Patent Application
20070177149
The present invention relates generally to the field of biochemical laboratory instrumentation for different applications of measuring properties of samples on e.g. microtitration plates and corresponding sample supports. More particularly the invention relates to more efficient and more accurate instrumental features of equipment used for measuring e.g. fluorescence.
The routine work and also the research work in analytical biochemical laboratories and in clinical laboratories is often based on different tags or labels coupled on macromolecules under inspection. The typical labels used are different radioactive isotopes, enzymes, different fluorescent molecules and e.g. fluorescent chelates of rare earth metals.
The detection of enzyme labels can be performed by utilizing its natural biochemical function, i.e. to alter the physical properties of molecules. In enzyme immunoassays colourless substances are catalysed by enzymes into colourful substances or non-fluorescent substances are catalysed into fluorescent substances.
The colourful substances are measured with absorption, i.e. photometric measurement. In the photometric measurement the intensity of filtered and stabilized beam is first measured without any sample and then the sample inside one plate is measured. The absorbance i.e. the absorption values are then calculated.
The fluorescent measurement is generally used for measuring quantities of fluorescent label substance in a sample. The most photoluminescence labels are based on molecular photoluminescence process. In this process optical radiation is absorbed by the ground state of a molecule. Due to the absorption of energy the quantum molecule rises into higher excited state. After the fast vibrational relaxation the molecule returns back to its ground state and the excess energy is released as an optical quantum.
A further measurement method is chemiluminescence measurement where emission is due to a chemical reaction, and emission of a substance is measured from a sample without excitation by illumination. Thus a photoluminometer can also be used as a chemiluminometer.
Further, there is an analysing method called Amplified Luminescent Proximity Homogeneous Assay or AlphaScreen™. The function of the AlphaScreen method is based on the use of small beads that attach to the molecules under study. There are two types of beads that are coated with a material acting either as a donor or acceptor of singlet-state oxygen. The measurement starts, when the liquid sample is activated by illuminating by light with a wavelength of 680 nm. After this the material in the donor bead converts ambient oxygen into singlet-state oxygen. The single-state molecules have a short lifetime and they can reach only about a 200 nm distance by diffusion in the liquid. If the chemical reaction in question has taken place, both the donor and acceptor beads are bound to the same molecule and so they are close to each other. In this case the singlet-state oxygen may reach the acceptor bead where a series of reactions is started. As the last phase of the reaction the coating material of the acceptor beads emits photons in the 500-700 nm range. If the chemical reaction has not taken place the singlet-state oxygen cannot reach the acceptor bead and the emission light is not detected. By measuring the intensity of light it is possible to conclude the efficiency of the chemical reaction.
The typical instruments in analytical chemical research laboratories are the different spectroscopic instruments. Many of them are utilizing optical region of electromagnetic spectrum. The two common types of instruments are the spectro-photometers and the spectrofluorometers. These instruments comprise usually one or two wavelength dispersion devices, such as monochromators. The dispersion devices make them capable to perform photometric, photo-luminescence and chemiluminescense measurements throughout the optical spectrum.
Patent documents U.S. Pat. No. 6,187,267 and U.S. Pat. No. 6,097,025 describe a device for detecting photoluminescence and chemiluminescence from samples.
The emission unit 145 receives photoluminescence emission radiation via fibre optic cable 110a or 110b either from the top measurement head 112a or from the bottom measurement head 112b, respectively. The emission beam is directed to the tips of the thin fibre optic cables with a confocal optical relay structure. The emission unit may comprise optical components, such as lenses, filters and detectors.
The instrument also comprises chemiluminescense measurement equipment. It includes a non-confocal optical relay structure 150, which guides the emission radiation to the thin fibre optic cable 156 by reflections and refraction. The emission radiation is guided via the fibre optic cable to the emission unit including a detector for measuring amount of radiation.
The measurement results achieved with prior art instruments are accurate when the sample to be measured is homogeneous. This means that the substance which is measured and which gives the emission is evenly distributed within the sample. This is the case for example when the substance is dissolved within a liquid sample. However, one important application for optical measurements relates to the measurement of biologic cells, i.e. measurement of substances which are inside the cells or attached to the cells. Usual measurements of cells include e.g. measuring concentrations of Ca2+ and GFP (Green Fluorescent Protein). The cells are typically within a liquid sample, and the cells are settled at the bottom of the sample well. In optical measurement of cells the emission is thus received from the bottom area of the sample within a sample well. In addition to biologic cells, measurements of other details within samples, such as various particles or beads is often necessary. The size of such details is typically 1-100 μm.
a,
2
b and 2c illustrate the measurement volume in confocal measurements of a sample.
There are certain limitations related to the prior art instrumentation when emission from small details within a sample is measured.
Let us consider a typical measurement where the measurement volume is 50 mm3, the number of cells within the sample is 10000 and the diameter of each cell is 10 μm. The total volume of the cells would be approx. 0.005 mm3 which is only 100 ppm of the whole measured volume within the sample. Therefore the intensity of the emission signal tends to be very low in such measurements. And further, noise signal is received also from a large sample volume outside the cells, which tends to make the signal-to-noise ratio of the measurement low.
Further, since the prior art measurement gives a value for the intensity of the total emission from the sample, the signal intensity depends on the number of cells within the sample. However, it is usually necessary to get information on the concentration of the measured substance within the details such as cells. In order to get this information the number of cells within the measurement volume should be known and constant, which is usually not possible to achieve. Even if the number of details, such as cells, would be approximately same in each sample, the varying location of the cells within the sample would cause the amount of cells inside the measurement volume to vary as well. For example, the measurement result in
An object of the present invention is to provide an optical instrument for laboratory measurements, wherein the described disadvantages of the prior art are avoided or reduced. The object of the invention is therefore to achieve a measurement instrument with improved versatility, accuracy and/or efficiency for performing measurements from both homogeneous samples and samples including details, such as cells.
The object of the invention is achieved by providing optical measurement instrumentation which comprises a point detector for the measurement of homogeneous samples, and an image detector for the measurement samples wherein the substance to be measured is inside or attached to details. The instrumentation has thus two measurement modes for the measurement of different types of samples.
The present invention has substantial advantages over prior art solutions. When the emission radiation is imaged using a sufficient resolution the details such as cells or beads can be distinguished from the background with a much higher signal-to-noise ratio. The measurement can be made based on only those areas of the image which include emitting details, and an average measurement result can be calculated for a detail or a pixel including emission. Therefore the number of the details or their location does not affect the measurement result.
It is also possible to use instrumentation according to the invention for simultaneous measurement of fluorescence both from details such as cells or particles, and from the sample liquid. It is further possible to use the location information of the emitting details within the sample for other purposes thus making multiparameter measurements possible.
In a preferable embodiment of the instrumentation a photomultiplier tube, PMT, is used as a point detector for measuring a measurement volume of a sample as a whole and a CCD is used as an image detector for measuring details from the sample. These detectors have the best sensitivity in different area of the spectrum; PMT is more sensitive blue side of the spectrum compared to the CCD, and the CCD is more sensitive on the red side of the spectrum compared to the PMT. When such detectors are used it is thus possible to have a good coverage of the whole spectrum of the emission radiation.
An optical measurement instrument according to the present invention for measuring samples, comprising
an illumination source for excitation or activation of a sample,
a point detector for measuring emission radiation in a first measurement mode, wherein the point detector outputs a signal corresponding to the radiation received within the whole sensor area of the point detector, and
means for projecting emission radiation from a first measurement volume of a sample to the point detector,
wherein the point detector outputs a signal which corresponds to intensity of the emission radiation received from the whole first measurement volume, is characterized in that the instrument further comprises
an image detector for measuring emission radiation in a second measurement mode, wherein a sensor area of the image detector includes a multitude of sensor pixels for providing signals which correspond to the radiation received by said sensor pixels, and
means for projecting emission radiation from a second measurement volume of a sample to the sensor area of the image detector,
wherein the image detector outputs a signal including information on intensity and spatial distribution of the emission radiation received from details within the second measurement volume of a sample.
A method according to the invention for optical measurement of samples with an optical measurement instrument, the method comprising a selectable first measurement mode, wherein
emission radiation is projected from a first measurement volume of a sample to a point detector of the instrument, and
emission radiation is measured with a point detector of the instrument, wherein the point detector outputs a signal corresponding to the radiation received within the whole sensor area of the point detector,
wherein the point detector outputs a signal which corresponds to intensity of the emission radiation received from the whole first measurement volume, is characterized in that the method further comprises a selectable second measurement mode, wherein
emission radiation is projected from a second measurement volume of a sample to the sensor area of an image detector,
emission radiation is measured with the image detector, wherein a multitude of sensor pixels of the image detector provide signals which correspond to the radiation received by said sensor pixels, and
wherein the image detector outputs a signal including information on intensity and spatial distribution of the emission radiation received from details within the second measurement volume of a sample.
Some preferred embodiments are described in the dependent claims.
In this patent application term “point detector” means a detector providing a signal, which substantially corresponds to the total radiation intensity received by the sensor area of the detector from a measurement volume.
In this patent application term “image detector” means a detector including several sensor pixels for providing a signal which includes information on the intensity of radiation received by the sensor pixels, including information on the spatial distribution of the received radiation between the pixels.
In this patent application term emitting “details” of a sample means cells, particles, beads etc. which are not homogeneously distributed within the sample and which include or attach emitting substance, the emission radiation of which is measured.
In this patent application term “measurement volume” means a volume within a sample from which the detector is adapted to receive radiation in the concerned measurement.
In this patent application “measuring” a substance or a sample may mean measuring the contents of a substance in a sample or measuring properties of a sample or properties of a substance in a sample.
The described and other advantages of the invention will become apparent from the following detailed description and by referring to the drawings where:
a illustrates the measurement volume in a prior art instrument when a homogeneous sample is measured from above or below the sample,
b illustrates the measurement volume in a prior art instrument when a sample including cells is measured from above the sample,
c illustrates the measurement volume in a prior art instrument when a sample including cells is measured from below the sample,
The measurement principle according to the
By using an imaging detector for imaging details thus improves the measurement accuracy when the number of the details in the imaging volume is small and the emission from the details only covers a part of the image on the sensor surface of the detector. The accuracy can also be improved by increasing the number of pixels in the detector. The number of pixels in the imaging detector can be e.g. as high as 1000×1000 pixels. In order to achieve improvement on the measurement accuracy, the number of pixels should be higher than the number of details within the measurement volume, preferably more than ten-fold or more preferably more than 100-fold higher.
The instrument comprises an illumination source 511 for the excitation of a sample in a photoluminescence measurement. The radiation from the lamp 511 is collimated with lens 515 and directed through an interference filter 514. Different filters can preferably be selected for different wavelengths. The excitation beam is reflected by a dichroic mirror 551 and further directed into the sample 581 through a lens system 563. In order to achieve a good accuracy of the intensity of the excitation beam it is also possible to use a reference detector (not shown in
The photoluminescence emission beam from the sample 581 is directed with the objective lens system 563 through mirrors 551 and 552. The dichroic mirror 551 can be designed for certain labels so that it reflects the excitation wavelength but transmits emission wavelength. Also, The dichroic mirror 552 can be designed for certain labels so that it transmits the wavelength of an emission beam received from a measurement from a homogeneous part of a sample, and reflects the wavelength of an excitation beam received from a sample detail measurement. The mirror may alternatively be an ordinary beam splitter mirror, which reflects 50% of the beam intensity, and transmits 50% of the beam intensity. The mirror may also be based on polarization etc. A beam splitter mirror can be produced e.g. by forming reflective coating for the mirror to be e.g. stripes or dots, which cover only a part of the mirror surface.
The emission beam further transmits an emission filter 534, and the beam is focused with a lens 535 into a point detector 531. The point detector measures a value for the total intensity received from the measurement volume of the sample. The point detector 531 is most preferably a photon multiplier tube (PMT), but alternatively other types of point detectors can also be used, such as a photo diode detector. The radiation reaches the window of the photo-multiplier tube, and after penetrating through the window the radiation reaches the active surface of the photo-multiplier tube. The block 531 includes the amplifier and other related electronics for the photo-multiplier tube. The amplified signal is preferably integrated over the reception time window, and the achieved signal is converted into a digital signal. The digital signal is led to the microprocessor controller 596, which determines the measurement result on the basis of the signal received from the PMT. This measurement result corresponds to the amount of the measured substance within the measurement volume. The measurement result is preferably stored in the memory 597 and displayed on the user interface 598. The measurement volume may correspond to
When imaging details from a sample the excitation can be performed in the arrangement of
The excitation filter 514 is selected according to the excitation wavelength of the substance to be measured. The excitation beam is reflected by a mirror 551 and further directed into the sample 581 through the lens system 563. The measurement volume may correspond to
The emission beam received from the details of a sample is also collimated with an objective lens 563. The emission beam further transmits the first dichroic mirror 551, which is designed to transmit radiation of emission wavelength and to reflect radiation of excitation wavelength. The purpose of the filter 594 is to prevent passing of light with a wavelength outside the emission radiation received from the sample details. The second dichroic mirror 552 is on the other hand designed to reflect the radiation of the emission wavelength which is received from the details of the sample. The mirror 552 transmits radiation on wavelength of the emission which is received when measuring the homogeneous volume of the sample. However, other than dichroic beam splitter mirrors can alternatively be used; radiation of unwanted wave lengths can be blocked by separate filters.
The emission beam reflected by the mirror 552 further transmits the filter 594, and the lens 595 focuses the emission beam to the sensor surface of a CCD detector 591. The CCD detector unit 591 advantageously comprises an amplifier and an analog-to-digital converter for providing digital data corresponding to the radiation intensity received by each pixel. The data is led to the microprocessor 596 which processes the data according to the program and parameters stored in the memory 597. The processor may perform image correction and determines locations of the emitting details within the sample measurement volume. The processor further calculates the average concentration of the measured substance within the details, such as cells or other particles. The result of the measurement can be stored in the memory 597 and displayed at the user interface 598.
The instrument of
The selection of the measurement type and measurement mode can be made using the user interface 598, which may comprise e.g. a keyboard and a display. The measurement sequence is controlled by the controller 596 according to the program and parameters which are stored in a memory 597. The controller thus controls the illumination source, the selection of filters and mirrors, as well as the acquisition of the measurement data from the detectors.
The measurement volume within a sample is preferably different in location and possibly in size when homogeneous substance and substance from details is measured. The centre of the measurement volume is preferably located near to the centre of the sample when homogeneous substance is measured. When substance from details is measured from the bottom of the sample well, the centre on the measurement volume is preferably at the lower part, possibly near to the bottom of the sample.
It is possible to measure details of each sample separately with the image detector. However, it is alternatively possible to make the measurement of details from two or large number of adjacent samples simultaneously with the image detector. In this case, it is required to design the optical components for a wider optical beam, and to use an image detector with higher resolution.
The instrument of
The instrument according to
An optical fibre 618T is used for guiding the excitation beam from the optical switch 617 to the optical module 640 of the top measurement head. An optical fibre 618B is used for guiding the excitation beam from the optical switch 617 to the optical module 650 of the bottom measurement head. The instrument may also have separate lamps for providing the excitation beam of the top head and the bottom head. For example, separate illumination sources may be used for the measurement of homogeneous substance from a first measurement volume, and for the measurement from details within a second measurement volume of a sample.
In the bottom measurement head the excitation beam is directed into the sample 681 via a collimating lens 692, mirror 651, and a lens system 663 of the bottom measurement head.
The equipment may also include a further pulse lamp 612b, 611b, which may be a low power lamp, e.g. for photometric measurements. The instrument has an optical fibre guide 612a for guiding the light from the second lamp. The light can be distributed for the photometric measurement into three filters 614h, 614j and 614k with fibre branches 677h, 677j and 677k. After filtering, the beams are collimated into ends of three optical fibre cables 678, which are led to the bottom measurement head for the photometric measurement. The light beams from the optical cables 678 are focused to three samples 684 with a lens system 679 including lenses for each three beams. After transmitting through the samples the beams are measured with three detectors 622d, 622e and 622f, which are e.g. photo diodes. The three ends of the fibre optic cables, three lenses, three simultaneously measured samples and three detectors are in this case located in a row perpendicular to the plane of the drawing and thus only one of them can be seen in the drawing.
It is also possible to use an instrument with same pulse lamp for photometric and photoluminescence measurements. For example, an optical switch 617 may have an output for an optical fibre 678a, which leads light from the lamp 612a to the photometric measurement optics 679. It is then possible to control the optical switch either to guide the light for providing excitation for an emission measurement or to guide the light the photometric measurement.
When the emission from a homogeneous substance is measured from above the sample, the emission beam from the sample 681 is directed with the lens system 623 into the optical module 640a. If emissions from two homogeneous substances are measured, the emission beam is divided into to two beams. A dichroic mirror in the optical module preferably functions as a filter so that a beam with a wavelength of a first emission is transmitted through a selectable filter 634 to the first point detector 631a, and a beam with a wavelength of a second emission is reflected by mirror 638 and directed through a selectable filter 634 to the second point detector 631b. The point detector can be e.g. a photo-multiplier tube, which may be used in analogue mode or in photon count mode, or in both modes simultaneously. When the equipment includes two point detectors they may be of different types and the detection modes may be different during a photoluminescence measurement.
The instrument also comprises an optical switch 637 for selecting the detected emission beam for a point detector from the top or bottom measurement head. An optical fibre 638 is used for guiding the first emission beam from the bottom measurement head 660 to the optical switch 637. When emission of a homogeneous substance is measured with a point detector from below the sample, the emission beam is first collimated with objective lens system 663. The emission beam then transmits the dichroic mirrors 651 and 652 as was shown in
The signals received from the point detectors are amplified and processed to achieve a value for the intensities of the homogeneous emissions. Measurement signals are amplified and read after each excitation pulse and possible signal corrections are calculated. Basic reference parameters are determined with standard solvents after the analyzer has been assembled. If there are more than one excitation pulses used for one sample well, the corresponding emission signals are preferably digitally integrated.
In a second measurement mode for measuring details an image detector 691 is used. The emission beam is collimated with the objective lens system 663, whereafter the beam transmits the dichroic mirror 651, and is reflected by the next dichroic mirror 652. Mirrors 651 and 652 may alternatively be of other type, based on e.g. 50%/50% separation or polarization. The emission beam is then filtered with a selectable filter 694 and focused to the sensor surface of the image detector. The image detector is preferably a charge coupled device, CCD.
It is possible to measure details of each sample separately with the image detector. However, it is alternatively possible to make the measurement of details from two or large number of adjacent samples simultaneously with the image detector. In this case, it is required to design the optical components for a wider optical beam, and to use an image detector with higher resolution.
The optical components such as the mirrors 651 and 652, filter 694 and lenses 692, 693 and 695 can be changeable, and they may be included in a changeable optical module. In
The instrument is also equipped with electronics for amplifying and processing the signals from the detectors, as well as electronics for driving the lamp(s). There is also control electronics provided for controlling the measurements, such as selecting filter(s), selecting the optical module(s), controlling optical switch(es), controlling the position of the sample tray 689, and controlling the positions of the measurement heads 620 and 660 relative to the sample platform 680. The electronics also includes processing means processing the image data received from the image sensor in order to obtain measurement results relating to the details of the sample. The main electronics is not shown in
The detectors and light sources including their electronics are shown reduced in size compared to other components in
Next an example of a measurement method according to the invention is described referring to
When a point detector measurement is performed, 71, a light source and suitable excitation and emission filters are next selected, 77, according to the excitation and emission wavelengths of the measurement. However, if chemiluminescence measurement is performed, no illumination source or excitation filter is required. After selecting those components the optical measurement is performed by transmitting an excitation light pulse to the sample (not in chemiluminescence measurement) and measuring the emission radiation received to the point detector, 84. The concentration of the measured substance can then be determined on the basis of the intensity of the signal outputted from the point detector.
When an image detector measurement is performed, 72, a light source as well as suitable excitation and emission filters are next selected, 73, according to the excitation and emission wavelengths of the measurement. Then the optical measurement is performed by transmitting an excitation light pulse to the sample and measuring the emission radiation received to the image detector, 74. However, if chemiluminescence measurement is performed, selection or use of illumination source and excitation filters is not necessary. Finally, concentration of the measured substance in details of the sample is determined based on the data outputted from the image detector, 75. This last step of determining the substance concentration on the basis of the image detector signal is shown in more detail in
The location of details within the measurement volume is next determined, 83. The location of details can be determined e.g. by comparing the intensity data from each pixel with the background signal intensity. When the data value of a pixel is higher than the background value by a predetermined amount, the pixel can be regarded as having received emission from a detail. It is then, using the location information of these pixels, possible to identify the location and size of the details within the measurement volume of the sample. The background signal level can be determined based on the lowest data values of the image pixels.
Next the intensity data values are determined from the pixels which are regarded as having received emission radiation from sample details, 84. The total intensity of these pixels can then be divided e.g. by the number of concerned pixels or the number of identified cells. Thus a value is achieved, which corresponds to the concentration of the emitting substance of the details, such as cells or other particles, 85.
In this patent specification the structure of the components in an optical measurement instrument is not described in more detail as they can be implemented using the description above and the general knowledge of a person skilled in the art.
An optical instrument includes control means for performing the optical measurement process. The control of the measuring process in an optical measurement instrument generally takes place in an arrangement of processing capacity in the form of microprocessor(s) and memory in the form of memory circuits. Such arrangements are known as such from the technology of analyzers and relating equipment. To convert a known optical instrument into equipment according to the invention it may be necessary, in addition to the hardware modifications, to store into the memory means a set of machine-readable instructions that instruct the microprocessor(s) to perform the operations described above. Composing and storing into memory of such instructions involves known technology which, when combined with the teachings of this patent application, is within the capabilities of a person skilled in the art.
Above, an embodiment of the solution according to the invention has been described. The principle according to the invention can naturally be modified within the frame of the scope defined by the claims, for example, by modification of the details of the implementation and ranges of use.
For example, the measurements with a point detector and measurements with an image detector can be made from the same samples, simultaneously or successively, or the different types of measurements can be made from different sets of samples.
It is also possible to make either measurements concerning the contents of a substance in a sample or properties of a sample based on the signals received from the point and/or image detectors. It is also possible that contents of a substance in a sample is measured with one of the detectors and a property of a same or other sample is measured with other one of the detectors.
Although the invention is described with an arrangement where the light source and the detector for the detail imaging function are located on the bottom measurement head, there is no reason why their location on the top measurement head should not work. It is also possible to use illumination from above and detection from below the sample or vice versa.
Also, although the invention has been described with reference to the various microtitration plates it is equally applicable to any form of sample matrixes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20051329 | Dec 2005 | FI | national |
