate 31 of polished black glass acts as the primary light trap. This plate 31 is angled to send the weak reflected beam to the end wall 109 at the end of hole 108. The black glass plate fits against the wall of the black plastic by means of a threaded plug 32. Another "O-ring" 33 seals against escape of the sample stream. A second plug 25 again confines the sample to the desired stream.
A short piece of stainless steel tubing 34 fitted into a hole 110 conveys the sample stream to a piece of Teflon (Polytetrafluoroethylene) tubing 35 which mates with still another piece 36 of stainless steel tubing by which the sample stream exits the containment vessel at 19. The plastic tubing 35 blocks only a negligible portion of the scattered rays from falling on the paraboloid.
The end cap 27 is dimensioned to slip through the hole in the paraboloid block 10 at its base.
The solid angle 112 of incident radiation is between about 45° and 135° relative to the axis 101. The end face 113 of the vessel 4 defines one limit of the angle 112. The end face 114 of the end cap 27 can define a second limit of the angle 112. Faces 113 and/or 114 form occlusion means about the tube defining radiating barrier means relative to the local point to define the solid angle. Alternatively to the end face 114, the limit 103 of the parabaloid surface 9 can limit the extent of the angle, as illustrated in FIGS. 1 and 2A and 2B.
FIG. 4 shows another embodiment of the containment vessel 4 and its end cap 27. In this case, the sample mixture is introduced to the generation point by means of a capillary tubing 41. A chemiluminescence reagent is introduced through entry 18. This embodiment is primarily intended for use with chemiluminescent samples. Exciting light source is not necessary in this application. It is presumed that the luminescence resulting from the chemical reaction is of short lifetime, hence, has a maximum intensity at the point of reaction which in this case occurs at the generation point.
FIG. 5 is similar to FIG. 2A and is useful in certain types of light scattering measurements. Plate 43 with an annular aperture 42 is inserted into the apparatus where the beam from the sample is collimated. The inner and outer diameters of this annular aperture determine the solid angle 112 of detected scattered light.
FIG. 6 is an extension of FIG. 2A and shows the focused radiation 106 passing through the aperture 13 in plate 14 and on to a Fery prism 44 which disperses the radiation 116 and focuses a spectrum on a curved plate 45. The location and size of the aperture 47 in plate 45 determines the wavelength and wavelength interval of radiation detected by detector 46. A multiplicity of apertures and detectors may be used when it is desired to monitor different spectral intervals simultaneously. Similarly, means other than a Fery prism may be used to disperse radiation 106.
Alternately, an array detector may be positioned in place of the aperture plate. The signal from the array of detectors 46 may be processed in the usual manner to provide the dynamic changing spectra as samples flow through the system. It should be apparent that any type of dispersing system may be employed in FIG. 6. However, the Fery prism 44 affords a high efficiency along with low stray light.
When there is no need to filter the light from the sample (by means of filter 111 shown in FIG. 1), surface 9 of block 10 may be in the shape of an ellipsoid. As shown in FIG. 7, the beam 106 is then focused directly onto aperture plate 14 and lens 12 is not needed.
Description of a few applications for the above equipment may help to understand the invention.
The apparatus in its light scattering configuration is useful in the study of antigen-antibody reactions. When the antigen specific to an antibody mixes with a solution containing the antibody, there is a reaction producing aggregates of rapidly increasing size. If the concentration of antibody and antigens is sufficiently high, the aggregates may become large enough to see with the naked eye. However, at low concentration, the limiting aggregate size may be submicroscopic and detectable only by very sensitive light scattering techniques.
The above invention is applicable to the screening of antibody-antigen reactions wherein a separation technique isolates fractions of either antibodies or antigens. As the components elute from the separations means 1 and mix with a potential conjugate steadily flowing from 2, the sample generates an elevated scattered light signal, indicative of an aggregating pair. The detector means 15 can be set up to respond to a spatial or temporal signal from the generating point 8.
Raman spectra often characterize particular classes of molecular structure. A detector can sense when sample fractions having that particular class of structure elute from a chromatograph column 11.
Fluorescence and phosphorescence techniques provide for extremely sensitive detection of classes of samples eluting from a separation system when certain classes of effluent can be tagged with a fluorescent dye or set of dyes. For example, DNA fragments of different size may be chromatographed to separate fragments by molecular weight. At the same time, there are only four possible end groups and these can be dyed with fluorophores specific to the end groups. The above apparatus can identify the end group of the eluting fractions.
Certain oxidation-reduction reactions can be sensitively detected by chemiluminescence. The above apparatus can provide a good detector when an eluting sample triggers a chemiluminescence reaction.
Many more examples of the invention exist, each differing from the other in matters of detail only. The invention is to be determined solely by the appended claims.