WO2017036483A1 - Cytomètre d'images continu - Google Patents

Cytomètre d'images continu Download PDF

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Publication number
WO2017036483A1
WO2017036483A1 PCT/DK2016/050289 DK2016050289W WO2017036483A1 WO 2017036483 A1 WO2017036483 A1 WO 2017036483A1 DK 2016050289 W DK2016050289 W DK 2016050289W WO 2017036483 A1 WO2017036483 A1 WO 2017036483A1
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WO
WIPO (PCT)
Prior art keywords
light
sample
images
image
array
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PCT/DK2016/050289
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English (en)
Inventor
Søren Kjærulff
Johan Holm
Frans Ejner RAVN HANSEN
Martin Glensbjerg
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Chemometec A/S
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Application filed by Chemometec A/S filed Critical Chemometec A/S
Publication of WO2017036483A1 publication Critical patent/WO2017036483A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/144—Imaging characterised by its optical setup

Definitions

  • the present invention relates to methods and systems for the determination of qualitative and quantitative properties of biological particles
  • Multi-channel images which are used to enhance the analysis, are collected by arranging light sources and/or optical filters to be used followed by the recording of an image. The process is then repeated for all images recorded.
  • This task requires careful planning, where selection of suitable light source and corresponding optical filters as well as the settings of the imaging device can affect the resulting image.
  • the biological material is sometimes fragile, sometimes being altered or damaged through prolonged exposure to the intensive light used. Further when analysing samples of biological particles in suspension the particles will move in the recorded images such that it is of importance to collect images during as short a timespan as possible, because otherwise it becomes necessary to locate particles in different places in different images.
  • the present invention attempts to solve these problems associated with Image Cytometry by offering methods and system which optimise analysis time and exposure of the sample to potentially damaging illumination. Summary of the Invention
  • Present invention includes methods and systems for performing optimised Image Cytometry analysis of biological samples.
  • the optimisation involves reducing time of analysis as well as reducing unnecessary exposure of the sample to high-intensity illumination, such as illumination needed for the generation of fluorescence signal.
  • the present invention comprises a method for the assessment of at least one quantity parameter and/or at least one quality parameter of particles in a biological sample, comprising applying a volume of the sample to a sample compartment having parallel wall part defining an exposing area, arranging two or more light sources in fixed arrangement to illuminate and/or transmit light onto and/or through the sample compartment, exposing, onto an array of active detection elements, light emitted from and/or having passed through the sample compartment, thus recording two or more images of spatial light intensity information of substantially the same portion of the sample, processing the image in such a manner that light intensity information from individual biological particles are identified as distinct from light intensity information from the background, and correlating the results of the processing to the at least one quantity parameter and/or the at least one quality parameter of biological particles in the sample.
  • each image may be changed, for instance by illuminating the sample with light of different wavelength and/or different intensity.
  • optical filter(s) When recording images of fluorescence it is further often an advantage to place one or more optical filter(s) in the optical path, thereby isolating light of relevant wavelength.
  • light interfering objects such as attenuating filter, aperture or an obstruction, thus improving conditions for microscopy such as Bright-Field, Dark-Field and Phase Contrast microscopy.
  • FIG. 1 illustrates sequence of operation.
  • FIG. 2 illustrates an image cytometer.
  • FIG. 3 illustrates sequence of operation
  • Figure 1 illustrates one sequence of operation, for the collection of two or more images of a biological sample.
  • the sequence is initiated by setting parameters such as number of collected images ( «), image, light source and optical component properties for each image (/ ' ).
  • Image properties ⁇ Image(i)) can be several properties, typically relating to operation of detection elements and/or electrical circuit transforming analogue image signals into digital image data. Typical properties are cycle time, the time interval between initiations of image recordation, exposure time, the time the detection elements are open for accumulation of light signals, and analogue signal condition, such as gain and/or offset, applied prior to digitalisation. Exposure time cannot exceed the cycle time but apart from that these parameters can be freely chosen among possible settings of the imaging system used. In some embodiments the image properties are identical for some or all recorded images, while in other equally preferred embodiments image properties of two or more images are different.
  • Light Source properties ⁇ LightSource(i)) identify which of the included light sources are to be activated. Often only a single light source is applied for the collection of a given image but in many embodiments two or more light sources are active during at least a part of a cycle time. The properties of each of the light sources concerned are typically the effect or power level as well as times for turning on and/or turning off, relative to the image cycle.
  • Optical Component properties ⁇ Opt.Comp.(i)) concern components affecting the optical system, such as lens(es) used for focusing, optical filters, mirrors, apertures or obstructions. Properties associated to a given image can concern none, 1 or more optical components. Typical properties could be to move a lens to a predetermined position for focusing, inserting or removing optical filter(s) and/or aperture(s) and/or obstruction(s).
  • the repeated sequence is synchronised by the cycle time of the imaging means, thus allowing the recording of images to take place during a short time.
  • At least two of the recorded images reflect different conditions, such as wavelength of light and/or intensity or exposure time.
  • Figure 2 illustrates a possible configuration of an Image Cytometer.
  • the illustration outlines 4 main groups of components, the sample holder (200), illumination means (210), imaging means (220) and detection means (230). Finally it illustrates the main optical axis of the Image Cytometer (240), along which majority of the optical components are arranged.
  • the sample holder can be moved along the optical axis relative to the imaging means, in order to assure that the sample is in focus alignment with the imaging means.
  • the sample compartment (201) is placed on the sample stage (202) in the optical path.
  • the sample compartment is typically attached to the sample stage but it can be released such that it can be removed from the Image Cytometer and replaced again through either manual or automated process.
  • the sample stage can move in two directions perpendicular to the optical axis. This allows different parts of the sample inside the sample compartment to be assessed.
  • the sample holder further comprises reference position means (203) to determine position and/or relative position of the sample compartment holder. Such reference position means allows reliable determination of position in the plane of the sample compartment as well as position along the optical axis of the image cytometer.
  • Insignia on the reference position means which can be read by the detection means, is used to determine position but the insignia can also be formed in such a way that it allows determination of the optical magnification of the optical system. Such determination of optical magnification can be based on a predetermined known pattern of the insignia, for instance when the distance between two or more identifiable insignia elements is known.
  • One preferred embodiment of reference position means is a grid of spots formed in a thin opaque layer deposited on transparent support, where position, relative position and/or size of said spots is known.
  • the illumination means can move along the optical axis in order to maintain the desired illumination of the sample compartment.
  • the illumination means contains usually 2 or more light sources (3 shown).
  • the illustration shows a light source located on the optical axis (21 1), such that illumination of the sample in the direction towards the detector, which is often only preferred when the purpose is to generate an image of passive light attenuation and/or scattering properties, such as Bright-field or Dark-field images.
  • the illustration shows two fluorescent light sources (212), which illuminate the sample compartment at an inclination relative to the optical axis, which has been found to improve conditions under which signal from particles is identified as being different from signal from the background. It further illustrates a light source emitting a single wavelength (212a) as well as a light source emitting two wavelengths (212b), by placing two Light Emitting Diodes in a single arrangement.
  • the imaging means comprise collection objective (221) in an arrangement where it is possible to interchange two or more collection objectives (221a and 121b) with different properties, such as nominal magnification.
  • the two or more collection objectives are interchanged either by linear or circular movement.
  • Light modulation means are preferably two or more and contain a number of optically active components labelled Yi (222) and Xi (223) respectively, such as filters, apertures, obstructions or phase contrast elements.
  • the light modulation means can be moved perpendicular to the optical axis such that each of the optical components can be placed in the beam of light emitted from the collection objective, the movement can either be linear or circular.
  • Each light modulation means may have a position without an optical component, such that if all light modulation means are arranged such that this empty position is located in the light beam then no modulation takes place.
  • the imaging means contain focusing means (224) which focus the light from the collection objective onto the detector.
  • the detection means can be moved along the optical axis in order record a focused image of light intensity information.
  • the information is gathered using an array of active detection elements (231), a light sensitive camera.
  • the operation of the Image Cytometer and collection of data is controlled by computer means (not shown).
  • the computer means preferably is equipped with image processing means which can be used for automatic identification and assessment of biological particles.
  • Figure 3 A illustrates an optical position sensor (300) included in several preferred embodiments.
  • the sensor includes a coherent light source (302), typically a laser or a laser diode, which illuminates the sample compartment (301), equivalent to sample compartment (201) in Figure 2, with a ray of coherent light (303).
  • the light is reflected from the surfaces of the sample compartment and the reflected rays (304) are measured using a detector (305), which is typically an array of detection elements such as a CCD or CMOS camera.
  • a typical sample compartment is illustrated in more details in Figure 3B.
  • the illustration shows that the sample compartment has an upper part or a window (301a) and a lower part or a window (301b), between which the sample is typically placed.
  • the coherent light (303) enters the sample compartment at an angle, typically about 45°, and is reflected off its surfaces at the same angle. Firstly it is reflected off the upper sample compartment window, forming a reflected ray or light (304a).
  • the part of the light not reflected enters the window and travels through to the opposite side, where a part of it is again reflected forming a ray of reflected light (304b) which is parallel to the first ray of reflected light but shifted due to the distance between the surfaces, i.e. the thickness of the upper part or window.
  • Figures 3C and 3D show typical images of the reflections as recorded by the detector.
  • the image in Figure 3C is typical for arrangement when a single beam of light, such as a laser or laser diode is focused into a small cylindrical beam of light. Such beam of light produces a reflection that resembles the shape of the beam of light, through producing a spot on the detector.
  • the image in Figure 3D shows a similar arrangement of coherent light that is focused in a line. The recorded reflection is therefore a line rather than a spot.
  • Implementation using linear coherent light offers the advantage of allowing for the simultaneous determination of position or distance at several locations, which is a clear advantage when position or distance across large area is to be determined.
  • a preferred embodiment comprises a method or a system where the array of active detection elements, e.g. array of active CCD or CMOS elements, is in continuous, uninterrupted operation during the capture or recording of a number of exposures of light, thus forming images of light intensity information.
  • the array of active detection elements e.g. array of active CCD or CMOS elements
  • the array of active detection elements is operated
  • recording of two images includes exposure of three images where the midmost image is omitted.
  • Such intermitted recording of images offers advantages in certain situations, such as when conditions of the measurement are changing at slower rate than the rate of capture of images.
  • the conditions for the recording of images are substantially identical for at least two images, where condition for the recording affect properties of the spatial image, for instance recorded intensity and/or level of noise in the image.
  • An example of preferred conditions for the recording is one or more of the following; integration time, readout time, sensitivity, amplification, excitation energy.
  • the conditions for the recording of the image of light intensity information is different in two individual images, preferably where such different conditions have no substantial influence on the rate of two or more recorded images, preferably consecutive images, since preferably these conditions have been altered before a subsequent or later image is recorded.
  • An example of preferred conditions for the recording is one or more of the following; integration time, readout time, sensitivity, amplification, excitation energy.
  • One preferred method of changing conditions while recording images of light intensity information is to alter the operation of a light source between the recordings of two consecutive images.
  • Light source could for instance be excitation light in
  • photoluminescence detection or transmission or reflection light source in scatter/attenuation detection It is often preferred to operate a light source in substantial synchronisation with the recording of images of light intensity information.
  • Such control can be the turning of a light source on or off, preferably where the light source is on during the exposure of an image, preferably where the light source is on during a predefined fraction of the duration of the exposure of an image of spatial light intensity information, further often preferred that the predefined fraction of duration where the light source is turned on is different in the of the two or more images thus reflecting different light intensities.
  • One advantage of such embodiments is the ability to increase the observed dynamic range of the recorded images by mathematically adjusting individual collected images and combining them to one recorded image, preferably such that the dynamic range exceeds that of the dynamic range and/or resolution of a single collected image, preferably where the dynamic range and/or resolution is increased by a factor of 2 or more, such as by a factor of 4 or more.
  • two or more images are recorded under substantially identical conditions, such as when differences in such two or more images reflect development in the light intensity due to changes in the sample or if differences between such two or more images improve distinction between light information and measurement noise.
  • Other equally preferred embodiments are characterised by slow rate of recorded images, preferably recording images at a rate of at least 1 second per image, such as 2 seconds per image or more, or even at rates such as such as 4 seconds per image or more. Further in specially demanding situations rates 16 seconds per image or more is preferred.
  • the rate of recording images is at the most 8 seconds per image, in other embodiments at the most 4 seconds per image and in yet other embodiments the rate of recorded images is at the most 2 seconds per image, the nature of the system under investigation determining the most suitable combination.
  • the rate may vary from 1-8 seconds per image, such as 1-4 or 1-2 seconds per image. Further, the rate may be from 2-8 seconds per image, such as 2-4 seconds per image.
  • a part of the recorded images are collected at high speed, e.g. high rate of images per second, while a part of the recorded images are recorded at low speed, e.g. high rate of seconds per image.
  • Several preferred embodiments comprise an interchangeable optical component placed in the light path from the sample compartment to the array of active detection elements.
  • interchangeable optical component is/are optical filter limiting the transmission of light to a predetermined band of wavelengths or components which contain predetermined regions which have different optical properties, such as components where different optical properties of regions of the optical component are transparency and/or optical phase.
  • preferred embodiments include optical interchangeable optical filters which facilitate the recording of images of fluorescence intensity, where such interchangeable optical filters can be removed from the optical path allowing light emitted from and/or through the sample to pass without substantial optical modulation or filtration.
  • the interchangeable optical filters are placed on a movable support, which through linear translation or rotation can introduce the optical filters into the light path.
  • at least one filter position of the movable support is void or containing optical component that allows light to pass without filtration.
  • Optical components typically can be moved into or out of the optical path of the image cytometer, so as to change optical properties of the light entering the array of active detection elements and/or the sample.
  • optical components are typically optical filters, selecting one or more wavebands or regions to be transmitted and/or modifying the intensity of transmitted light, or obstructions, defining spatial limitations of the optical path such as aperture or a light blocking elements, thus spatially modifying the transmitted light and/or causing phase alteration.
  • Further optical components can be lenses, altering the optical magnification of the illumination light and/or the collected image. In many of these embodiments the optical components are moved into or out of the light path at a
  • the time it takes to move such optical component is 100 ms or less, such as 50 ms or less. In other embodiments, typically embodiments intended for fast image acquisition, it takes 20 ms or less to move optical components, preferably 10 ms or less.
  • optical components are moved into and/or out of the optical paths of the image cytometer at speeds such that the movement takes less than the cycle time of the image recordation, such as time that is 50 % of the image cycle time or less.
  • the cycle time of the image recordation typically when high rate of image recordation is preferred, such movement is performed during a time period that is 20 % or less of the image cycle time, such as 10 % or less of the image cycle time.
  • the period of image recordation cycle can be divided up into one fraction, during which one or more optical component is moved and another fraction, during which optical signal intensity is generated, preferably by turning a light source on during such fraction of the image cycle time.
  • Such operation preferably allows an image to be recorded during each image recordation cycle of the active detection elements, not being affected by the movement of an optical component, which typically would take the time of one or more image recordation cycle.
  • images of spatial light intensity information are recorded where all of the two or more light sources are used.
  • the number of images corresponds to the number of light sources, while in preferred embodiments at least one image is recorded with the sample illuminated by two or more light sources and/or where at least two images are recorded with the sample illuminated by two or more light sources.
  • Such embodiments offer several advantages, such as the ability to obtain a full description of the sample being analysed in a single sequence of operation, preferably where such full description of the sample is obtained without any prior knowledge about the sample being analysed.
  • one or more light source can be omitted, thus reducing the number of collected images accordingly, since this can reduce analysis time and/or reduce the exposure of light onto the sample.
  • Several preferred embodiments comprise means which can determine relative and/or absolute position of surfaces, mainly surfaces of walls defining the sample compartment.
  • These means include a light source and an optical arrangement which allow coherent or a substantially coherent light to be reflected from a surface onto a position sensitive device, such that the arrangement of the reflecting surf ace/surf aces relative to the position sensitive device can be determined.
  • the coherent or substantially coherent light used to produce reflection on the position sensitive device is preferably generated by a laser diode. In other equally preferred embodiments the light is generated by a light emitting diode (LED).
  • LED light emitting diode
  • the position sensitive device used to determine position of reflected light is preferably a two dimensional array of active detection elements, such as CCD sensor or a CMOS sensor.
  • Such two dimensional array of active detection elements allows the determination of the spatial position of the reflected light by comparing intensities on the different detection element to the know position of said detection elements on the position sensitive device.
  • position at the position sensitive receiver is determined by determining position of light information detected by the receiver. Since the reflection of light detected by the position sensitive device often gives rise to signal on plurality of active detection elements it is often preferred to calculate position based on the determination of intensity at plurality of detection elements. Such determination can be based on locating the detection element with the highest light intensity or by determining the centre of intensity using methods commonly used to determine centre of mass.
  • the determination of position of the wall part is used to determine difference in position of surfaces of two parallel wall parts, preferably where the surfaces are the surfaces closest to each other defining the thickness of the sample compartment.
  • the two parallel wall parts are the surfaces of the bottom and top boundaries of the sample compartment, which allows the determination of the thickness of the sample compartment.
  • This information about thickness of the sample compartment can be combined with information concerning the area size of the sample compartment that is exposed onto the array of active detection elements to determine the volume of a liquid sample being analysed. In order to improve the determination of the sample volume being analysed it is often preferred to determine differences in position of surfaces of parallel wall parts of the sample compartment at two or more locations.
  • such determination at two or more locations is done sequentially while in other equally preferred embodiments the determination at two or more locations is done simultaneously. Simultaneous determination is preferably facilitated through the use of optical elements such as reflecting surfaces or diffracting media. Preferably determination at two or more locations is obtained using a single position sensitive device. [0043] In embodiments where the position and/or difference in position is determined at more than two locations simultaneously it is further preferred that such position is determined along a line segment. Preferably the position is determined along such a line segment with a short interval, such as at 1 mm intervals or less, such as 0.5 mm intervals or less.
  • position is determined along a line segment with intervals of 0.2 mm or less, such as 0.1 mm or less. It is typically preferred that position and/or difference in position is determined at 10 or more locations along a line segment, such as 50 locations or more and in some preferred embodiments, typically when position information of an extended region is of interest it is preferred that position is determined at 100 or more locations, such as at 200 or more locations.
  • One preferred task of the method of determining position of a wall part is to locate the sample relative to the light source.
  • the light from the light source is preferably focused onto the sample and preferably it is possible to improve the focusing when location of the sample compartment is determined.
  • the position of the bottom wall part of the sample compartment is used to locate the sample compartment relative to the light source, preferably it is the upper part of the bottom of the sample compartment that is used to determine the location. This information may be used to position the sample compartment and the light source in a predetermined relation to each other.
  • Another preferred task of the method of determining position of a wall part is to locate the sample relative to the array of active detection elements.
  • the light from the sample compartment is focused on the array of active detection elements and it is therefore of interest that this focusing is at optimum when generating a spatial image of light intensity information.
  • Information about the position of the sample in the sample compartment can be used to position the sample compartment and the array of active detection elements relative to each other in such a manner that optimum or substantially optimum focus is obtained. It is typically preferred to use the position of the lower surface of a transparent plate forming the top part of the sample compartment when arranging the sample compartment and array of active detection elements relative to each other.
  • the light source illuminating and/or transmitting light onto and/or through the sample compartment have an active light emitting area greater than 1mm 2 in size. It has been found that such large-size light sources can be used to produce high-intensity illumination where the light intensity is substantially even across the illuminated area of the sample in the sample compartment.
  • the active exposing area of a light source may be less than 20 mm 2 , preferably in the range between 1 mm 2 and 20 mm 2 , such as in the range between 2 mm 2 and 6 mm 2 .
  • the intensity from a light source with active exposing area greater than 1 mm 2 may be substantially uniform, such that the that variations in intensity is less than 35 %, preferably less than 20%, more preferably less than 10 %.
  • optical means which decrease variation in light intensity exposed onto and/or through the sample compartment. Such means are preferably a diffuser or an array of micro lenses. In embodiments including a diffuser it is often preferred that such diffuser changes the wavelength of emitted light, preferably through a process of photoluminescence.
  • the aspect ratio of the exposing area is substantially identical to the aspect ratio of the array of active detection elements.
  • the shape of the active exposing area of the light source is such that when focused onto the sample compartment its image forms a substantial rectangular illumination area.
  • the aspect ratio of such rectangular illumination is substantially identical to the aspect ratio of the array of active detection elements.
  • the sample compartment holder can be moved in such a manner that the optical compartment is illuminated with light from a light source and is in the field of view of the imaging system.
  • the optical component comprises insignia that produces a detectable pattern in the detection system and by recording one or more images of the optical component and analysing the detectable pattern, it is possible to determine the position of the optical component and thereby the position of the sample compartment holder and/or a position of the sample compartment holder relative to the detection system.
  • such transparent optical component contains insignia which can be on the form of any or all of the following, engraving on the surface or the interior, printing on the surface of the optical component thus making it less transparent in regions, adhesion or deposition of material that has different transparency properties from that of the optical component, to form an optical component where varying transparent regions are formed on a surface or in a plane.
  • the image of recorded insignia of the optical component reflects transparency properties of the optical component.
  • the insignia of the optical component is formed by transparent/opaque and/or illuminating region(s), such as transparent regions, such as holes in an opaque film, it is preferred that such transparent regions have the dimension of between 2 and 20 ⁇ .
  • the regions are smaller in size, such as between 1 and ⁇ or even between 1 and 5 ⁇ in diameter.
  • the biological sample being assessed may be a suspension of biological particles in liquid. Such suspension of biological particle can be a portion of a larger sample volume where the purpose of the assessment can be to determine or estimate the property of the larger sample volume.
  • the biological sample is a sample of cells grown and/or growing on a substrate.
  • the substrate is suspended (e.g. beads), while in other equally preferred embodiment such substrate is, or can become, an integrated part of the sample compartment.
  • adherent cells may be growing inside the sample compartment on the wall part of the sample compartment defining an exposing area.
  • substrate placed in the sample compartment is substantially transparent.
  • the assessment of at least one quantity parameter and/or at least one quality parameter of biological sample may be the analysis of individual cells. Such individual cells are often single, either in suspension or on a substrate, but such individual cells can also be in a clump of cells, such cells adhering to each other. In other embodiments the assessment of at least one quantity parameter and/or at least one quality parameter of biological sample is the analysis of a bulk of cells, such as a tissue sample.
  • the first light source may be a light emitting diode (LED) and/or a diode laser and/or laser.
  • LED light emitting diode
  • a diode laser and/or laser Several of the properties of LED's and laser diodes offer substantial advantages in the design and operation of system, such as small physical size and power efficiency.
  • the biological particles being assessed are sensitive to light, to a degree where the illumination can alter the properties of the particles.
  • One preferred method to reduce the effect of the light is to limit the duration of exposure of the sample to the light.
  • an illumination period is between 0.0001 and 0.1000 seconds, such as between 0.0001 and 0.0500 seconds.
  • the sample is illuminated with 200 nJ/mm 2 or less, such as 100 nJ/mm 2 or less, preferably 50 nJ/mm 2 or less, such as 20 nJ/mm 2 or less.
  • a light source may be used for the attenuation of light, for instance through refraction and/or reflection. Often it is preferred that the light from such a light source is transmitted through the sample compartment in a substantially collimated manner, such that a substantial portion of the light is parallel or substantially parallel. It has been surprisingly found that such substantially parallel light can enhance the contrast of the attenuation, i.e. the ratio of attenuation of light by the biological particle to the intensity of transmitted light in a region of the background.
  • a portion of the light transmitted through the sample is not exactly parallel, but may deviate from parallelism at an angle less than 45° relative to parallel, such as less than 30°, such as 15°. Even less divergence, such as 10° or less, is often preferred, such as divergence of light of no more than 5° relative to the optical axis.
  • the high contrast recorded under collimated conditions can be maintained while a moderate divergence often contributes positively to evening out the intensity of light transmitted through the sample compartment and exposed onto the array of active detection elements.
  • a moderate divergence often contributes positively to evening out the intensity of light transmitted through the sample compartment and exposed onto the array of active detection elements.
  • these embodiments have the focus point of the light source brought about by optical components such as lens(es) or mirror(s), outside the sample compartment.
  • the light from a light source passing through the sample compartment for attenuation of light is substantially focused on the sample compartment.
  • a substantial portion of the light transmitted through the sample compartment can be recorded by the array of active detection elements.
  • the light transmitted through, and/or onto, the sample compartment is substantially even in intensity across the field of view of the array of active detection elements.
  • deviation from even illumination for instance expressed as the ratio of the variation of intensity to the mean intensity, is less than 25%, more preferably less than 10 % and even more preferably less than 5%.
  • Such property generally has the effect of reducing variation in an optical property of a sample as recorded on the array of active detection elements, which often results in lower variation in the expression of a property, which is dependent on the intensity of illuminated light. This is for instance apparent for both attenuation of light and emission of fluorescence.
  • Optical means consisting of number of lenses arranged in an array, can be arranged to focus multiple images of the light-emitting element of a light source onto and/or through the sample compartment.
  • Such arrays of micro lenses are preferred in several embodiments for the purpose of effectively illuminating a portion of the sample
  • micro lenses includes an array of cylindrical micro lenses. Such a micro lens array is preferably used in combination with a second or further array(s) of cylindrical micro lenses, which typically are oriented perpendicular to each other. Such an arrangement is included in several preferred
  • the properties of such two or more arrays of cylindrical micro lenses are substantially identical, while in other often-preferred embodiments the properties are different, such as to produce illumination which is adapted to the shape of the array of active detection elements. Properties of arrays of micro lenses that are varied to produce such shape are for instance pitch of the lenses and/or the focal length.
  • optical components such as lens(es), optical filter(s), aperture(s) and obstruction(s) are movable
  • many preferred embodiments have one or more of the light sources in fixed arrangement.
  • Such fixed arrangement can be relative to the general body of the image cytometer or relative to any other component of the image cytometer, such as the sample compartment or the array of active detection elements.
  • optical means to focus light exposed onto an array of detection elements such as one or more lens(es) or mirror(s), where such optical means have the ability to focus exposed light signals, expressed as depth of focus in the object plane.
  • such focusing means have focus depth larger than 5 ⁇ , preferably in the range from ⁇ to 150 ⁇ .
  • the one or more lens(es) used for focusing of light exposed onto an array of detection elements are substantially transparent to light in the wavelength regions of between 200 nm and 1,000 nm, preferably where it is transparent in the wavelength region of between 300 nm and 1,000 nm more preferably where it is transparent in the wavelength region of between 350 nm and 850 nm.
  • the one or more lens(es) used for focusing are optically aberration-corrected in the range.
  • the transparency of the one or more lens(es) is such that attenuation of light is less than 3 OD in the region, more preferably less than 1 OD.
  • the array of active detection elements in embodiments is typically either a CCD or CMOS sensor.
  • the optical magnification of light exposed onto an array of active detection element is less than 20: 1, defined as the ratio of size of projection of any dimension of an object in the sample compartment to the size of the object.
  • Other equally preferred embodiments have optical magnification of between 1 : 1 and 20: 1, preferably between 1 : 1 and 1 : 10.
  • the optical magnification is less than 4: 1, such as in the range from 1 : 1 to 4: 1.
  • Some preferred embodiments include means for variable optical
  • magnification e.g. zoom.
  • any movement of a sample or particle in a sample is kept under control, more preferably such movement should be kept at minimum. Therefore it is preferred that the volume of liquid sample is at stand still during the exposure, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during one recordation of an image.
  • conditions of stand-still are maintained, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during time of two image recordings, preferably such that it is contained substantially within the boundary of the same detection elements during time of more than two recordings, such as during three, four, five or even six recordings. Most preferably conditions of stand-still are maintained during the recording of all images of light intensity information processed for the assessment of property of biological particle. In many instances the sample compartment is characterised by the absence of flow of the liquid sample, once the sample has been loaded into the sample compartment.
  • the volume of liquid sample is at stand still during the exposure of light intensity information, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during one image recording with the light source through the time of a second image recording with a second light source, preferably where the result of the exposure of the second light source is the recording of an image of spatial fluorescence light intensity information.
  • the volume of sample analysed usually relates to the statistical quality of the assessment of biological particles, since the size of the volume typically correlates directly to the number of individual particles that are analysed. For instance when the assessment of biological particles concerns the counting of individual particles the total number of counted particles determines the precision of the results.
  • the thickness of the sample compartment defined by it's wall parts, and therefore the interior of the sample compartment may have an average thickness of between 20 ⁇ and 1,000 ⁇ . In these and other preferred implementations, the average thickness is between 20 ⁇ and 100 ⁇ . Ideally the thickness of the sample compartment is uniform, but it has surprisingly been found that a substantial deviation from uniform thickness does not compromise the results of the assessment, as long as the average thickness of the portion of the sample compartment that is analysed is known. Further, in embodiments where the assessment of biological particles is performed in a disposable sample compartment, such as sample compartments intended for only a single analysis of a sample, it is preferred that the average thickness of each sample compartment is known, or preferably determined by means of the system.
  • the volume of the liquid sample from which electromagnetic radiation is exposed onto the array of detection elements is in the range between 0.01 ⁇ _, and 20 ⁇ ., such as between 0.05 ⁇ _, and 5 ⁇ ..
  • the total volume of the sample being assessed, which is exposed onto the array of detection elements depends on several factors, including the sample thickness and area of the part of the sample
  • the total volume of the assessment can preferably be further increased by exposing an additional portion of the sample, either by replacing the portion of the sample in the sample compartment with a different portion or by moving the sample compartment and thus exposing a different part of the sample compartment onto the array of sample compartment.
  • One generally preferred embodiment, which typically has a significant contribution to the determination of the volume of sample exposed onto the array of detection elements is the view area of the optical arrangement exposing light intensity information.
  • the view area is fixed, and in equally preferred embodiments the view area can be determined on the basis of the adjustment of the optical components.
  • One preferred method for the determination of the volume analysed in a single exposure is to combine information concerning the thickness of the sample compartment with information about the active view area of the exposing system.
  • Biological particles are diverse in type and properties, but it is generally preferred in several of the embodiments that the size of the particles, the parameter or parameters of which is/are to be assessed, are of a size between 0.1 ⁇ and 100 ⁇ . Such size of a particle is typically the average diameter of a particle, and in several equally preferred embodiments this average size of a biological particle is between 0.1 ⁇ and 20 ⁇ . [0076] As biological particles and the properties of these are very diverse, embodiments can be used to assess a great number of quantity and/or quality parameters of biological samples and/or particles of a biological sample.
  • the number of the biological particles per volume of a liquid sample the diameter, area, circumference, shape, asymmetry, circularity of biological particles, determination of adhesion and/or degree of clumping of biological particles, preferably where degree of clumping allows the substantial determination of the number of individual cells in a clump of cells.
  • Other equally preferred parameters are; the species of biological particles, the metabolic status of biological particles, intracellular property, such as number, size, shape of nucleolus and organelles.
  • the assessment comprises the recording of two or more images of spatial light intensity information, where one image represents attenuation of light, is the substantial location of a biological particle in the spatial light intensity image.
  • This location of a particle is preferably used to correlate other light intensity information to the particle, such as fluorescence. This is for instance often preferred when among plurality of individual particles it can be expected that some particles reflect such other light intensity information while other particles substantially do not reflect this light intensity information. The substantial absence of such light intensity information can make it difficult to determine the presence of such particle based solely on the light intensity information, as it sometimes shows no
  • the location of a particle can be derived on the basis of other image of spatial image intensities.
  • the location of a particle can be determined by using transmission or bright field imaging.
  • Preferred embodiments include methods for the assessment of properties of biological samples and/or biological particles, where an image of spatial light attenuation information is recorded. Many of these embodiments preferably further include the steps of recording additional images of spatial light intensity information, included in the processing of image information, where the additional spatial light intensity information is information about fluorescence.
  • this additional fluorescence image information is generated by excitation light from the light source passed through the wall parts of the sample compartment and used to generate attenuation image information, where an emission filter is employed thus producing fluorescence.
  • such additional fluorescence information image is generated by excitation light from an additional light source.
  • more information is generally recorded by recording more than one or two images of spatial light intensity information, it is generally preferred that in the addition to an image of attenuation information, two additional fluorescence information images are included in the processing of images, and typically it is more preferably to use three, four or five additional fluorescence information images.
  • the assessment of a parameter of a biological sample and/or biological particle includes acquiring and processing multiple images
  • the number and nature of the light sources which are usually two, three, four or five individual light sources, reflects properties such as wavelength and intensity of light, attenuation and scatter, and in the case of fluorescence background suppression.
  • spatial information about location of biological particles is an integrated feature of the processing of images of spatial light intensity information.
  • parameters of biological particles that can be assessed can preferably be one or several of the following; assessment of species biological particle, condition of biological particle, preferably wherein condition of biological particle is metabolic condition such as cell cycle, viability, vitality, apoptosis, or motility.
  • a light source used is a tuneable solid-state light source.
  • the presence of a tuneable light source can significantly simplify the design of system, and would also allow for greater flexibility, such as when the tuneable solid-state light source is used for excitation of fluorescence light.
  • a tuneable solid-state light source is either a light emitting diode (LED) or a laser diode.
  • a method for the assessment of at least one quantity parameter and/or at least one quality parameter of particles in a biological sample comprising
  • the conditions for the recording is one or more of the following; integration time, readout time, sensitivity, amplification, and excitation energy.
  • controlling of light sources include on/off periods of the light source.
  • interchangeable and/or movable optical component is moved at a speed such that it is entirely moved into or out of the optical path within 20 ms or less, preferably within 10 ms or less.
  • a wall part defining an exposing area is a transparent plate defining top or bottom part of the sample compartment.
  • the wall part is the upper or lower surface of the transparent plate defining top or bottom part of the sample compartment.
  • the position sensitive receiver is an array of detection elements, preferably a CCD sensor or a CMOS sensor.
  • position of the wall part is used to determine difference in position of surfaces of two parallel wall parts, preferably where the surfaces are the surfaces closest to each other defining the top and bottom of the sample compartment.
  • the wall part is the upper surface of a transparent plate forming the bottom part of the sample compartment. 52. The method according to items 33 through the preceding item, wherein the determination of position of a wall part is used to determine relative position of the array of active detection elements and the sample.
  • one or more light source(s) to illuminate and/or transmit light onto and/or through the sample compartment, where the active exposing area of at least one light source is greater than 1 mm 2 .
  • the means which decrease variation in light intensity is a diffuser.
  • the diffuser substantially changes the wavelength of the light, preferably through the process of photoluminescence.
  • the sample compartment holder being movable such that it can be moved and thereby placing the optical component in a location where it is illuminated with light from a light source and in the field of view of the imaging system comprising an array of active detection elements,
  • the optical component comprising an insignia producing a detectable pattern on the array of active detection elements
  • optical component comprises transparent material such as glass.
  • the transparent location(s) is a hole, preferably a hole of about 2 to 20 ⁇ in diameter.
  • the substrate is a transparent substrate, preferably where such transparent substrate is, or can become, an integrated part of the sample compartment, such as wherein the substrate is a wall part of the sample compartment defining an exposing area.
  • a light source is a light emitting diode and/or a diode laser and/or laser.
  • duration of illumination of light from a or one light source passed through the wall parts of the sample compartment is less than 1 second, preferably where it is less than 0.1 second.
  • the method according to any of the preceding items wherein the light exposed onto the array of active detection elements is focused by focusing means, preferably where the focusing means produce a depth of focus that is more than 5 ⁇ , such as between 10 ⁇ and 150 ⁇ . 99.
  • the light exposed onto the array of active detection elements is focused by focusing means, preferably where the focus means are substantially transparent to light in the wavelength region of between 200 nm and 1,000 nm, more preferably in the wavelength region of between 350 nm and 1000 nm, more preferably in the wavelength region of between 350 nm and 850 nm.
  • the array of active detection elements is an array of CCD or CMOS sensor elements.
  • a volume of liquid sample is at stand still during the exposure, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during recordation of one image.
  • a volume of liquid sample is at stand still during the exposure, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during time of recordation of two images, preferably such that it is contained substantially within the boundary of the same detection elements during time of recordation of four images.
  • volume of liquid sample is at stand still during the exposure, where stand-still is defined as the situation where at least a part of the image of a biological particle does not move any more than it is contained substantially within the boundary of the same detection elements during time of recordation of a first image with the first light source through the time of a second recordation with a second light source, preferably where the result of the exposure of the second light source is the recording of an image of spatial fluorescence light intensity information.
  • compartment is intended for a single analysis of a sample.
  • the volume of the liquid sample from which electromagnetic radiation is exposed onto the array is in the range between 0.01 ⁇ _, and 20 ⁇ ⁇ .
  • the volume is between 0.05 ⁇ _, and 5 ⁇ ⁇ .
  • the parameter to be assessed is a morphological property, such as the diameter, area, circumference, asymmetry, circularity of the biological particles.
  • the parameter to be assessed is the determination of adhesion and/or degree of clumping of biological particles, preferably where the degree of clumping allows the substantial determination of the number of individual cells in a clump of cells.
  • the parameter to be assessed is the metabolic status of biological particles.
  • the parameter to be assessed is intracellular property, such as number, size, shape of nucleolus or organelle.
  • the parameter to be assessed is location of biological particles in the spatial light intensity image.
  • a second image of second spatial light intensity information is recorded, where the second spatial light intensity information is information about fluorescence caused by excitation light from the first light source passed through the wall parts of the sample compartment, or caused by excitation light from a second light source.
  • the light source is a tuneable solid-state light source.
  • the tuneable solid-state light source is tuneable light emitting diode, LED.
  • a system for the assessment of at least one quantity parameter and/or at least one quality parameter of particles in a biological sample comprising
  • a sample compartment having wall part parallel to the sample compartment and defining an exposing area configured to hold a volume of a biological sample comprising particles
  • two or more light sources in fixed arrangement configured to illuminate and/or transmit light onto and/or through the sample compartment
  • a computer configured to process the images in such a manner that light intensity information from individual biological particles are identified as distinct from light intensity information from the background, and to correlate the results of the processing to the at least one quantity parameter and/or the at least one quality parameter of biological particles in the sample.
  • the system of item 135, further comprising means for operating the array of active detection elements substantially continuously during the exposure and recording of two or more images of spatial light intensity information.

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Abstract

La présente invention concerne des procédés et des systèmes pour effectuer une analyse d'échantillons biologiques par cytométrie d'image optimisée. L'optimisation consiste à réduire le temps d'analyse ainsi que l'exposition inutile de l'échantillon à un éclairage de haute intensité, tel que l'éclairage nécessaire pour générer un signal de fluorescence.
PCT/DK2016/050289 2015-08-28 2016-08-29 Cytomètre d'images continu WO2017036483A1 (fr)

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CN107091800A (zh) * 2017-06-06 2017-08-25 深圳小孚医疗科技有限公司 用于显微成像粒子分析的聚焦系统和聚焦方法
CN107389537A (zh) * 2017-08-31 2017-11-24 许金泉 一种新型多通道细胞计数仪及多通道细胞计数系统
CN108183860A (zh) * 2018-01-19 2018-06-19 东南大学 基于粒子群算法的二维片上网络自适应路由方法
CN114174799A (zh) * 2019-05-30 2022-03-11 贝克曼库尔特有限公司 用于固定生物标本以进行显微成像的方法和系统

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WO2014191003A1 (fr) * 2013-05-28 2014-12-04 Chemometec A/S Cytomètre à formation d'images

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WO2014068003A1 (fr) * 2012-10-30 2014-05-08 Commissariat à l'énergie atomique et aux énergies alternatives Dispositif d'acquisition d'une image d'un échantillon, comportant un organe de régulation du chauffage d'un support de réception de l'échantillon, et système d'imagerie associé
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CN107091800A (zh) * 2017-06-06 2017-08-25 深圳小孚医疗科技有限公司 用于显微成像粒子分析的聚焦系统和聚焦方法
CN107389537A (zh) * 2017-08-31 2017-11-24 许金泉 一种新型多通道细胞计数仪及多通道细胞计数系统
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