CH705318B1 - Apparatus and method for time-resolved fluorescence detection. - Google Patents
Apparatus and method for time-resolved fluorescence detection. Download PDFInfo
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- CH705318B1 CH705318B1 CH01220/11A CH12202011A CH705318B1 CH 705318 B1 CH705318 B1 CH 705318B1 CH 01220/11 A CH01220/11 A CH 01220/11A CH 12202011 A CH12202011 A CH 12202011A CH 705318 B1 CH705318 B1 CH 705318B1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
- G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
Abstract
Die Ausführungsformen der vorliegenden Erfindung offenbaren ein Fluoreszenzdetektionssystem, das betriebsfähig ist, um eine Fluoreszenzstrahlung in zeitgesteuerter Synchronität zu erkennen. Gemäss den Ausführungsformen der Erfindung kann das Fluoreszenzdetektionssystem einen Bildsensor umfassen, der betriebsfähig ist, um eine Fluoreszenzstrahlung zu erkennen, und der eine Vielzahl von Photodetektoren umfasst. Jeder Photodetektor ist betriebsfähig, um eine erkannte Fluoreszenzstrahlung in eine entsprechende Anzahl von Ausgangsladungen umzuwandeln. Das Fluoreszenzdetektionssystem kann dadurch gekennzeichnet sein, dass jeder der Vielzahl von Photodetektoren mindestens einen Ladungsintegrierer umfasst, der betriebsfähig ist, um während eines vorherbestimmten Zeitintervalls die Ausgangsphotoladungen, die als Reaktion auf die erkannte Fluoreszenzstrahlung generiert werden, selektiv zu integrieren.
Description
Description: FIELD OF THE INVENTION The disclosed technique relates to the field of fluorescence imaging, and more specifically the field of time-resolved fluorescence imaging.
Brief Description of the Drawings [0002] The disclosed technique will be better understood from the following detailed description when taken in conjunction with the drawings. Show it:
FIG. 1 is an illustration of a curve of the amplitude as a function of the decay time of autofluorescence, labeling fluorescence and the sum thereof (hereinafter: total fluorescence); FIG.
FIG. 2 is an illustration of a schematic block diagram of a time-resolved fluorescence detection device constructed and functioning according to an embodiment of the invention; FIG. and
FIG. 3 shows an illustration of a sequence diagram of a method for time-resolved fluorescence detection according to an embodiment of the invention.
[0003] For the sake of simplicity and clarity, the elements shown in the figures are not necessarily drawn to scale. For example, the dimensions of some elements relative to other elements may be exaggerated for the sake of clarity. Further, reference numerals may be repeated in the figures to indicate identical or similar elements without, however, being mentioned in the description of all the figures.
BACKGROUND OF THE INVENTION [0004] Fluorescence detection systems and methods are among the most important tools in the life sciences and are used to investigate the properties of test specimens by using the effect of fluorescence.
[0005] In order to investigate the properties at different points of the test bodies, an optoelectronic imaging system, such as a light microscope, is used. Interesting organic or inorganic substances in a test specimen are specifically labeled with fluorescent markers, which are hereinafter referred to as "fluorophors". The labeled test piece is illuminated with light of one or more specific wavelengths which are able to excite the fluorophores. These fluorophores then emit fluorescent radiation (hereinafter, "labeling fluorescence") at a longer or shorter wavelength than the excitation light. In the case of two-photon fluorescence microscopy, the emitted fluorescent light has a shorter wavelength than the excitation light.
[0006] Many biological test bodies contain considerable amounts of organic compounds, several of which are self-fluorescing, so that they have so-called autofluorescence properties. In addition, the container or the holder of the test body could also exhibit autofluorescence properties, in particular if the container comprises autofluorescence molecules, for example when the container or the holder is made of glass or a polymer material. This type of fluorophore is hereinafter referred to as "autofluorophores". Accordingly, the total fluorescent radiation generated in a test body may contain both autofluorescence and tagging fluorescence components, respectively, of autofluorophores and labeling fluorophores.
[0007] Hitherto, various types of fluorescence systems and methods have been proposed in an effort to keep apart the autofluorescence and the labeling fluorescence in the total fluorescence radiation.
[0008] The following publications address this problem by attempting to achieve a reduction in the background autofluorescence light.
[0009] US Patent No. 3,013,467, in the name of Minsky, entitled "Microscopy apparatus", discloses an optical system comprising means for producing a point-shaped light source. Light from this point-shaped source is focused onto a specimen to be enlarged to illuminate a point-shaped observation field contained in the specimen. The illuminated dot is then focused as an image of the spot on a pinhole, and the light intensity of the image is measured by a light sensitive device. As the optical system remains stationary, means are provided to move the specimen in a selected pattern across the focus of the illumination such that a selected area of the specimen passes through the light spot and is examined therefrom. This scan pattern, Which is traversed by the test specimen, is reproduced by an identical scanning pattern or raster on a display device, such as a cathode ray tube, which is also supplied with the signal from the light-sensitive device. The screen area extends far beyond the selected area of the test body. Therefore, an image of the second test body region is reproduced on a greatly enlarged scale in the grid of the cathode ray tube.
US Pat. No. 6,248,988, in the name of Krantz, entitled "Conventional and confocal multi-spot scanning microscope", discloses an optical microscope system comprising a spot array illumination system that is a microscope lens Array or a pinhole array (or both) to produce an array of a plurality of separate focused light points that illuminates an object, an imaging and detection system comprising an array detector having detection elements arranged to simultaneously scan the array of detectors For each point mapped by the illuminated object, means for scanning the position of the array of separated points relative to the object that scan each pixel to acquire an image of a whole field of the object in a contiguous pass.A processor creates the image from the pixel data received successively from the detector array.
[0011] The following publication discloses an alternative solution approach to distinguish between autofluorescence and tagging fluorescence based on fluorescence systems and methods in which the directions of illumination and observation are no longer collinear to avoid or at least increase the detection of autofluorescence to reduce.
[0012] International patent application WO 2009/088 874, in the name of Brown et al., Entitled "Optical Substrate for Microscopic Imaging of a Sample with Reduced Background Fluorescence", discloses a substrate shape which allows an illumination beam to reach the target area Without going through unnecessary surfaces within the optical system. The thickness and the surface angles of the substrate are carefully selected to provide both beamed and emission beam separated beam paths.
[0013] Still other attempts to reduce the influence of autofluorescence on the labeling fluorescence are based on the utilization of the relatively short decay time of the autofluorescence compared to the decay time of the labeling fluorescence of the fluorescence labels. Autofluorescence typically has a decay time in the range of 1 to 100 nanoseconds. If a fluorescence marker is used in which the decay time of the labeling fluorescence is considerably longer than the decay time of the autofluorescence of an autofluorophore, the autofluorescence fraction can be distinguished from the labeling fluorescence in the total fluorescence by using a gate-controlled detection of the optical signal representing the total fluorescence, Which is converted with a photodetector,
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIG. 1, a curve of the amplitude as a function of the decay time of autofluorescence, marker fluorescence and the sum of autofluorescence and labeling fluorescence is depicted, where the X axis is the decay time (in arbitrary time units) and the Y axis is the amplitude (In arbitrary energy units). In the example of FIG. 1, the autofluorescence has a decay time that is seven times shorter than the tag fluorescence, whereas the emission amplitude of the autofluorophore is initially ten times higher than the emission amplitude of the tag fluorophore. Thus, in this particular example, it may be sufficient to wait for about 50 time units during which the autofluorescence has decreased to such a low level,
The following publications attempt to reduce the influence of the autofluorescence by exploiting the previously explained phenomenon of the different decay times between autofluorescence and labeling fluorescence.
[0016] Die japanische Patentanmeldung Nr. 2006 194 770 A im Namen von Tawara et al., mit dem Titel «Time-resolved fluorescence microscope», offenbart einen Laserstrahl, der aus einem Impulslaserstrahl-Generierungsmittel stammt, wobei der Laserstrahl durch einen Halbspiegel halbiert wird und ein geteilter Strahl als Anregungslicht auf eine Probe gestrahlt wird. Ein nicht lineares optisches Element zum Erzeugen eines optischen Kerr-Effekts wird zwischen einem Paar Polarisationsplatten eingeschoben und angeordnet, deren Ablenkungsrichtungen sich in einem optischen Abbildungssystem orthogonal schneiden, um die Fluoreszenz zu kondensieren, die von der Probe generiert wird, und das Fluoreszenzbild der Probe zu bilden, wobei der andere Laserstrahl, der von dem Halbspiegel geteilt wird, als Ansteuerlicht auf ein nicht lineares optisches Element gestrahlt wird. Der optische Pfadunterschied zwischen dem Anregungslicht und dem Ansteuerlicht wird durch eine optische Verzögerungsschaltung angepasst, und das Fluoreszenzbild der Probe wird von einem zweidimensionalen Abbildungsmittel aufgenommen.
[0017] US Patent Application No. 2008/0265177, in the name of Connally et al. (Entitled "Fluorescence Detection", discloses a fluorescence detection system comprising a radiation source to generate a light emission to detect fluorescence in a fluorescently labeled manner, and a detector for detecting fluorescence, wherein either the fading of the light emission from the radiation source Is such that it enables a measurement of the fluorescence at a time when the fluorescence is distinguishable from the autofluorescence or the detector is a chip-mounted amplified detector (CCD) for detecting the fluorescence or both.
Beschreibung der Ausführungsformen [0018] Es versteht sich, dass die Begriffe «enthaltend», «umfassend» und ihre grammatikalischen Varianten nicht das Hinzufügen eines oder mehrerer von Komponenten, Merkmalen, Schritten, Ganzzahlen oder Gruppen derselben aus-schliesst.
[0019] Die Erwähnung oder Identifizierung einer beliebigen Referenz in der Beschreibung einiger Ausführungsformen der Erfindung ist nicht als Zugeständnis zu deuten, dass eine derartige Referenz als Stand der Technik für die vorliegende Erfindung zur Verfügung steht.
The terms "upper", "lower", "right", "left", "lower", "below", "lowered", "low", "top", "above", "raised" High, "" vertical, "and" horizontal, "as well as their grammatical variations, as used herein, do not imply that, for example, a" lower "component is below an" upper "component, or a component" "Is actually" below "another component, or that a component that is" above "is actually" above "another component since these directions, components, or both are exchanged, reversed, spatially moved, in a diagonal direction Orientation or position, can be used horizontally or vertically, or similarly modified. Accordingly,
[0021] Although some exemplary embodiments of the invention are not limited in this regard, discussions that include terms such as "process," "calculate," "calculate," "determine," "analyze," " Identify, or use the same, refer to one or more processes and / or processes of a computer, computer platform, computer system, or other electronic computer device that manipulate and / or transform data as physical (eg electronic) variables within The register and / or memory or other information storage medium of the computer which may store instructions to perform operations and / or processes and / or applications.
It should be noted that the use of the undefined articles "ein, ein, ein", when they initiate a feature, is in no way to be interpreted such that there is only one specimen of this feature unless otherwise stated. Accordingly, the indeterminate articles, as used herein, include the meaning of the expression "at least one" of the same feature.
[0023] Although state diagrams, flowcharts, or both may be used to describe embodiments, the invention is not limited to these diagrams or to the corresponding descriptions. For example, an operation does not have to go through each box or state shown in exactly the same order as described and described.
The term "method" refers to methods, techniques, and procedures for accomplishing a particular task, including, without limitation, methods, techniques, and procedures that are either known or readily known by those skilled in the art from known methods, Techniques and procedures can be developed.
[0025] Although state diagrams, flowcharts, or both may be used to describe embodiments, the invention is not limited to these diagrams or to the corresponding descriptions. For example, an operation does not have to go through each box or state shown in exactly the same order as described and described.
[0026] The descriptions, examples, methods and materials presented in the specification are not to be construed as limiting, but as purely exemplary.
The term "autofluorescence" encompasses any other type of excited fluorescence radiation (eg background illumination) than the marking fluorescence, which relates to the fluorescence radiation, which is of interest for the analysis of the marked test piece.
The term "radiation" as used herein can refer to both visible and invisible electromagnetic radiation, such as, for example, light in the ultraviolet spectrum.
It should be noted that although the term "excitation light pulses" is referenced throughout the description with the same number to be emitted from a specific light source, this is in no way to be construed as limiting, inasmuch as it is clear to the person skilled in the art, That these excitation light pulses could undergo changes, for example, with respect to the spectral bandwidth and / or intensity as it propagates through an optical medium.
[0030] It should be noted, however, that although the term "total fluorescent radiation" is referenced throughout the description with the same number to be emitted from the sample, this is in no way to be construed as limiting, as is clear to the skilled artisan Is that the total fluorescence radiation can undergo changes, for example with respect to the spectral bandwidth and / or intensity as it propagates through an optical medium.
[0031] The present invention discloses a fluorescence detection device operable to detect fluorescence radiation in timed synchronism.
[0032] According to the invention, the fluorescence detection device comprises an image sensor which is operable to detect a fluorescence radiation. The image sensor comprises a plurality of photodetectors, each photodetector being operable to convert a detected fluorescence radiation into a corresponding number of photocads, also referred to as photocads.
[0033] According to the invention, each of the plurality of photodetectors comprises at least one charge integrator operable to integrate the photocads generated in response to the detected fluorescence during a predetermined time interval.
[0034] In some embodiments, the fluorescence detection device comprises a plurality of electric drift field generators each operatively coupled to the plurality of photodetectors.
[0035] In some embodiments, the plurality of electric drift field generators are operable to selectively transmit the photocads provided by each of the photodetectors to the at least one charge integrator during a predetermined time interval.
[0036] In some embodiments, each of the plurality of photodetectors comprises an amplifier operable to amplify the signal representing the photocads generated in response to the detected fluorescence radiation.
[0037] In some embodiments, the amplifier comprises a loop amplifier.
[0038] According to the invention, the fluorescence detection device comprises a radiation source operable to emit a sequence of excitation light pulses having respective lighting periods, and a controller. The photodetectors, the radiation source and the controller are operatively coupled to each other.
[0039] According to the invention, the image sensor is selectively operable in a detection mode in which fluorescence-induced photocads are stored and integrated and in a non-detection mode, in which the fluorescence-induced photocads are discarded.
[0040] According to the invention, the controller is operable to alternate between the detection mode and the non-detection mode of the photodetectors in timed synchronism with the radiation source.
In some embodiments, each of the photodetectors comprises a photosensitive member for converting photons into photocads, and a light-insensitive member comprising at least one electrical switch, and wherein the at least one charge integrator is assigned to the at least one electrical switch, respectively; And a clock for controlling the electric switch such that photocads generated in the photosensitive member are given to the at least one charge integrator in timed synchronism with respect to the excitation light pulses.
[0042] In some embodiments, each photodetector comprises a linear time-conversion element of elongate extent and a lateral electric field generator.
In some embodiments, the linear time conversion element and the lateral electric field generator are operatively coupled to each other so that a lateral drift field is generated in the linear time conversion element whereby the scanning of photocads in the time conversion element from a first end to a second end at the Lengthwise extension of the linear time conversion element.
[0044] In some embodiments, the charge integrators are arranged in at least one row along the longitudinal extent of the linear time conversion element.
[0045] In some embodiments, the charge integrators are electrically coupled to the time conversion element, respectively, at a plurality of spaced sampling points of the time conversion element.
In some embodiments, at least one light-sensitive region for converting photons into photocads is coupled to the linear time conversion element such that the generated photocads are fed into the linear time conversion element.
In some embodiments, the at least one charge detection circuit can be electrically connected to the charge integrators in order to detect a photocaddition which has passed along its longitudinal extent through at least a part of the linear time conversion element.
[0048] In some embodiments, the plurality of photodetectors each comprise a plurality of reset transistors.
In some embodiments, each reset transistor is controllable by a reset signal such that as long as the reset signal holds the reset transistor open, photocouples generated in response to detected light are simultaneously derived so that the photodetectors are in the non-detection mode ; And wherein when the reset transistor is closed, the photocads are cumulated so that the photodetectors are placed in the detection mode.
[0050] In some embodiments, each of the photodetectors further comprises a pixel-internal amplifier.
[0051] The present invention further discloses a method of operating a fluorescence detection device.
In some embodiments, the method comprises, for example, the step of depleting the photodetectors from previously generated photocads.
[0053] In some embodiments, the method includes, for example, the step of setting the photodetectors into a non-detection mode.
In some embodiments, the method comprises, for example, the step of illuminating at least one fluorophore-labeled species with an excitation light pulse.
In some embodiments, the method comprises, for example, after a predetermined gate delay starting at the end of the illumination time, the step of setting the photodetectors into a detection mode for a predetermined photon exposure period.
In some embodiments, the duration of the gate delay is selected such that the ratio between the autofluorescence and the desired fluorescence excited by the excitation light pulse is low enough to ensure reliable detection of the desired fluorescence by the photodetectors.
In some embodiments, the method comprises, as long as the at least one predetermined termination criterion is not met, the depletion of the photodetectors from the previously generated photocads; Adjusting the photodetectors to the non-detection mode; Illuminating at least one fluorophore-labeled species with an excitation light pulse; And after the predetermined gate delay starting at the end of the illumination time, the photodetectors are set to the detection mode during the predetermined photon exposure period.
[0058] In some embodiments, the method comprises, for example, the step of scanning at least twice during the exposure period of the signal which represents the photocads of the detected fluorescence radiation.
[0059] In some embodiments, the time difference between each sampling step is selected to correspond to the difference in decay time between at least two fluorophore labels.
[0060] In some embodiments, the method includes, for example, the step of providing information about the fluorescence ablation properties of the light-emitting fluorophore label.
Detailed Description of the Invention The disclosed invention overcomes the disadvantages of the prior art by providing an alternative device and method for time-resolved fluorescence detection that allows operability at comparatively low voltages, eg, equal to or less than 10 volts And a short gate delay lasting for example equal to or less than 1 microsecond and a high sensitivity for fluorescence detection. The device and the method for time-resolved fluorescence detection according to the embodiments of the invention are operable for the analysis of structures, features and properties of microscopic or macroscopic test bodies.
The fluorescence device according to the embodiments of the invention comprises a radiation source which is operable to emit a sequence of excitation light pulses, each of which has a duration of, for example, equal to or less than 1 microsecond, an image sensor and a controller. The fluorescence detection device according to the embodiments of the invention can comprise a dichroic mirror, an imaging optics, an ocular and an emission filter.
The radiation source is operable to emit excitation light pulses on one or more wavelengths which excite fluorescence molecules (hereinafter: fluorophores) in a test body.
The fluorescence device is operable and configured such that at least a portion of the fluorescent light emerging from the fluorophores falls onto the image sensor and is converted by the latter into corresponding photoconducts, cumulated and stored in the respective photodetectors. More specifically, the image sensor comprises a plurality of photodetectors, each photodetector comprising at least one charge integrator operable to selectively integrate during a predetermined time interval the photocads generated in the at least one charge integrator in response to the detected fluorescence radiation , Accordingly, each photodetector has capabilities for scanning and integrating photosignals with respect to each of the sampled electrical signals,
The controller is operatively connected to the radiation source and the image sensor so as to enable the emission of the excitation light pulses and the scanning intervals of the photodetectors in a time-controlled synchronization so that the proportion of the autofluorescence as a proportion of the marker fluorescence is slight from the total fluorescence detected , Thereupon background noise, which would otherwise be caused by the autofluorescence, is at least reduced.
The method according to the embodiments of the invention can include the step of excitation light pulse emission, time-controlled fluorescence detection and the repeated and simultaneous integration of a photocoupling in all photodetectors until a sufficiently high electrical signal level is reached, whereupon the respective electrical signals of the at least one, A charge integrator can be read out for further processing. The electrical signals represent the integrated photocads.
2, in accordance with one embodiment of the invention, a fluorescence detection device 100 operable to detect fluorescence detection in a time-controlled or time-controlled manner, for example a radiation source 110, which is operable, To emit excitation light pulses 112; A narrowband excitation filter 130; A dichroic mirror 140; An imaging lens 150; An emission filter 160; An eyepiece 170; An image sensor 180 comprising a plurality of photodetectors 185, all of which may be optically coupled to one another, as discussed below, and a controller 190. The fluorescence detection device 100 may further comprise a container (not shown)
The radiation source 110 can, for example, be implemented as adjustable optical parametric oscillator (OPO) lasers, pulsed laser diodes, pulsed light-emitting diodes (LEDs), mode-coupled or Q-switched lasers, or as another suitable radiation source, as is known, for example, in the art ,
The wavelength of the excitation light pulses 112 is selected such that at least some of the marking fluorophores 121 are excited to emit a total fluorescence radiation 114 which can include a marker fluorescence and an autofluorescence radiation component. In some embodiments, the bandwidth of the excitation light pulses 112 may at least partially superimpose or incorporate the absorption bandwidth of the tag fluorophores 121. In order to reduce the bandwidth of the excitation light pulses 112 in such a way that it excludes the absorption bandwidth from the excitation light pulses 112 by at least 50%, the excitation filter 130 can be used, for example, between the radiation source 110 and the dichroic mirror 140. This might be necessary, for example,
[0070] Ferner können die Anregungslichtimpulse 112, die von der Strahlungsquelle 110 emittiert werden, eine Impul-sabklingzeit aufweisen, die kürzer ist als die charakteristische Abklingzeit der emittierten Markierungsfluoreszenzstrahlung, die als Reaktion auf einfallende Anregungslichtimpulse 112 aus den Markierungsfluorophoren 121 angeregt wird, so dass der Anregungslichtimpuls nach einem gewissen Zeitraum die Markierungsfluoreszenz-Strahlungskomponente der Gesamtfluoreszenzstrahlung 114 nicht stören kann. Dadurch wird diese messbar und von den Anregungslichtimpulsen 112 unterscheidbar.
[0071] Je nach den charakteristischen Abklingzeiten der Markierungsfluorophore 121. kann die notwendige Dauer der Anregungslichtimpulse 112 beispielsweise von 10 Pikosekunden bis 1 Mikrosekunde reichen.
The excitation filter 130 may be positioned between the radiation source 110 and the dichroic mirror 140 relative to the propagation direction of the excitation light pulses 112 emitted from the radiation source 110. Further, the imaging lens 150 can be positioned between the container (not shown) and the dichroic mirror 140 relative to the direction of propagation of the excitation light pulses 112. In other words, the radiation source 110 can be optically coupled to the dichroic mirror 140 such that the dichroic mirror 140 deflects the emitted excitation light pulses 112 toward the imaging lens 150; And the imaging lens 150 may be optically coupled to the container (not shown) such that excitation light pulses 112,
Excitation light pulses 112 incident on marker fluorophores have a strength and a spectral bandwidth which cause excitation of the tag fluorophores 121, ie, the tag fluorophores 121 may be triggered in response to incident excitation light pulses 112 to emit a marker fluorescent radiation. However, as explained herein, the excitation light pulses 112 can also trigger the excitation of autofluorophores, the sum of which is referred to herein as "total fluorescence radiation". The total fluorescent radiation 114 is collected by the imaging lens 150 and the dichroic mirror 140 and propagates therethrough. The dichroic mirror 140 may have a cut-off wavelength which allows the transmission of a marking fluorescent radiation,
According to some embodiments of the invention, the fluorescence detection device 100 may be operable to control the total fluorescence radiation 114 transmitted through the dichroic mirror 140 using, for example, an emission filter 160 which is disposed relative to the propagation direction of the total fluorescence radiation 114 below the dichroic mirror 140 , The emission filter 160 correspondingly having suitable spectral band width filter characteristics.
[0075] Gemäss einer Ausführungsform der Erfindung kann das Okular 170 unterhalb des dichroitischen Spiegels 140 oder, wenn es auf das Emissionsfilter 160 angewendet wird, im Verhältnis zur Ausbreitungsrichtung der Gesamtfluoreszenzstrahlung 114, d.h. zwischen dem Bildsensor 180 und dem dichroitischen Spiegel 140 oder dem Emissionsfilter 160, positioniert sein, so dass die Gesamtfluoreszenzstrahlung 114 dann von dem Okular 170 geführt werden kann, um auf die Oberfläche des Bildsensors 180 einzufallen.
In some embodiments of the invention, the fluorescence detection device 100 may be operable such that at least a portion of the autofluorescence radiation (emanating eg from the surface of the container and / or the test body 120) excited by the excitation light pulses 112 may not occur The image sensor 180, ie, being excluded, can be imaged on the image sensor 180 to at least reduce the negative effects caused by the autofluorescence radiation. For example, in embodiments where the fluorescence detection device 100 comprises the imaging lens 150 and / or the eyepiece 170, they may be configured and positioned, for example, relative to the container (not shown) Such that at least a portion of the autofluorescence radiation which can be excited is left by the imaging lens 150 and / or the eyepiece 170 as not shown. More specifically, a reduction of the imaged portion of the autofluorescence radiation may be formed in some embodiments by, for example, using a confocal multispot scanning device and / or by using a large field optical imaging device having a large numerical aperture, for example, greater than 0.2 ,
[0077] According to one embodiment of the invention, the image sensor 180 includes a plurality of photodetectors 185 that comprise at least one charge integrator (not shown). Each photodetector 185 has a timed operability in relation to the emission of the excitation light pulses 112. Further, each photodetector 185 is operable to integrate photocads (not shown) generated in response to the total fluorescence radiation 114 detected by the photodetectors 185 during a predetermined time interval. In other words, the image sensor 180 has integration capabilities with respect to locally generated photocads generated by the photodetectors 185.
In some embodiments, each photodetector 185 may be operable to perform a plurality of scanning steps during the predetermined time interval and to integrate the photocads with respect to the various scanning steps in the respective charge integrators. The sampling intervals that the image sensor 180 can reach may be less than the decay time of the tag fluorophores 121 in some embodiments. Accordingly, the image sensor 180 may be operable to map mark fluorescence with respect to the tag fluorophores 121 having different decay times for the marker fluorescent radiation.
The image sensor 180 can, for example, be implemented as described in US Pat. No. 5,856,667, in the name of Seitz et al., Entitled "Device and method for the detection and demodulation of an intensity-modulated radiation field (667), for example, with reference to the section entitled "Brief Summary of the Invention", FIG. 2, FIG. 3, and the claims. The conversion of the image sensor 180 according to FIG. 667 allows sampling intervals of, for example, equal to or less than 10 nanoseconds at drive voltages of equal or less than 5 V.
In some embodiments of the invention, the image sensor 180 can be implemented, for example, as described in European patent application No. 2,182,523 (523) in the name of P. Seitz, entitled "Batch sampling device and method" The section entitled "Brief Description of the Invention", "Description of the Figures", "Detailed Description of the Drawings", the "Claims", are disclosed in connection with the corresponding figures. Embodiments of the image sensor 180, as illustrated in FIG. 523, may be operable to achieve time sampling intervals, eg less than 1 nanosecond, which is generally less than the decay time of the marker fluorescence radiation.
[0081] Bei einigen Ausführungsformen kann der Bildsensor 180 als Komplementär-Metalloxid-Halbleiter(CMOS)-Bildsen-sor umgesetzt sein, bei dem alle (nicht gezeigten) Rücksetztransistoren der individuellen Photodetektoren 185 von einem (nicht gezeigten) gemeinsamen Rücksetzsignal gesteuert werden. Solange dieses Rücksetzsignal die Rücksetztransistoren offen hält, werden die Photoladungen, die sich aus dem erkannten Licht ergeben, gleichzeitig abgeleitet. Erst wenn die Rücksetztransistoren ausgeschaltet sind, werden die Photodetektoren 185 lichtempfindlich und beginnen die Expositionsperiode. Dadurch, dass das Rücksetzsignal zum selektiven Öffnen und Schliessen der Rücksetztransistoren gesteuert wird, sind die Photodetektoren 185 in zeitgesteuerter Synchronität, die notwendig ist, um eine zeitgesteuerte Funktionalität der Fluoreszenzdetektionsvorrichtung 100 gemäss den Ausführungsformen der vorliegenden Erfindung zu ermöglichen, betriebsfähig.
In embodiments of the invention, the sensitivity of the image sensor 180 can be implemented, for example, as described in European patent application No. EP 2 160 012 (012), in the name of Lotto et al., Entitled "Highly sensitive single photon imaging device ", For example, in the sections entitled" Description of the Figures ", excluding FIG. 1," Brief Description of Some Embodiments of the Invention "," Description of the Embodiments of the Invention "," Claims "," Amended Claims ... "and in the corresponding Figures are disclosed. Accordingly, the image sensor 180 based on the CMOS technology as explained above, for example, can be further amplified by using a pixel-internal gain circuit. Such amplification circuitry obviates the need to use increased voltages, As is the case with photodetectors 185, which function on the basis of the utilization of an avalanche effect multiplying the photocadication. The reading-out of the image sensor 180, which can be referred to as a "pixel-amplified semiconductor image sensor", if it is implemented as explained in 012, can be equal to or smaller than, for example, 1 electron at a room temperature which is, for example, from 20 ° C. to 30 ° C. -effective value (rms). Since the photodetectors 185 of the image sensor 180 are implemented as disclosed in 012 using a local reset transistor, such a pixel-enhanced semiconductor image sensor can be used to form a highly sensitive fluorescence detection device 100 operable in timed synchronism according to the embodiments of the present invention.
The time-controlled operational capabilities of the fluorescence detection device 100 according to the embodiments of the invention are explained in more detail below.
The photodetectors 185 of the image sensor 180 and the radiation source 110 are operatively coupled to the controller 190 so that the latter can operate the photodetectors 185 and radiation source 110 in a time-controlled (or in other words, time-resolved) synchronism. The controller 190 is operable to synchronize the operation between the radiation source 110 and the image sensor 180 so that there is a time gate between the emission of excitation light pulses 112 and the beginning of the sequence of integrating and reading the photocads. In other words, the starting point of the exposure period during which the detection of photons is enabled is synchronized in timed synchronism with the initiation of the emission of an excitation light pulse 112, The controller 190 uses a gate delay between each initiation of the emission of an excitation light pulse 112 and the beginning of the exposure period (integration period) of the photodetectors 185. This gate delay is selected according to the decay time of the autofluorescence so that the ratio between marker fluorescence and autofluorescence is high enough to produce one Reliable detection of the labeling fluorescence, the ratio having one of the values as described, for example, in US Patent Application No. US 2008/0265177, in the name of Connally et al., Entitled "Fluorescence detection" (177 ) Will be disclosed in the section entitled "Brief Summary of the Invention". The gate delay can, for example, be selected according to the gate delay, Which is disclosed in the section titled "Short Summary of the Invention" at '177. In any event, the gate delay is selected according to the decay characteristics of the autofluorophore and the tag fluorophore.
Accordingly, the controller 190 may include a memory (not shown) storing instructions (not shown) and a processor (not shown), wherein the execution of the instructions by the processor results in a fluorescence detection application that performs the time-controlled synchronized operation Between the radiation source 110 and the photodetectors 185.
In addition, with reference to FIG. 3, this fluorescence application according to an embodiment of the invention can implement a method for fluorescence detection, for example by carrying out the following steps: As indicated in block 300, the method may comprise the step of depleting the photodetectors 185. Thereupon, photocads which have been generated in the photodetectors 185 and previously stored may be removed therefrom.
As indicated in block 302, the method may then comprise the step of setting the photodetectors 185 to a non-detection mode, resulting in the photodetectors 185 deriving all photocads produced by incident photons.
As indicated in block 304, the method can comprise the emission of a first excitation light pulse of predetermined length and spectral wavelength, for example by switching on the radiation source 110. The emission of a first excitation pulse can be directed such that the test body 120 is illuminated by the first excitation light pulse becomes. In view of the fact that the duration of the first excitation light pulse is predetermined and thus known, the illumination period during which the test specimen 120 is illuminated is also known in duration.
As indicated at block 304, the process may then include the step of setting the photodetectors 185 to the detection mode during a predetermined photon exposure period upon expiration of a predetermined gate delay which may begin at the end of the illumination period. The gate delay is selected such that the autofluorescence radiation decays to sufficiently low levels which enable the reliable detection of the marking autofluorescence. The gate delay may, for example, be selected such that the autofluorescence radiation which may be present in the total fluorescent radiation decays to equal or less than one of the following percentages of its original strength: 15%, 14%, 13%, 12%, 11%, 10% , 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% Or equal to or less than 0.01%. During the predetermined exposure period, the photodetectors 185 of the image sensor 180 integrate the locally generated photocads locally in at least one charge integrator (not shown) included in each of the photodetectors 185. The photodetectors 185, In embodiments where each photodetector 185 comprises a plurality of charge integrators, photocads generated during the exposure period may be sampled during respective sampling periods and sequentially transmitted to the plurality of charge integrators with respect to each sampling period The photodetectors 185 of the image sensor 180 receive the locally generated photocads locally in at least one charge integrator (not shown) included in each of the photodetectors 185. In embodiments where each photodetector 185 comprises a plurality of charge integrators, photocads generated during the exposure period may be sampled during respective sampling periods and sequentially transmitted to the plurality of charge integrators with respect to each sampling period The photodetectors 185 of the image sensor 180 receive the locally generated photocads locally in at least one charge integrator (not shown) included in each of the photodetectors 185. In embodiments where each photodetector 185 comprises a plurality of charge integrators, photocads generated during the exposure period may be sampled during respective sampling periods and sequentially transmitted to the plurality of charge integrators with respect to each sampling period
Claims (12)
1. Fluoreszenzdetektionsvorrichtung (100), umfassend: einen Bildsensor (180), der ausgebildet ist, um eine Fluoreszenzstrahlung zu erkennen; einen Controller (190); eine Strahlungsquelle (110); wobei der Bildsensor (180) eine Vielzahl von Photodetektoren (185) umfasst, wobei jeder Photodetektor (185) ausgebildet ist, um eine erkannte Fluoreszenzstrahlung in eine entsprechende Anzahl von Photoladungen umzusetzen; wobei die Strahlungsquelle (110), die ausgebildet ist, eine Sequenz von Anregungslichtimpulsen zu emittieren, jeweilige Beleuchtungsperioden aufweisen; und wobei die Photodetektoren (185), die Strahlungsquelle (110) und der Controller (190) betriebsfähig miteinander gekoppelt sind; wobei jeder der Vielzahl von Photodetektoren (185) mindestens einen Ladungsintegrierer umfasst, der ausgebildet ist, während eines vorherbestimmten Zeitintervalls die Photoladungen, die als Reaktion auf die erkannte Fluoreszenzstrahlung generiert werden, zu integrieren; wobei die Photodetektoren (185) in einem Detektionsmodus, in dem fluoreszenzinduzierte Photoladungen gespeichert und integriert werden, und in einem Nicht-Detektionsmodus, in dem fluoreszenzinduzierte Photoladungen verworfen werden, betrieben werden können; wobei der Controller (190) ausgebildet ist, zwischen dem Detektionsmodus und dem Nicht-Detektionsmodus der Photodetektoren abzuwechseln; und wobei der Controller (190) ausgebildet ist, die Photodetektoren in den Beleuchtungsperioden in den Nicht-Detektionsmodus einzustellen und nach einer vorherbestimmten Gatterverzögerung nach dem Ende der jeweiligen Beleuchtungsperiode in den Detektionsmodus einzustellen.
2. The fluorescence detection device as claimed in claim 1, comprising: a plurality of electric drift field generators each coupled to the plurality of photodetectors (185), the plurality of electric drift field generators being adapted to receive the photocads received from each of the plurality of photodetectors Photodetectors to which at least one charge integrator can selectively transmit during a predetermined time interval.
3. The fluorescence detection device (100) as claimed in claim 1 or 2, wherein each of the plurality of photodetectors comprises an amplifier configured to amplify the signal representing the photocads generated in response to the detected fluorescence radiation.
4. The fluorescence detection device (100) as claimed in claim 3, wherein the amplifier is a control loop amplifier.
5. The fluorescence detection device as claimed in claim 1, wherein each of the photodetectors comprises a light-sensitive part for converting photons into photocads and a light-insensitive part comprising at least one electrical switch, and the at least one charge integrator is assigned to the at least one electrical switch Switch; And comprising a clock for controlling the electric switch so that photocads generated in the photosensitive member are given to the at least one charge integrator after the predetermined delay.
6. The fluorescence detection device as claimed in claim 1, wherein each photodetector comprises: a linear time conversion element of elongation and a lateral electric field generator; Wherein the linear time conversion element and the lateral electric field generator are coupled to one another in such a way that a lateral drift field is generated in the linear time conversion element which enables the scanning of photocads in the time conversion element from a first end to a second end along the longitudinal extent of the linear time conversion element ; The charge integrators being arranged in at least one row along the longitudinal extent of the linear time conversion element; Wherein the charge integrators are electrically coupled to the time conversion element, respectively, at a plurality of discretely spaced sampling points of the time conversion element; At least one light-sensitive region for converting photons into photocads which is coupled to the linear time conversion element such that generated photocads can be supplied to the linear time conversion element and at least one charge detection circuit electrically connected to the charge integrators which is suitable for photocadication Which has passed through at least a part of the linear time conversion element along its longitudinal extent.
7. The fluorescence detection device (100) as claimed in one of claims 1 to 4, wherein the plurality of photodetectors each comprise a plurality of reset transistors; Each reset transistor being controllable by a reset signal such that as long as the reset signal keeps the reset transistor open, photocouples generated in response to detected light are simultaneously derived, the photodetectors thus being in the non-detection mode; And wherein when the reset transistor is closed, the photocouplings are cumulated, the photodetectors thus being in the detection mode.
8. The fluorescence detection device (100) as claimed in claim 7, wherein each of the photodetectors further comprises a pixel-internal amplifier.
9. A method for operating a fluorescence detection device according to claim 1, comprising the following steps: a) reducing previously generated photocads in the photodetectors; B) (302) setting the photodetectors to a non-detection mode in which fluorescence-induced photocads are discarded; C) (304) illuminating at least one fluorophore-labeled test body with an excitation light pulse, the excitation light pulse having an illumination period; And d) (306) after a predetermined gate delay beginning at the end of the illumination period, adjusting the photodetectors to a detection mode for a predetermined photon exposure period in which the fluorescence-induced photocads are stored and integrated;
10. The method as claimed in claim 9, further comprising the following steps: e) determining whether at least one termination criterion is fulfilled, and if not, f) repeating steps a) to d). And if so, (312) reading the cumulative photocads.
11. The method as claimed in claim 9, comprising the following step: at least twice scanning during the exposure period of the signal which represents the photocads of the detected fluorescence radiation.
12. The method according to claim 11, wherein the time difference between each sampling step is selected to correspond to the difference in the decay time between at least two fluorophore markings.
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| DE4440613C1 (en) * | 1994-11-14 | 1996-07-25 | Leica Ag | Device and method for the detection and demodulation of an intensity-modulated radiation field |
| US6992761B2 (en) * | 1997-09-20 | 2006-01-31 | Molecular Devices Corporation | Broad range light detection system |
| US7633616B2 (en) * | 2003-06-02 | 2009-12-15 | Sensovation Ag | Apparatus and method for photo-electric measurement |
| CA2584186A1 (en) * | 2004-10-18 | 2006-08-31 | Macquarie University | Fluorescence detection |
| US7738086B2 (en) * | 2005-05-09 | 2010-06-15 | The Trustees Of Columbia University In The City Of New York | Active CMOS biosensor chip for fluorescent-based detection |
| EP2160012B1 (en) * | 2008-09-01 | 2013-11-27 | CSEM Centre Suisse d'Electronique et de Microtechnique SA - Recherche et Développement | Single photon imaging device |
| EP2182523B1 (en) * | 2008-10-31 | 2013-01-09 | CSEM Centre Suisse d'Electronique et de Microtechnique SA -Recherche et Développement | Charge sampling device and method based on a MOS-transmission line |
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