JP2003526767A - Wide-area light detection system - Google Patents

Abstract

(57) [Summary] The present invention relates to a wide-area light detection system. In some embodiments, the system includes devices and methods that can detect light with high accuracy over a wide range of intensities. In other embodiments, the system includes devices and methods that automatically switch the scale of the detection range in response to the intensity of the detected light, thereby improving detection. In yet another embodiment, the system includes an apparatus and method that can detect light at an increased speed, particularly in applications to the analysis of continuous samples.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

This application is related to U.S. patent application Ser. No. 09/06, filed April 17, 1998.
No. 2,472, US patent application Ser. No. 09/1 filed Sep. 24, 1998
No. 60,533, PCT application No. US9 filed on October 30, 1998
8/23095, PCT application No. US9 filed on January 25, 1999
It is a continuation application of 9/01656, which claims these priorities.
These applications are incorporated herein by reference.

This application is based on US Patent Preliminary Application No. 60 / filed on Feb. 20, 1998.
075,414, US Patent Preliminary Application No. 60 / 082,253, filed April 17, 1998, and US Patent Preliminary Application No. 60 / 100,951, filed September 18, 1998. , And claims the rights of these applications under 35 USC 119. These applications are incorporated herein by reference.

For reference purposes, this application includes PAUL HOROWITZ & WINFIELD HILL, THE ART OF ELECTRONICS (1980) and JOSEPH R. LAKOWICZ, PRINCIPLES OF FLUORESCENCE.
The document SPECTROSCOPY (1983) is incorporated.

The present invention relates to light detection. More specifically, the present invention relates to a wide range photo detection system.

[0005]

[Prior art]

Systems that include light detection are used in a variety of fields. In particular, systems involving photodetection and subsequent analysis are used in performing optical spectral analysis such as emission and absorption analysis. These analyzes can be used to characterize the components and properties of molecular systems. Recently, these analyzes have been used in high throughput screening methods to recognize drug candidate compounds.
For optical spectrum analysis, fluorescence polarization method (FP), fluorescence resonance energy conversion method (FR)
ET), fluorescence lifetime method (FLT), total internal reflection by fluorescence (TIR), fluorescence correlation spectroscopy (FCS), fluorescence recovery method after light bleaching (FRAP), and those methods that perform phosphorescence. is there. Optical spectrum analysis also includes absorption spectroscopy.

[0006]

[Problems to be Solved by the Invention]

Unfortunately, photodetection systems have many drawbacks. That is, such systems are limited in range. That is, such a system can only accurately detect light within a relatively narrow intensity range. Further, in such a system, when the detection range is changeable, the user often operates manually to change the detection range. Also, such systems are limited to either discrete or analog detection. Therefore, in such a system, individual quanta, that is, photons are discretely counted, or an analog value corresponding to such quanta is integrated. However, you cannot do both. Also, such systems require a significant amount of time to make measurements. These drawbacks occur individually or in combination, and in the field of high-throughput screening in which tens, hundreds, or thousands of measurements must be performed per day, Especially serious.

[0007]

[Means for Solving the Problems]

The present invention provides a wide range light detection system. In some embodiments, the system comprises an apparatus and method that can detect light with high accuracy over a wide intensity range. In another embodiment, the system comprises a device and method that automatically switches the scale of the detection range depending on the intensity of the detected light, thereby improving detection. In yet another embodiment, the system has particular application in the analysis of serial samples.
An apparatus and method is provided that allows light to be detected with increased speed.

[0008]

DETAILED DESCRIPTION OF THE INVENTION

The features of the present invention will be clearly understood in view of the accompanying drawings and the following detailed description of the preferred embodiments.

FIG. 1 is a schematic diagram of fluorescently labeled molecules, showing how molecular reorientation affects fluorescence polarization. FIG. 2 is a schematic graph showing temporal measurements in the frequency domain,
The definition of the phase angle (phase) φ and the demodulation factor (modulation) M is shown. FIG. 3 is a diagram schematically showing a photodetector according to the present invention. FIG. 4 is a perspective view schematically showing a part of the device shown in FIG. FIG. 5 is a diagram schematically showing an optical member for photoluminescence in the device of FIG. FIG. 6 is a diagram schematically showing an optical member for chemiluminescence in the apparatus shown in FIG. FIG. 7 is a partial exploded view showing a portion of a housing for the device. FIG. 8 is a schematic view of an alternative photo-detecting device according to the present invention. FIG. 9 is a block diagram showing a photo detection device according to the present invention. FIG. 10 is a block diagram showing an alternative photodetection device according to the present invention. FIG. 11 is a schematic diagram of an integrator similar to that of the devices of FIGS. 9 and 10, but with an additional automatic scale switching mechanism. 12 and 13 are graphs showing response curves showing the non-linearity of a digital counting circuit for the device shown in FIGS. FIG. 14 is a graph showing a response curve showing the saturation characteristic of the integrating capacitor in the analog counting circuit for the device shown in FIGS. FIG. 15 is a graph showing a response curve showing the saturation characteristic of the analog counting circuit used together with the comparator in the devices shown in FIGS.

The present invention provides an apparatus and method for detecting light, as described below. For clarity, the following description of the invention is divided into the following three parts. That is, it is divided into (1) outline of emission analysis, (2) description of light emitting device, and (3) detection method.

1. Outline of luminescence analysis In luminescence analysis, the luminescence from the luminescence analyte is used to study the properties and environment of the analyte. In addition, we will study the binding reaction, enzyme activity, etc., including the analyte. In this sense, the analyte can act as a reporter providing information about other materials or target substances that are the focus of the analysis. As the light emission, various light emission forms such as intensity, polarization and life can be used. Also, in luminescence analysis, the properties of time-independent (steady-state) and / or time-dependent (temporal) luminescence can be used. Steady-state analysis is usually simpler than temporal analysis, but provides less information.

[Intensity Analysis] In the emission intensity analysis, the intensity of emission (or the amount of emission) from the complex is observed. The emission intensity depends on the extinction coefficient, the quantum efficiency, the number of luminescent analytes in the complex, etc. These physical quantities depend on the environment in which the analyte is placed, such as the proximity and efficiency of the quencher and energy conversion partner. Thus, emission intensity analysis is used to study binding reactions, among other applications.

[Polarization Analysis] In emission polarization analysis, polarized light is absorbed or emitted. Molecular rotation is typically studied by using polarimetry. (Polarized light means the electric field direction of light. This electric field direction is usually perpendicular to the light propagation direction.)

FIG. 1 schematically shows how the emission polarization is affected by molecular rotation. In emission polarization analysis, a particular molecule 30 within complex 32 is labeled with one or more luminescent materials. The complex is irradiated with polarized excitation light. The polarized excitation light preferably excites a luminescent material having an absorbing dipole oriented parallel to the polarization of the excitation light. Thereafter, the composite is irradiated with polarized excitation light. Excitation light preferentially excites a luminescent material having an absorption dipole that is parallel to the polarization of the excitation light. Such molecules are attenuated by preferentially emitting light polarized parallel to the emitting dipole. The degree of polarization of the total emission depends on the degree of molecular reorientation in the time interval between emission excitation and emission, called emission lifetime τ. The degree of molecular reorientation depends on the emission lifetime, size, shape and environment of the reorientated molecule. Thus, emission polarization analysis can be used to quantitatively analyze binding reactions, enzyme activity and other quantities. In particular, the molecule rotates through diffusion with a rotational correlation time τ _rot that is proportional to the molecular size. Thus, within the emission lifetime, relatively large molecules are less likely to reorient. Therefore, the entire light emission has a relatively large degree of polarization. On the other hand, even at the same time interval, if the molecule is relatively small, the degree of reorientation is large, and therefore, the entire emission has a relatively small degree of polarization.

[0015]   The relationship between polarization and intensity is represented by Equation 1 below.

[Equation 1] Here, P is the polarized light, I ∥ is the emission intensity parallel to the polarization of the excitation light,
I⊥ is the emission intensity perpendicular to the polarization of the excitation light. If there is only a slight rotation between the excitation and the emission, then I.parallel. Is relatively large and I ..
(P is less than 1 even if there is no rotation.
For example, P is less than 1 if the absorbing and emitting dipoles are not parallel. ). Conversely, if there is a significant rotation between excitation and emission, then I / | will be equal to I /, and P will approach zero. Polarization is in millimeter P units (10
It is often reported as 00 × P). Since P is in the range of 0 to 1, millimeter P
Units vary over the range 0-1000.

Polarization can also be described by other equivalent physical quantities such as anisotropy. The relationship between anisotropy and strength is expressed by the following Equation 2.

[Equation 2] Here, r is anisotropic. Polarization and anisotropy contain the same information. However, anisotropy can more simply represent a system containing one or more luminescent materials. In the following description and claims, these terms are used interchangeably. It is to be understood that a general expression for one of these terms also includes a general expression for the other.

[0017]   The relationship between the polarization and the rotation is expressed by the following Equation 3 which is a Perrin equation.

[Equation 3] Here, P ₀ is the polarized light when there is no molecular movement (intrinsic polarized light), τ is the emission lifetime (the reciprocal of the decay rate), and τ _rot is the rotational correlation time (the rotational speed Reciprocal).

The Perrin equation shows that emission polarimetry is most sensitive when the emission lifetime and rotational correlation time are equivalent. The rotation correlation time is proportional to the molecular weight, and the molecular weight (for spherical molecules) is 2400 (2 for the reference unit).
Every 400 Daltons), it increases by about 1 nanosecond. For short-lived luminescent materials, such as fluorescein, which has an emission lifetime of approximately 4 nanoseconds, emission polarimetry is most useful for molecular weights below about 40,000 (40,000 daltons). Be sensitive. For example, Ru (bpy) 2dcbpy (ruthenium, 2,2'-dipyridyl, 4,4) having an emission lifetime of about 400 nanoseconds.
In the case of long-lived probes such as 4'-dicarbonyl-2,2'-dibipyridine), emission polarimetry is about 70,000 (70,000 daltons)
It is most sensitive to a molecular weight of 4,000,000 (4,000,000 Daltons).

[Temporal Analysis] In the temporal analysis, the time change of luminescence emission is measured. The temporal analysis can be done in the time domain or in the frequency domain. Both are functionally equivalent. In the measurement in the time domain, the time change of light emission is directly observed. Although various procedures are possible,
Typically, the complex containing the luminescent analyte is irradiated with a short pulse of light and the resulting change in luminescence intensity over time is observed. In the case of simple molecules, the emission usually decays according to a single exponential function.

In the measurement in the frequency domain, the time change of light emission is indirectly observed in the frequency space. Typically, the composite is illuminated with light whose intensity is sinusoidally modulated at a single modulation frequency f. However, other methods (
For example, transforming data in the time domain into the frequency domain) can be used. The resulting emission intensity is modulated at the same frequency as the excitation light. However, the light emission is delayed from the excitation light by the phase angle (phase) φ, and the emission intensity is demodulated by the demodulation factor (modulation) M as compared with the excitation light intensity.

FIG. 2 shows the relationship between emission and excitation for experiments in the frequency domain at a single frequency. Phase φ is the phase difference between excitation and emission. Modulation M is the ratio of the ratio of the AC intensity and the DC intensity of the emitted light to the ratio of the AC intensity and the DC intensity of the excitation light. The phase and the modulation are related to the emission lifetime τ by the following equations 4 and 5.

[Equation 4]

[Equation 5] Here, ω is the angular frequency of modulation and is equal to the product of the modulation frequency and 2π. At maximum sensitivity, the modulation angular frequency is approximately the reciprocal of the emission lifetime. Lifetime in high throughput screening varies from less than 1 nanosecond to greater than 10 microseconds. Therefore, an instrument for high throughput screening should be able to cover modulation frequencies of 20 kHz to 200 MHz.

2. Description of Light Emitting Device FIGS. 3-6 show an apparatus 90 for detecting light emitted from an analyte within a complex. The device 90 includes (1) a stage for supporting the complex, and
2) one or more light sources for supplying light to the complex, and (3) one or more light sources for receiving the light transmitted from the complex and converting the received light into a signal. A detector, (4) first and second optical relay structures for directing light between the light source, the complex and the detector, and (5) a processor for analyzing the signal from the detector. It is equipped with. In general, a subset of these components is used in the present invention.

The device 90 can be used to perform a variety of emission and absorption analyses, such as, but not limited to, the analyzes described above. Generally, in such analyses, luminescence emission, which is the emission from an excited electronic state of an atom or molecule, is detected. Luminescent light emission generally refers to all types of light emission except incandescent light emission, and includes photoluminescence, chemiluminescence, electrochemiluminescence, and the like. In photoluminescence involving fluorescence and phosphorescence, excited electronic states are formed by absorption of electromagnetic radiation. In chemiluminescence including bioluminescence, excited electronic states are formed by the conversion of chemical energy. In electrochemiluminescence, excited electronic states are created through electrochemical processes.

The components of the optical system are selected to optimize sensitivity and dynamic range for each analysis supported by the analyzer. For this purpose,
An optical element that inherently has low luminescence emission is selected. In addition, some components are shared in various modes. On the other hand, other elements are specific to a particular mode. For example, in the emission intensity mode and the steady-state emission polarization mode, the light source is shared. On the other hand, in the temporal emission mode, a unique light source is used. In the chemiluminescence mode,
No light source is used. Similarly, different detectors are used in the photoluminescence mode and the chemiluminescence mode.

The remainder of this term is divided into five parts. That is, it is divided into (1) photoluminescence optical system, (2) chemiluminescence optical system, (3) housing, (4) alternative device, and (5) methodology.

[Photoluminescence Optical System] In this embodiment, the device 90 includes a continuous light source 100 and a time-modulated light source 102. The device 90 includes a light source slot 103 for four light sources.
a to 103d. However, the number of light source slots and the number of light sources are
Other numbers are possible. The light source slots 103a-103d function as a housing for covering at least a portion of each light source to provide protection from radiation and explosion. The direction of light transmission in the photoluminescence optical system is indicated by an arrow.

Continuous light source 100 provides light for emission intensity analysis and steady state emission polarization analysis. The continuous light source 100 can be a lamp, a laser, a laser diode, a light emitting diode, or the like. A preferred continuous light source is a high intensity, high color temperature xenon arc lamp, such as the Model LX175F CERMAX xenon lamp available from ILC Technology. The color temperature is an absolute temperature in which a unit in which a black body radiator having the same chromaticity as that of a light source operates is Kelvin. Lamps with a high color temperature produce more light than lamps with a low color temperature. A lamp having a large color temperature can shift its maximum output toward or within a visible wavelength range or an ultraviolet wavelength range where many luminescent materials absorb. A preferred continuous light source has a color temperature of 5600 Kelvin. This is well above the color temperature of about 3000 Kelvin in a tungsten filament light source. Preferred light sources provide more light per unit time than flash-type light sources. This increases sensitivity and reduces reading time. The device 90 can include a modulation mechanism configured to change the incident light intensity on the composite without changing the intensity of the light generated by the light source.

The time-modulated light source 102 provides light for temporal emission analysis such as emission lifetime or temporal emission polarization analysis. As a preferable time-modulated light source,
There are xenon flash lamps, such as the Model FX-l 160 xenon flash lamps available from EG & G Electro-Optics. A preferred light source is one that produces a "flash" of light with a short time interval before signal detection, which is particularly suitable for measurements in the time domain. Other time-modulated light sources include pulsed lasers and continuous lamps whose intensity can be externally modulated using a mechanism such as a Pockels cell or Kersel. The latter light source is particularly suitable for measurements in the frequency domain.

In the device 90, the continuous light source 100 and the time-modulated light source 102 generate polychromatic, non-polarized, and non-coherent light. Continuous light source 100 produces substantially continuous light emission. On the other hand, the time-modulated light source 102 generates time-modulated light emission. Light from these sources can be delivered to the sample without modification. Alternatively, the light from these light sources can be filtered to modify the intensity, spectrum, polarization, and other properties before being delivered to the sample.

The light generated by the light source is guided to the test site through the optical path for the excitation light. Such light can be passed through one or more "spectral filters." Spectral filters are generally equipped with any mechanism for altering the light spectrum delivered to the sample. A spectrum is a wavelength distribution of light. By using a spectral filter, white light or polychromatic light having many colors of light can be converted into red light, blue light or green light having one color or only a few colors of light. Alternatively, it can be converted to other substantially monochromatic light. In the device 90, the spectrum is changed by the excitation light interference filter 104. The filter 104 selectively transmits light having a preselected wavelength and selectively absorbs light having another wavelength. For simplicity, the pump light interference filter 104 may be housed within the pump light filter wheel 106. The filter wheel 106 can change the excitation light spectrum by rotating a preselected filter into the optical path. Spectral filters can also spatially split light by wavelength. Examples of such include diffraction gratings, monochromators, and prisms.

Spectral filters are not needed for monochromatic (“monochromatic”) light sources, such as some lasers, that output only light of a single wavelength. Therefore, the excitation light filter wheel 106 includes several light source slots 103a,
Can be installed in the optical path of 103b, and other light source slots 103c, 103
It may not be installed in the optical path of d.

The light then passes through an excitation light optical shuttle (ie, switch) 108. The pump light optical shuttle 108 includes a pump light optical fiber cable 1 in front of a suitable light source.
10a and 110b are positioned and guide light to the upper optical head 112a and the lower optical head 112b. Both optical heads are equipped with various optics for directing light into the examination volume and for receiving light transmitted from the examination volume. Light is guided through fiber optic cables just as water is guided through hoses. Fiber optic cables can be easily used to direct light to corners and to bypass opaque members of the device. Further, fiber optic cables provide light with a more uniform intensity distribution. The preferred fiber optic cable is a fused silicon cable with low self-luminous. Despite these advantages, light can also be directed to the optical head using other mechanisms such as mirrors.

The light reaching the optical head passes through one or more “polarizing filters” for excitation light. The excitation light polarization filter generally has an arbitrary mechanism capable of changing the polarization of light. The polarization filter for excitation light can be provided in one or both of the upper optical head and the lower optical head. In the device 90, the polarization can be changed by the excitation light polarizer 114. The excitation light polarizer 114 is provided only in the upper optical system 112a. The excitation light polarizer 114 includes an s-polarizer S that allows only s-polarized light to pass, a p-polarizer P that allows only p-polarized light to pass, and a blank O that allows substantially all light to pass. , Can be provided. The pump light polarizer 114 may also comprise a standard switching system, ie a polarization switching system in a ferroelectric liquid crystal display (LCD).
Such systems are faster and more economical than mechanical switches. The pump light polarizer 114 may also include a continuous mode LCD polarization rotator with sync detection to improve the signal to noise ratio in polarization analysis. The excitation light polarizer 114 can also be installed in a light source that inherently produces polarized light, such as some lasers.

Light in one or both optical heads can pass through the “confocal optical member” for excitation light. A confocal optical member for excitation light generally comprises any mechanism for focusing light within the "test volume". In the apparatus 90, the confocal optical member includes a set of lenses 117a to 117c and an excitation light aperture 116 arranged in an imaginary plane conjugate with the examination volume as shown in FIG. There is. Opening 11
6 can be formed directly as an opening or indirectly as the end of a fiber optic cable. The lenses 117a and 117b have the aperture 1
Sixteen images are projected on the sample. This illuminates only the preselected test volume of the sample.

Light passing through both optical heads is reflected and passes through the beam splitter 118. Beam splitter 118 directs the reflected light to composite 120 and the transmitted light to optical monitor 122. Both the reflected light and the transmitted light are reflected by the beam splitter 11
8 and the composite 120 through a lens 117b operatively arranged.

By using the beam splitter 118, the excitation light is directed towards the sample and the optical monitor and the emission is directed towards the detector. The interchangeability of the beam splitter allows the beam splitter to be optimized for different analysis modes and for different complexes. In the study of many or many types of luminescent molecules, the beam splitter needs to be compatible with a wide range of wavelengths of light. In this case, a "50:50" beam splitter that reflects half of the incident light and transmits half of the incident light regardless of wavelength is optimal. Such beamsplitters can be used with many types of molecules, directing most of the excitation light onto the complex and transmitting most of the emission to the detector. For the study of one or a few related luminescent molecules, it is sufficient for the beam splitter to be compatible with light in a limited wavelength range. In this case, a “dichroic” or “polychromatic” beam splitter is optimal. Such a beam splitter can be configured to have a cutoff frequency for the appropriate set of molecules, reflecting most or substantially all of the excitation and background light and most of the emission. Or it makes virtually everything transparent. This is possible because the reflectivity and transmissivity of the beam splitter can be changed depending on the wavelength.

The use of the light monitor corrects for disturbances in the intensity of the light introduced by the light source. Such a correction is made by reporting the detected intensity as a ratio of the corresponding multiple of the emission intensity measured by the detector and the excitation light intensity measured by the optical monitor. The light monitor can also be programmed to alert the user when the light source shuts down. A preferred optical monitor is a silicon photodiode with a self-illuminating small quartz window.

The composite (ie, sample) 120 can be held in a sample holder supported by a stage 123. Complexes are compounds, mixtures, surfaces, solutions, emulsions, suspensions, cell clusters, fermentation clusters, cells, tissues, secretions,
And / or their derivatives and / or extracts.
In the analysis of complexes, the presence of the luminescent analyte in such complexes,
Measure concentration and physical properties (including interactions). The sample holder can be a microplate, a biochip, or a sample of arbitrary sequence in a known format. In device 90, the preferred sample holder is a microplate 124 having a plurality of microplate wells 126 for holding each complex. The complex can be in a single microplate well or in multiple microplate wells, depending on the analysis. In some embodiments, such as a handheld analyzer, the stage is
It can be integrated with the instrument.

The examination volume is typically hourglass shaped with a cone angle of about 25 ° and a minimum diameter of 0.1 mm to 2.0 mm. For 96 well and 384 well microplates, the preferred minimum diameter is about 1.5 mm. For a microplate with 1536 wells, the preferred minimum diameter is about 1.0 mm. The size and shape of the sample container can be adapted to the size and shape of the test volume.

The position of the examination volume can be precisely moved within the complex so as to optimize the signal to noise ratio and the signal to background ratio. For example, the test volume can be moved away from the wall within the sample holder. This makes it possible to reduce spurious signals due to the light-emitting material that is immobilized on the wall and immobilized, and optimizes the signal-noise ratio and the signal-background ratio. In device 90,
The position in the X, Y plane, perpendicular to the optical path, is controlled by moving the stage supporting the complex, and the position along the Z-axis, parallel to the optical path, is , Is controlled by moving the optical head using a Z-axis adjustment mechanism as shown in FIGS. However, any mechanism capable of aligning the test volume to the appropriate composite location can be used.

By combining the upper optical system and the lower optical system, the following combinations of analyzes are possible. That is, (1) irradiation from the upper side and detection on the upper side, or (2) irradiation from the upper side and detection on the lower side, or (3) irradiation from the lower side and detection on the upper side. Or
(4) Irradiate from the lower side and detect on the lower side. The methods (1) and (4) in which irradiation and detection are performed from the same side are referred to as “epi” mode and are preferred in emission analysis. The methods (2) and (3) of irradiating and detecting on the opposite side are referred to as "trans" mode and are preferred in absorption spectroscopy. The epi mode is supported in the device 90. Therefore, the excitation light and the light emission move along the same path in the optical head. However, the directions of movement are opposite to each other and antiparallel. However, it is also possible to support the trans mode, which is substantially the case in the case of absorption spectroscopy. In general, the upper optics can be used with open top sample holders, while the lower optics can be used with sample holders that have an optically transparent bottom, such as a glass bottom or a thin plastic bottom. Can only be used.

Light is emitted from the complex in multiple directions. Some of the emitted light will pass through the emission path towards the detector. The light emission passes through the lens 117c, and further passes through the light emitting aperture 131 and / or the light emitting polarizer 132. In the device 90, the light-emission aperture is arranged in an imaginary plane that is conjugate with the examination volume and allows light from the examination volume to be substantially exclusively transmitted. In device 90, both the upper and lower optics emission apertures are sized the same as the associated (corresponding) excitation light apertures. However, other sizes are possible. The light emitting polarizer is provided only for the upper optical head 112a. The light emitting aperture and the light emitting polarizer are
Each is substantially the same as that for the corresponding excitation light. Light emitting polarizer 1
32 may be included in the detector in the case of a detector essentially for detecting the polarization of light.

Excitation light polarizer 114 and emission polarizer 132 can be used together in non-polarization analysis to eliminate certain background signals. It is considered that the light emitted from the sample holder and the light emitted from the light emitting molecule attached to the sample holder are polarized. Because the rotational mobility of these molecules should be hindered. Such a polarized background signal can be removed by "crossing" the excitation light polarizer and the emission light polarizer, that is, by arranging them so that they form an angle of 90 ° around the transmission axis. it can. As mentioned above, such a polarized background signal can also be reduced by moving the test volume away from the sample holder wall. In order to increase the signal level, the beam splitter 118 should be optimized to reflect some polarized light and transmit other polarized light. This method is effective when the luminescent molecule to be analyzed emits light having a relatively small degree of polarization. This is the case for small luminescent molecules in solution.

The light then travels through the light emitting fiber optic cables 134 a, 134 b to the light emitting optical shuttle (ie, switch) 136. This shuttle positions the appropriate emitting fiber optic cable in front of the appropriate detector. In the device 90, these members are substantially similar to the corresponding members for excitation light. However, those of other mechanisms can also be used.

Light exiting the fiber optic cable may pass through one or more emission “intensity filters”. The intensity filter is generally provided with any mechanism capable of reducing the light intensity. The intensity is represented by the amount of light per unit area and per unit time. In the device 90, the intensity is modified by the neutral density filter for emission 138. The neutral density filter for light emission 138 absorbs light substantially irrespective of the wavelength and emits the absorbed energy as heat. Neutral density filter for light emission 1
38 may comprise a high density filter H that absorbs most of the incident light, an intermediate density filter M that absorbs some of the incident light, and a blank O that does not substantially absorb the incident light. These filters can be replaced manually. Alternatively, other replacement methods such as filter wheels can be used. The intensity filter can divert some of the light away from the sample without absorbing it. Examples of this are beam splitters that direct some light along one path and reflect other light along the other path, and Pockels cells that deflect light into various paths by diffraction.

The light then passes through the emission interference filter 140. The emission interference filter 140 can be housed within the emission filter wheel 142. In the device 90, these members are substantially similar to the corresponding members for excitation light. However, those of other mechanisms can also be used. The emission interference filter blocks stray excitation light entering the emission path through various mechanisms such as reflection and scattering. If the blocking is not performed, such stray excitation light will be detected and will be mistakenly recognized as light emission, and the signal background ratio will be reduced. Since the interference filter for light emission has a longer wavelength of light emission than the related excitation light, it is possible to distinguish the emission light from the excitation light. The emitted light typically has a wavelength of 200-2000 nm.

The relative positions of the spectral filters, intensity filters, polarization filters and other filters exemplified in this specification can be changed without departing from the spirit of the invention. For example, filters such as intensity filters that are used here only within one optical path may be used in other optical paths. In addition, filters, such as polarization filters, which are used here only in one of the upper or lower optics, can also be used in the other optics,
Alternatively, it can be used in both optical systems. The optimal placement and combination of filters for a particular experiment will depend on the mode of analysis, the complex, and other factors.

The light finally reaches the detector. The detector is used in absorption and emission analysis. In device 90, there is one emission detector 144 that detects light from all emission modes. A preferred detector is a photomultiplier (
PMT). In device 90, detection slots 14 for four detectors
5a to 145d are provided. However, the number of detection slots and the number of detectors are
Other numbers are possible.

More generally, the detector comprises any mechanism capable of converting the energy of the detected light into a signal that can be processed by a device such as a processor in particular. Suitable detectors include photomultipliers, avalanche photodiodes, charge coupled devices (CCDs), enhanced CCDs, and the like.
Depending on the detector, the light source and the mode of analysis, such a detector can be used in various detection modes. Such detection modes include (1) discrete (eg, photon count) mode, (2) analog (eg, current integration) mode, and / or (3) imaging mode, etc., as described below. .

[Chemiluminescence Optical System] FIGS. 3, 4, and 6 show a chemiluminescence optical system of the analyzer 50.
Chemiluminescence optics do not require a light source or, for example, excitation optics, because chemiluminescence occurs after a chemical event rather than after light absorption. Instead, chemiluminescent optics require only selected luminescent optical elements. In the analyzer 50, an isolated lensless type chemiluminescence optical system is used.
It is optimized for maximum sensitivity for chemiluminescence detection.

In general, the constituent elements of the chemiluminescence optical system function in the same way as the corresponding members in the photoluminescence optical system, and receive the same warning and the like. The chemiluminescence optical system can also be used for other analysis modes that do not require irradiation, such as electrochemiluminescence.

The chemiluminescence optical path starts from the chemiluminescent complex 120 held in the sample container 126. The complex and sample container are similar to the complex and sample container used in photoluminescence optics. However, in the analysis of the complex, the intensity of the light produced by the luminescent chemical reaction within the complex is measured rather than by the photoluminescence induced by the light. A similar example of chemiluminescence is the incandescent glow of fireflies.

Chemiluminescent light is typically emitted from the complex in all directions. However, most of the light will be absorbed or reflected by the container wall. A portion of the light emitted from the top surface of the well is collected using a chemiluminescent head 150 as shown in FIG. 3 and directed through a chemiluminescent optical path to a detector. The direction of light transmission through the chemiluminescent optics is indicated by the arrow.

The chemiluminescent optical head comprises a non-confocal mechanism for the light transmitted from the examination volume within the complex. The detection from the test volume reduces the contribution of light due to "crosstalk", which is taken from adjacent wells, to the chemiluminescence signal. The non-confocal mechanism includes a chemiluminescence baffle 152. The baffle 152 is formed with unevenness 153 for absorbing and reflecting light from other wells. The non-confocal mechanism also includes a chemiluminescent aperture 154, which further limits detection to the examination volume.

Next, the light passes through the chemiluminescence optical fiber cable 156. The fiber optic cable 156 can be replaced with any suitable mechanism that can direct the light from the composite towards a detector. The optical fiber cable 156 is similar to the excitation light optical fiber cables 110a and 110b and the light emission optical fiber cables 134a and 134b in the photoluminescence optical system. The fiber optic cable 156 can include a lumen with a transparent open end that can be filled with fluid. This lumen allows the optical fiber to both transmit luminescence from the microplate well and supply fluid into the microplate well. The effect of such a lumen on the optical properties of an optical fiber can be minimized by using a transparent fluid with an index matching that of the optical fiber.

The light then passes through one or more chemiluminescent intensity filters. The intensity filter is generally provided with any mechanism capable of reducing the light intensity. In the analyzer 50, the intensity is changed by the chemiluminescence neutral density filter 158. If desired, the light can pass through other filters.

The light finally reaches the detector. The detector converts the light into a signal that can be processed by the analyzer 50. In the analyzer 50, one chemiluminescence detector 160 is provided. This detector 16
0 can be chosen to optimize the detection of blue / green light, which is the most abundant type of chemiluminescence. A preferred detector is a photomultiplier tube selected for high quantum efficiency and low dark count at chemiluminescent wavelengths (400-500 nm).

Housing FIG. 7 shows a housing 150 and other accessories for the device of FIGS. The housing 150 substantially contains the device, is continuous, and forms two protective layers around the xenon arc lamp having a large color temperature (in cooperation with the light source slots 103a to 103d). . The housing 150 is capable of automatic sample introduction, automatic light source switching, and automatic detector switching.
It protects the operator from the xenon arc lamp and other system components.

Alternative Device FIG. 8 shows an alternative device 160 for detecting the luminescence from the analytes within the complex 162. The device 160 includes a substantial part of the device 90, and includes an optical head 164 connected by an optical fiber, an excitation light filter 166, an emission filter 168, a dichroic beam splitter 170, and a sample. A mechanism for position control and focusing control. However,
Device 160 includes an alternative light source 172, an alternative sample detector (“S” detector) 174, an alternative reference detector (“R” detector) 176, and an alternative detection detector. And electronic circuitry 178. Although the replaceable members 172-178 are shown external to the device 90 in FIG. 8, it is easy to incorporate them into the housing 150 of the device 90 if desired.

The device 160 can excite emission in a variety of ways, such as using an LED light source or a laser diode light source. For example,
For blue light absorbing analytes, a high brightness blue LED from NICHIA (Model NSPB500; Mountvlle, PA) can be used to excite.
This LED produces excitation light over a wide spectral range. Therefore, the excitation light filter 166 is typically used to block the red band in the spectrum. If the laser diode excites the analyte, the excitation light filter is not necessary.

The device 160 can detect the emitted light and can convert the detected emitted light into a signal in various ways. Luminescence can be detected using the sample-side PMT174. The PMT 174 can be a gain modulation PMT (Champaign, IL) from ISS. High frequency emission can be frequency down converted to a low frequency signal using a technique called heterodyne. The phase and modulation of the low frequency signal can be determined using a lock-in amplifier 180, such as a lock-in amplifier (model number SR830; Sunnyvale, CA) from STANFORD RESEARCH SYSTEMS. The lock-in amplifier 180 uses the light source 17
Phase lock loop 182 may be used to phase lock to a modulation frequency of two. In order to correct the drift of the light source, the output of the light source 172 is the reference PMT.
It can be observed using 176. The reference-side PMT 176 can be a PMT (model number H6780; Bridgewater, NJ) from HAMAMATSU. If the reference PMT 176 is capable of responding to high frequency signals, the heterodyne step can be performed using the external mixer 184. The phase and modulation of reference PMT 176 can be obtained by lock-in amplifier 180 and can be used to normalize the signal from sample PMT 174.

A computer or processor controls the device, including external components. The computer also controls sample handling and data collection. Generally, phase and modulation depth data are collected at one or more frequencies as appropriate for the lifetime of the analyte. In some cases, the phase and the degree of modulation are measured at one or several frequencies and processed by a computer or processor to serve to reduce the detected background signal.

Methodology Both device 90 and device 160 can be used to perform various steady state analyzes and various temporal emission analyses. In steady state analysis, luminescence is measured under constant irradiation using a continuous light source. In temporal polarization analysis, luminescence is measured as a function of time by using a continuous light source with appropriate intensity modulation or by using a time varying light source.

Intensity analysis can be performed by observing the intensity of luminescence emitted from the complex.

Polarization analysis can be performed as follows. Excitation light from the continuous light source can be guided through the excitation light filter, the low-emission optical fiber cable, and the excitation light polarization filter. Then, the excitation light is guided to the beam splitter. At the beam splitter, most of the light is reflected back into the composite, and the remaining small portion of the pump light is directed into the optical monitor. The emission from the complex is
It is returned to the beam splitter, guided through another low-emission optical fiber cable, an emission filter, and a polarization filter (in the S or P direction), and finally, a photomultiplier tube 174. Detected by. Two measurements are made for each complex. One measurement was performed with the excitation light polarizer and the emission polarizer aligned with each other, and the other measurement was with the excitation light polarizer and the emission polarizer crossed with each other. Done in the state. Each polarizer can be static or dynamic, and each polarizer can be in the S or P orientation. However, typically, the excitation light polarizer is set in the S direction.

Other emission analyzes such as FRET, FLT, TIR, FCS, FRAP and their analysis in phosphorescence mode are described in PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
Can be carried out using procedures described in the above-mentioned document entitled, and known to those skilled in the art.

3. Detection Mode FIG. 9 is a block diagram schematically showing a photo detection device 200 according to the present invention. In this embodiment, the light 202 from the composite 204 is directed to the appropriate optics 20.
It is led to the detector 208 via 6. The optical system 206 and the detector 208 can take various forms, including the forms illustrated in FIGS. 3-8 and described above. In general, the detector 208 has an input 209 that receives light and an output 210 that corresponds to the received light. The output 210 can be in various forms such as a current signal or a voltage signal. Depending on the intensity of the light received by the detector,
The output can be discrete pulses corresponding to individual photons, or it can be an analog voltage or current proportional to the input light.

In device 200, output 210 is directed through selector switch 212. The selector switch 212 selects an output path toward various detection elements such as a pulse (photon) counter 214 and an analog integrator 216. The selector switch 212 can be of the manual type, in which case the user can select the appropriate one from the plurality of detection elements. Alternatively, the selector switch 212 may be passive, in which case both detection elements may be used based on light intensity, analysis type, etc. The passive selector switch removes the switch 212 and connects a capacitor 218 between the detector output and the pulse counter 214 and an inductor 220 between the detector output and the analog integrator 216. Due to
Can be formed. The capacitor 218 is connected to the pulse counter 214,
The AC component of output 210 is passed, while inductor 220 passes the DC component of output 210 toward analog integrator 216. The typical capacitance of the capacitor 218 is 1-10 nanofarads and the typical inductance of the inductor 220 is 0.1-1 microHenry. In another variation, the selector switch can be an electronically controlled switching device such as a solid state electronic switch or relay. In this case, the switch can be automatically controlled according to the expected light level or the measured light level.

The pulse counter 214 is used as a discrete accumulator or a discrete integrator, and can detect or sample the detection light by counting the number of photons in the detection light. Typically, detectors are selected that produce an output corresponding to each detected photon. For example, photomultiplier tubes (PMTs) generate a current pulse for each photon that strikes the photocathode in the PMT. The discrete output from the detector is aggregated over the sampling or integration time and the amount of detected light is measured in units such as counts, counts per second, relative fluorescence units (RFUs), etc., and associated output ports 222. These results, which are output from, can be corrected for the source intensity perturbations by using a reference detector as described above in the case of fluorescence.

The analog integrator 216 is used as an analog integrator, and can detect the detected light by generating a signal corresponding to the output of the detector. For example, P
Current pulses from MT or other detectors can be stored (integrated) using capacitors 224 or other storage members in the electronic circuit. As the capacitor charges, the analog voltage value of the capacitor will change to PMT over the integration time.
Increase in proportion to the number of photons collected by. Typical capacitances range from 0.22 to 100 nanofarads. Typical capacitors are low leak polystyrene and polyester capacitors to minimize drift errors. Results from analog integrators are usually reported in units of RFUs. However, in the present invention, it is possible to report in units such as the number of counts and the number of counts per second. If desired, these results can also be corrected using a reference detector.

The size of the capacitor 224 is such that the detection accuracy and the detection range can be optimized.
Should be selected manually or automatically. In general, maximum accuracy is obtained with the smallest capacitors. Also, the largest detection range is obtained with the largest capacitors. The rating of the capacitor is determined by the total counts, not the counts per second. This is because saturation is determined by the total count or analog counterpart. In the case of a continuous light source, the total counts are given by the integration of the counts per second over the integration time. In the case of a flash lamp, the total number of counts is given by the product of the number of counts per flash and the number of flashes. Generally, the capacitors are zeroed using reset 226 before each sampling time. The capacitor is charged to 1/2 to 3/4 of its full capacity at each sampling. in this case,
Charging to 2/3 of full capacity is the preferred value. To increase flexibility, the device can include multiple capacitors or other storage members with varying capacities. Alternatively, an amplifier or attenuator can be selectively placed between the detector output and the integrator input. This allows the scale of the detector output to be adjusted to the range over which the expected light intensity can be integrated without exceeding the capacity of the storage member. The output can be scaled to account for amplification and attenuation effects caused by the amplifier and attenuator, respectively.

The output signal from the integrator is provided to range observing device 228. Range observing device 228 may include a threshold detecting device in the form of a differential amplifier or comparator 229. The comparator 229 has a + side input 230, a − side input 232, and an output 234. The + side input 230 is connected to the output signal from the integrator. The-side input 232 is connected to a full scale reference value 236, which is typically a voltage reference value. The reference value can be adjustable over a range, for example by using a potentiometer configured as a voltage divider. The reference value can also be fixed at a specific value, for example by using a Zener diode. If the voltage value at the-side input 232 exceeds the voltage value at the + side input 230, the output of the comparator is low. However, when the output signal voltage value from the integrator exceeds the reference voltage value 236, the output of the comparator becomes large. Therefore, by observing the output of the comparator, it is possible to detect whether or not the reference value is exceeded and further when the reference value is exceeded. The output signal of the integrator can also be observed in other ways by using other digital circuits or other analog circuits.

The timing of the detection may be by using one or more timers 238 and the system controller 240 to allow the system controller 240 to zero the timer 238 prior to detecting light from the sample. Moreover, the analog integrator 216 can be zeroed by using the trigger controller 242 and the reset 226. After completion of the sampling time, the system controller 240 can use the analog-to-digital converter 244 to process the integrated signal stored in the analog integrator 216 and also use the associated output port 246. Then, the data and the sampling time can be output. Of course, other mechanisms that can form an analog integrator for data collection and that output data from the analog integrator after data collection can also be used.

FIG. 10 is a block diagram schematically showing an alternative photo-detecting device 300 according to the present invention. Device 300 is similar to device 200 of FIG. 9 except for a few differences. The first difference is that the selector switch 212 in the device 200 is replaced with the high-speed input amplifier 312 in the device 300. The high speed input amplifier 312 should have an accurate signal response from DC to 0.5 or 1 GHz AC. This allows the pulse counter 314 and the analog integrator 316 to be used simultaneously.
The second difference is that in the device 300, unlike the case of the device 200,
The output from the pulse counter is connected to the system controller 340. Due to these differences, the range is expanded in the case of the device 300 as compared with the case of the device 200. The reason is that the device 300 can automatically switch or select between the pulse counter and the analog integrator depending on the level of received light and / or in response to a command from the system controller. is there.

The sampling time or reading time in the device 200, 300 can be determined by at least two methods. First, when determined by the system controller and / or timer, the device can be instructed to integrate the detector output over a predetermined time (fixed time). Second, it can instruct the device, when determined by the comparator and the full-scale reference value, to integrate the detector output until a predetermined integrated signal (fixed integrated signal) is obtained. it can.

The use of fixed-time detection mode and fixed-amount signal detection mode can provide overflow protection as well as a dynamic range beyond what is available only with discrete or analog detectors. Can be expanded to. In analog detection, information is lost if the integrating capacitor or other storage member reaches its full-scale value before the completion of the sampling time, even if the PMT or other detector did not saturate. Will end up. This is because the electronics for analog counting cannot respond to signals that exceed the full-scale count for each capacitor setting. In standard analog detection, each sample that appears brighter than the saturation level will give the same full scale result.

In the present invention, the intensity of the detection light can be determined even when the integrated signal over the entire sampling time substantially exceeds the storage capacity of the capacitor. The light intensity can be expressed as the amount of light per unit time. In the present invention, the time required to fully charge the storage member is measured by a timer operatively associated with the comparator. Therefore, even when the capacitor is fully charged before the sampling time is completed, the storage capacity of the capacitor is related to the light amount detected by the detector, and the light amount is divided by the ratio of the charging time to the sampling time. The intensity can be determined by calculation. If the capacitor is not fully charged within the sampling time, the strength can be determined by using the actual charge and the sampling time.

By using the time fixed detection mode and the signal amount fixed detection mode, the reading time can be shortened and underflow can be protected. In general, optical analysis is affected by various sources of error such as photon noise (PN), pipetting error (PE), and so on. The coefficient of variation (CV) related to the optical analysis result affected by such an error source can be expressed by the following equation 6.

[Equation 6] Here, CV (PN) is (the number of detected photons) ^-0.5 , and CV (PE) is typically in the range of 1 to 5%. Thus, in order to obtain results limited by pipetting error, it is only necessary to collect enough photons so that the CV (PN) can be reduced to about 0.5-1%. This noise level corresponds to a collection of 10,000-20,000 photons. For many analyses, collecting this number of photons can give good results. For example, in fluorescence polarization analysis, the polarization noise in units of millipoise (mP) is given by equation 7.

[Equation 7] Generally, polarization analysis requires less than 10 mP of polarization noise, corresponding to collecting at least about 5,000 photons.

The read time can be shortened by allowing the read time to vary as a function of signal strength. In this case, the sample is analyzed long enough to obtain results that are not limited by the amount of detected light. In discrete detection, the pulse counter can be configured to count pulses only up to a predetermined number of pulses, or a threshold number of pulses. In analog detection, the analog integrator can be configured to integrate the detector output until a preset threshold is obtained. Here, the threshold value is a value corresponding to the collection of a predetermined light amount. More specifically, the integrator can be zeroed and the time required to integrate the detector current enough to trigger the comparator can be measured. The integration time describes the number of photons collected and thus the signal level.

The integral detector current required to activate the comparator can be changed by changing the electronic gain, threshold voltage, integrating capacitor, etc. Such changes in the comparator activation value will correspond to changes in the number of photons that can be collected (
And will respond to changes in the associated signal-to-noise ratio). Larger capacitors and / or smaller gains can be used to increase the number of photons collected. Conversely, smaller capacitors and / or shorter read times can be used to reduce the number of photons collected.

In general, if the sample is bright, it takes a short time to acquire a desired amount of light (the number of photons), and if the sample is dark, the time to acquire the desired amount of light (the number of photons) is long. Underflow occurs if the sample is so dark that it does not collect the desired number of photons within a preset time limit, ie within the time limit. In this case, the measurement will be "timed out" and the integrator voltage will be measured by an analog-to-digital converter, or the integrator voltage will be set to zero, or
The integrator voltage is set to some other suitable value indicating an underflow condition. Alternatively, if analog integration and photon counting are performed simultaneously, the counter output determined in the photon counting mode can be used to determine the intensity.

Table I shows the possible reduction in read time using variable read, assuming the read time is reduced from 100 ms to 1 ms per sample. Reducing read time is important when large numbers of samples have to be analyzed.
For example, for high throughput screening applications, the samples can be contained in microplates, each containing 96 or 384 or 1536 samples. Furthermore, high throughput screening applications typically require the analysis of large numbers of such microplates.

[Table 1]

The reading time per microplate decreases as shown in Table I as the reading time for each sample decreases. Alternatively, in some cases, the read time per microplate can be kept constant, independent of the read time for each sample. The latter aspect is more effective when the analysis of the microplate is combined with other processes performed at fixed time intervals.

FIG. 11 is a diagram schematically showing an integration part similar to the integration part shown in FIGS. 9 and 10, and the difference is that an additional automatic scale switching mechanism is provided. .

The signal output from the PMT is supplied to the preamplifier. The preamplifier is P
The current output from MT is converted to a voltage output. As mentioned above, this preamplifier should have good performance from DC to radio frequency AC.

The output from the preamplifier is fed in parallel to multiple integrating devices. One integrating device is a photon counter such that it is effective for minimum photon intensity. The other integrating device is four analog integrators having substantially the same configuration as each other and different integrating capacitor sizes. In particular, each of the first three analog integrators comprises a capacitor having a capacitance that is about an order of magnitude less than the capacitor of the latter integrator.

The output of each integrating device is provided to the identification part. The identification portion observes the output voltage between the preselected high voltage V _H and the preselected low voltage V _L as shown. By using the output of the individual identification portion for each integrating device, the counter corresponding to that integrating device can be triggered. The counter for each integrating device is such that the output from each integrating device is V _L.
The count is started when the value exceeds V _H , and the count is stopped when the output from the individual integrating device exceeds V _H. Thus, the counters can be used to determine the time required to saturate each associated integrating device. Thus, for a medium intensity signal, the time required for the minimum range integrating device to saturate is short compared to the total sampling time. The time required for the next larger range of integrating devices to saturate is approximately an order of magnitude longer. The maximum range integrating device does not actually saturate at a particular sampling time for medium strength signals.

When calculating the light intensity for a given sampling time, it is usually preferable to select the output of the most recently saturated or near-saturated integrating device. The signal from such an integrating device, or in some cases, the time taken for saturation, provides the most accurate data for intensity calculations.

The advantage of this system is that the intensity values can always be calculated based only on the time it takes for the integrating device corresponding to the largest range of saturated integrating devices to saturate. The intensity value can be calculated by dividing the number of photons required for saturation by the time required for saturation. If neither analog integrator device saturates, the integrated signal from the counter can be used. Alternatively, the analog value of the integrator device, which has not reached saturation but is closest to saturation, can be measured using an analog-to-digital converter and the intensity can be calculated from this measurement.

Additional types of detectors (eg, photodiodes) can be used in parallel with the PMT to further extend the measurable intensity range. The output from the photodiode can be fed to a series of analog integrators similar to those described above. In this case, the photodiode is within the proper range even if the intensity that can be accurately measured by the PMT is exceeded.

As mentioned above, the sampling time can be ended prematurely if desired by the user when a predetermined number of photons have been collected. Termination occurs when the corresponding integrated signal is obtained as measured by the integrating device.

12 and 13 are response curves showing the non-linearity of the digital counting circuit for the device shown in FIGS. FIG. 12 was generated using photoluminescence optics and shows 10% non-linearity at 1.5 × 10 ⁶ counts / second and 15% at 2 × 10 ⁶ counts / second. The non-linearity of 3% is shown at 2.5 × 10 ⁶ counts / second, and the non-linearity of 3% is shown.
It shows a non-linearity of 27% at × 10 ⁶ counts / second. FIG. 13 was generated using chemiluminescence optics and shows 5% non-linearity at 2 × 10 ⁶ counts / second and 1 at 3 × 10 ⁶ counts / second.
It shows 0% non-linearity and 20% non-linearity at 4 × 10 ⁶ counts / sec.

FIG. 14 is a response curve showing the saturation characteristic of the integrating capacitor in the analog counting circuit for the device shown in FIGS. These curves were generated using a sampling time of 100 ms. Data is shown for three capacitors, including a relatively large capacitor (sensitivity 0) and a relatively small capacitor (sensitivity 2). Large capacitors are better than small capacitors
Saturates at higher light intensity. The response for the maximum capacitor is 10 × 1
It is linear up to 0 ⁶ counts / second. This is about 10 times larger than that of the digital count circuit.

FIG. 15 is a response curve showing the saturation characteristic in the analog count circuit when the comparator is used. FIG. 15 was produced using the same equipment and the same sample as in FIG. 14 and the same sampling time of 100 ms. The response is linear up to about 1 × 10 ⁹ counts / second, at least for capacitors corresponding to sensitivities 0-3.

Table II provides guidelines for selecting an appropriate counting method and unit depending on the analysis, light source (when used), detector and light intensity. Such guidelines apply only for the embodiments shown in FIGS. 3-6 and 9. Because it assumes separate chemiluminescence and photoluminescence optics, and because it is assumed that photon counting and analog integration are selectable, and that analog This is because it is assumed that the presence or absence of the comparator can be selected in the case of detection.

[Table 2]

Discrete (photon) counting sacrifices dynamic range for sensitivity at very small signal levels. Saturation (˜1 × 10 ⁶ counts / sec) is determined by the rate at which the detector and counting circuits receive photons. This method is optimally used for low intensity detection in chemiluminescence, temporal emission analysis and the like.

In the analog count (PMT current integration), the sensitivity at the low intensity signal level and the expansion of the dynamic range have an exchange relationship (inverse proportion relationship). Saturation has a much higher count rate (about 0.5 to 1) than discrete counts.
X 10 ⁹ counts / sec). Usually, the maximum integrable signal is determined by the size of the integrating capacitor. This method is optimally used for emission intensity analysis and emission polarization analysis performed using a flash lamp.

In counting using a comparator (analog + time measurement to full scale), sensitivity at low signal level and maximum dynamic range are in a trade-off relationship with a single device setting. Saturation is similar to analog counts. Compared with the case of analog count only, even if the capacitor is fully charged, it automatically measures the time it takes for the capacitor to reach full scale. Will detect. This method
It is best used for low intensity analysis and dynamic range readings in emission intensity and emission polarization analysis performed using a continuous lamp.

While the present invention has been illustrated and described with reference to preferred embodiments, several specific embodiments of the invention disclosed and illustrated herein limit the invention by virtue of various modifications. It should not be regarded as a thing. Applicants view the subject matter of the invention as novel and nonobvious combinations and subcombinations of the various components, features, functions, and characteristics disclosed herein. No single feature, function, member or characteristic of the disclosed embodiment is essential. The accompanying claims define various novel and nonobvious combinations and subcombinations of such features, functions, components and characteristics. Other combinations and sub-combinations can be claimed by amending existing claims or by presenting new claims in this or a related application. Such claims, whether broader, narrower, or the same as the original claims, are considered to be encompassed within the subject matter of Applicants' invention.

[Brief description of drawings]

FIG. 1 is a schematic diagram of fluorescently labeled molecules, showing how the reorientation of the molecules affects fluorescence polarization.

FIG. 2 is a schematic graph showing temporal measurements in the frequency domain, showing the definitions of phase angle (phase) φ and demodulation factor (modulation) M.

FIG. 3 is a diagram schematically showing a photo-detecting device according to the present invention.

4 is a perspective view schematically showing a part of the apparatus shown in FIG.

5 is a diagram schematically showing an optical member for photoluminescence in the device of FIG.

6 is a diagram schematically showing an optical member for chemiluminescence in the apparatus of FIG.

FIG. 7 is a partial exploded view showing a portion of a housing for the device.

FIG. 8 is a schematic view of an alternative photo-detecting device according to the present invention.

FIG. 9 is a block diagram showing a photo detection device according to the present invention.

FIG. 10 is a block diagram illustrating an alternative photodetection device according to the present invention.

FIG. 11 is a schematic diagram of an integrator similar to that in the device of FIGS. 9 and 10, but with an additional automatic scale switching mechanism.

FIG. 12 is a graph showing a response curve showing the non-linearity of a digital counting circuit for the device shown in FIGS.

FIG. 13 is a graph showing a response curve showing the non-linearity of a digital counting circuit for the device shown in FIGS.

FIG. 14 is a graph showing a response curve showing a saturation characteristic of an integrating capacitor in an analog counting circuit for the device shown in FIGS. 3 to 6;

FIG. 15 is a graph showing a response curve showing a saturation characteristic of an analog counting circuit used together with a comparator in the device shown in FIGS. 3 to 6;

[Explanation of symbols]

144 detector 160 detector 174 detector 176 detector 200 photo detection device 208 detector 214 pulse counter 216 Analog integrator 228 Range observation device 238 timer 240 system controller 300 photo detection device 314 pulse counter 316 Analog integrator 340 system controller

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 09 / 062,472 (32) Priority date April 17, 1998 (April 17, 1998) (33) Priority claiming countries United States (US) (31) Priority claim number 60 / 100,951 (32) Priority date September 18, 1998 (September 18, 1998) (33) Priority claiming countries United States (US) (31) Priority claim number 09 / 160,533 (32) Priority date September 24, 1998 (September 24, 1998) (33) Priority claiming countries United States (US) (31) Priority claim number PCT / US98 / 23095 (32) Priority date October 30, 1998 (October 30, 1998) (33) Priority claiming countries United States (US) (31) Priority claim number PCT / US99 / 01656 (32) Priority date January 25, 1999 (January 25, 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SZ, UG, ZW), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GD, GE, GH, GM , HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, L T, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, U A, UG, US, UZ, VN, YU, ZW (72) Inventor David Pee Stambo             United States, California,             94002 Belmont Sixth Ave             New 1561 (72) Inventor Rick Buoy Stelmeisha             United States, California,             95132 San Jose Drywood Ray             3155 F-term (reference) 2G043 AA01 DA02 DA05 DA06 EA01                       EA06 EA13 FA03 GA08 GB18                       HA05 HA07 HA09 JA02 KA01                       KA02 KA03 KA07 KA09 LA01                       LA02 MA04 NA01 NA04 NA14

Claims (41)

[Claims] 1. A device for observing light intensity, comprising a detector configured to detect light; operably connected to the detector and during a sampling time. An integrator configured to generate an integral value corresponding to the integral of the output from the detector; and an integration time during the sampling time at least until the integral value reaches a preselectable value. A timer configured to be measurable; an analyzer configured to calculate light intensity based on the integrated value; wherein the integrated value is the preselectable value within the sampling time If not, the analyzer functions to calculate the light intensity using the integrated value and the sampling time, the integrated value being at the sampling time. If the preselectable value is reached within, the analyzer is operable to calculate the light intensity using the integration time measured before reaching the preselectable value. Characterized device. 2. The device of claim 1, wherein the detector is selected from the group consisting of photomultiplier tubes, photodiodes, avalanche photodiodes, and charge coupled devices. Characterized device. 3. The device according to claim 1, further comprising an amplifier arranged between the detector and the integrator for adjusting the scale of the detector. device. 4. The device according to claim 1, wherein the integrator is of a discrete type, and the integrated value is calculated by counting pulses from the detector corresponding to the quantum of detected light. A device characterized by producing. 5. The device according to claim 1, wherein the integrator is of an analog type, and the integration value is generated by charging an integration capacitor with a charge amount corresponding to the amount of detected light. A device characterized by: 6. The device of claim 5, wherein the integrator comprises a plurality of integrating capacitors having capacitances that are substantially different from each other and is required to produce an integrated value equal to the preselectable value. A device having a different amount of detected light can be selected by selecting a specific one of the plurality of integration capacitors. 7. The device according to claim 5, wherein the integrator further comprises a discrete integrator that integrates a signal by counting pulses from the detector by an amount corresponding to a quantum of detected light. The device switches between the discrete integrator and the analog integrator according to the amount of detected light. 8. The device according to claim 1, wherein the integrator is of a digital type, and the output of the detector is digitally measured at a plurality of discrete timings during the sampling time. A device which generates the integrated value by totaling. 9. The device of claim 1, further comprising a plurality of integrators connected in parallel and simultaneously operable to the detector, each integrator during the sampling time. Configured to generate an integral value proportional to the integral of the output of the detector, wherein the proportional relationship between the integral value and the amount of detected light is substantially different for different integrators. A device characterized by being said to be. 10. The device of claim 1, wherein the device is configured to sequentially detect light from a plurality of samples at periodic time intervals. 11. The device according to claim 1, which is configured to sequentially detect light from a plurality of samples, and the time interval between start timings in the case of sequentially detecting light from the samples is variable. The device is characterized in that the variable time interval is short if the integrated signal exceeds the preselectable value within the sampling time. 12. The device of claim 1, further comprising a first light source configured to direct light onto a sample, the detector being configured to detect light from the sample. A device characterized by being. 13. The device of claim 12, wherein the first light source is capable of producing polarized light and the detector is sensitive to the polarization of light from the sample. A device characterized by the above. 14. The device according to claim 1, wherein the wavelength of the light is 200 nm to 2000 nm. 15. The device of claim 1, wherein the analyzer reports the intensity in units selected from the group consisting of counts per unit time and relative emission units per unit time. A device characterized by being able to. 16. The device of claim 1, further comprising a stage configured to support a sample holder, the sample holder having a plurality of individual sample positions. Device to do. 17. A device for observing light intensity, said detector being configured to detect light; operably connected to said detector and during a sampling time. An accumulator configured to generate an accumulator signal corresponding to all the light received by the detector, wherein the accumulator signal is provided with an associated predetermined threshold value. An accumulator; an analyzer provided in association with the accumulator for calculating the light intensity; and, wherein the accumulator signal reaches the predetermined threshold value within the sampling time, , The analyzer is, at least in part, the arrival time until the accumulator signal reaches the predetermined threshold. Device characterized by calculating a light intensity Zui. 18. The device of claim 17, wherein if the predetermined threshold is not reached within the sampling time, the analyzer is based, at least in part, on the light intensity based on the accumulator signal. A device characterized by calculating. 19. A device for sampling light, said detector having an input for receiving light and an output corresponding to the received light; said detector being connected to said detector and said detector. An integrator configured to generate an integrated signal corresponding to integration of an output of the integrated signal; and, if the integrated signal is smaller than a predetermined value at the end of the sampling time, Value is used to calculate the amount of light received by the detector at the time of the sampling, and if the integrated signal reaches a predetermined value within the sampling time, the predetermined value and the predetermined value. A processor configured to calculate the amount of light received by the detector at the time of sampling using the time taken to reach the value; Device, wherein the door. 20. The device of claim 19, further comprising a stage configured to support a sample holder having a plurality of individual sample locations, ie associated with each of a series of sampling times. At each sample position
The detector receives the light emitted from the sample position within the sampling time, and when the integrated signal reaches a predetermined amount within the sampling time, the movement to the next sample position is caused. And the device to be. 21. A system for determining the amount of light received by a detector within a sampling time, wherein the detector produces an output as a function of received light. Is operably connected to the output of and is proportional to the amount of light received by the detector within the sampling time if the amount of light received during the sampling time is less than a first predetermined amount. A first integration device configured to generate a first integration signal during the sampling time; and operatively connected to the output of the detector and within the sampling time. If the amount of light received by is less than a second predetermined amount that is at least several times larger than the first predetermined amount, the A second integrating device configured to generate a second integrating signal during the sampling time that is proportional to the amount of light received by the detector; and for the first integrating device and the second integrating device. When the light amount received by the detector is determined, one of the two integrated signals is automatically determined according to the light amount received within the sampling time expressed as the integrated signal. A range selection system configured to be selectable; 22. The system of claim 21, wherein the range selection system is, at least in part, dependent on whether the first integrated signal within the sampling time exceeds the first predetermined amount. A system for selecting from the two integrated signals. 23. The system of claim 21, wherein the first integrating device discretely counts pulses from the output of the detector that correspond to photons received by the detector. System to do. 24. The system of claim 21, wherein the first integrating device produces the first integrated signal by performing analog integration corresponding to the output of the detector. 25. The system of claim 21, further comprising operably connected to the output of the detector,
Proportional to the amount of light received by the detector during the sampling time if the amount of light received during the sampling time is less than a third predetermined amount that is at least several times greater than the second predetermined amount A system comprising a third integrating device configured to generate a third integrating signal as described above during the sampling time. 26. The system of claim 21, further comprising: providing a time associated with the first integrating device to reach the first threshold of the first integrating signal. A system comprising a timer configured to obtain. 27. A device for observing light intensity, the detector having an input for receiving light and configured to produce an output corresponding to the received light; If the sampling time is shorter than the preselectable value, the integrated signal corresponding to the integration of the output from the detector during the sampling time is set within the sampling time. An integrator configured to generate; when the integrated signal reaches the preselectable value within the sampling time, the integrator is configured to generate a threshold and The time taken to reach the threshold is used to determine the light intensity, and if the integrated signal does not reach the threshold within the preselectable time, the integrator , The integrated signal Device characterized by determining the light intensity by using the length of the serial sampling period. 28. A device for detecting light, the detector being capable of detecting light and generating an output representative of the detected light; and a signal corresponding to the output of the detector. An analog data acquisition device configured to calculate a total amount of the detected light based on; and to calculate a total amount of the detected light based on a signal corresponding to the output of the detector A discrete data acquisition device configured; a system controller configured to select the analog device and the discrete device based on a preselectable criterion. And the device to be. 29. The device of claim 28,   Furthermore, it has at least two light sources,   A device characterized in that the criterion is based on a light source type. 30. The device of claim 28,   The device, wherein the criterion is based on the amount of detected light. 31. The device of claim 28,   A device characterized in that the criterion is based on the intensity of detected light. 32. The device of claim 31, wherein the analog device is used to determine if the amount of light exceeds the threshold value. 33. The device of claim 31, wherein the discrete device is used to determine if the amount of light exceeds the threshold value. 34. The device according to claim 28, wherein when the amount of detected light exceeds a threshold value, an analog processor is used to automatically determine the amount of light. 35. A device for detecting light from a series of complexes, comprising a stage for supporting the complexes at a test site; said complex for sequential analysis of said complex. An automatic alignment device for automatically aligning the light beams from the complex to a position registered at the inspection site; a detector; and a relay structure for guiding light from the complex toward the detector. A relay structure for causing the detector to detect light from the complex and use the detected light to generate a signal corresponding to the detected light; An analyzer for quantitatively analyzing the amount of light transmitted from the complex by using the information in the signal, wherein the analyzer performs a quantitative analysis of the amount of light in a substantially continuous manner. When Device characterized in that it comprises a; the analyzer and discrete mode for quantitative analysis of the amount of light by counting the pulses corresponding to the quantum of the detection light, and a is a selectable analyzer. 36. The device of claim 35, wherein the stage is configured to support a microplate, the complex being retained in a well formed in the microplate. Characterized device. 37. The device of claim 35, wherein the complex is located at successive sites on a biochip. 38. The device of claim 35, wherein the composite is held in continuous position along the gel. 39. A method for measuring light, the method comprising: detecting photons incident on a detector during a sampling time; collecting data representative of the cumulative number of photons detected during the sampling time. Terminating the sampling time at the time when any one of the reaching of a predetermined threshold value of the cumulative number of photons detected during the sampling time and the lapse of a predetermined time as the sampling time occurs; How to characterize. 40. A method for measuring the intensity of a light source, the method comprising: detecting photons incident on the detector from the light source; wherein a cumulative number of photons incident on the detector during the sampling time is a first. If it is less than the range value, a first integrated signal proportional to the cumulative number of photons is generated; at the same time as the generation of the first integrated signal, the cumulative number of photons incident on the detector during the sampling time is A second integral signal proportional to the cumulative number of photons when the second range value is smaller than the first range value by at least one digit; for use in calculating the intensity of the detected light And selecting one of the two integrated signals depending on whether the cumulative number of photons incident on the detector during the sampling time exceeds the first range value; By using the signals to calculate the intensity of the detected light; wherein the. 41. The method of claim 40, wherein the cumulative number of photons incident on the detector during the sampling time is the second range value at the same time as the generation of the first and second integrated signals. A third integrated signal proportional to the cumulative number of photons is generated when the third range value is smaller than the third range value by at least one digit.