Apparatus for determining information relating to components of a body fluid and method for determining said information

Abstract

Apparatus and method for determining information associated with particles, such as cells, in a fluid, in which images are generated at various focal lengths / depths and various times, from which particle motion over time, and therefrom density and / or type, is determined for a plurality of individual cells, bacteria, platelets, etc.

Description

[Technical Field]

[0001] The present method relates to an apparatus and method for determining information associated with components of bodily fluids, particularly for determining information associated with generally individual components of bodily fluids, such as cells, bacteria, etc. [Background technology]

[0002] Historically, cell identification and classification has been performed visually by human operators or experts, and other techniques such as DNA staining have also been used.

[0003] Image-based cell identification is a widely known technique, and the density of each cell type is known.

[0004] The related art can be seen in the following: [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] BERNHARDT I et al., "Application of digital holographic microscopy to investigate the sedimentation of intact red blood cells and their interaction with artificial surfaces," BIOELECTROCHEMISTRY, vol. 73, no. 2, August 1, 2008 (2008-08-01), pp. 92-96, XP023784160, ISSN: 1567-5394, DOI: 10.1016 / J.BIOELECHEM.2007.12.001, Elsevier, Amsterdam, The Netherlands [Non-patent document 2] YU XIAO et al., "Review of digital holographic microscopy for three-dimensional profiling and tracking," OPTICAL ENGINEERING, vol. 53, no. 11, November 1, 2014 (2014-11-01), pp. 112306, XP060048121, ISSN: 0091-3286, DOI: 10.1117 / 1.0E.53.11.112306, SOC. OF PHOTO-OPTICAL INSTRUMENTATION ENGINEERS, Bellingham [Non-patent document 3] SANG-HYUK LEE and others, "Characterizing and tracking single colloidal particles with video holographic microscopy", December 11, 2007 (2007-12-11), XP080 345670, ARXIV.ORG, Cornell University Library, 201 OLIN LIBRARY CORNELL UNIVERSITY ITHACA, NY 14853 Summary of the Invention

[0006] In a first aspect, the invention relates to an apparatus as claimed in claim 1.

[0007] In this context, the elements of the device may be attached to one another or simply positioned appropriately relative to one another.

[0008] The bodily fluid may be, for example, blood, saliva, milk, and / or urine, and the components therein may be cells, such as blood cells, such as white blood cells (WBC) and red blood cells (RBC), and pathological cells, such as circulating tumor cells (CTC) and nucleated red blood cells (nRBC), epithelial cells, platelets, bacteria, parasites, proteins, or other components of the bodily fluid.

[0009] The sample container is configured to provide and contain a sample of the bodily fluid containing the component. The sample container can be of any desired type. Often, the sample container includes or is made of a translucent material that allows light or radiation to pass through the sample and the sample container so that an imaging element can provide imaging information. When containing the sample, the sample container can allow the sample to have a predetermined or minimum height along the vertical direction, thereby allowing cells present in the sample to move along the vertical direction, for example, up to a predetermined distance without colliding with the sample container.

[0010] A sample can be an untreated or unfiltered body fluid, or it can contain only a portion of the body fluid, for example, where the body fluid has been filtered or prepared by removing components such as red blood cells. A sample can be prepared by adding to the body fluid one or more agents configured to alter one or more parameters or components of the body fluid. For example, red blood cells can be filtered or lysed using a lysing agent.

[0011] The sample can be a sample of a collected bodily fluid containing a component having a relevant characteristic, the relevant characteristic substantially corresponding to the relevant characteristic of the component of the bodily fluid before collection. The sample can be a bodily fluid sample from which a specific type of cell has been removed or lysed. The sample can be a diluted bodily fluid sample. An imaging element is provided and configured to generate a series of imaging information over time. Thus, the imaging element can be suitably positioned at or around the sample container. The sample container and the imaging element can be adapted to one another to facilitate output of the desired imaging information.

[0012] The imaging element is provided to generate a series of imaging information, such that a plurality of imaging information is provided, each imaging information being generated at a distinct time point, such as every second, every 2 seconds, every 4 seconds, every 5 seconds, every 10 seconds, every 15 seconds, or every 20 seconds, etc.

[0013] Each imaging information includes information representing images of the sample in the sample container at at least two different focal lengths / depths. Thus, each imaging information can itself include multiple images, each having a specific or distinct focal length / depth. Thus, imaging information is provided over a period of time, and thus, preferably, the periods of different imaging information do not overlap, thereby providing multiple imaging information over time. Alternatively, it may be desirable for all represented images in one of the imaging information, representing a specific focal length / depth, to be provided at different times, thereby allowing represented images at other depths to be provided at different times.

[0014] Imaging information includes information representing images of a sample at at least two different focal lengths / depths. Such information can be provided by an actual image obtained using an optical system that provides the desired focal depth / distance. In other situations, the information can be configured in the form of a hologram. From a hologram, information can be obtained that represents an image obtained focused at a desired plane, which is often a horizontal plane, so that the plane has a height / depth. This information resembles an image obtained at a desired focal depth / distance, in which not only are components at or within a particular plane or depth / distance visible, but other components may also be present. However, components within or at that plane are more focused, which can be used to determine which components are present at that depth / distance. The represented image can alternatively resemble or be a cross-section of the sample.

[0015] In the different imaging information, each of the plurality of imaging information is identified and its position is determined. The positions of the components are then tracked or determined in the different imaging information, and thus over time. From any movement thereof, the type or other parameters of each component can then be determined. Essentially, the plurality of components is more than one component, such as all recognizable components in the imaging information, but often the number of components is 5-1000 components, such as 15-100 components, or 10-500 components.

[0016] Thus, from information representing the image, it is possible to determine which components are present in or at the depth / distance of interest.

[0017] In one embodiment, the imaging information is generated using variable optics or multiple sets of stationary optics, such that different focal lengths / depths can be achieved using such optics.

[0018] In some embodiments, a sample can be recorded with a hologram. From a single hologram, images or representations can be generated having any focal length / depth. Any number of such images or representations can be generated for any number of focal lengths / depths of the sample. Thus, a series of holograms, each representing distinct imaging information, can be recorded, and then distinct information representing each focal length / depth can be generated from the holograms.

[0019] A controller is provided, which can be hardwired or software controlled and can be, for example, a processor, DSP, ASIC, FPGA, etc. The controller can be formed by several elements communicating with each other.

[0020] The controller is configured to receive the set of imaging information. Thus, the controller can communicate with the imaging element. This communication can be wired or wireless and can occur via any protocol, bus, or even the World Wide Web. The controller can be provided as a cloud server, if desired.

[0021] The controller is configured to identify the same component in different imaging information acquired at different times. This determination can be based on several parameters of the imaging information and the sample. In a preferred embodiment, the component is assumed to move along a vertical path, and therefore, the component is assumed to always be at a specific location in a horizontal plane. Furthermore, the component is assumed to move downward, and therefore, the component's location is also assumed to be deeper at later times. Alternatively or additionally, other parameters of the component, such as shape, contour, edge, color, reflectance, emission, fluorescence, or content, can be used to identify the same component in different imaging information. Cells may have a nucleus or other components with a specific shape, location, or number, which can be used to distinguish between cells.

[0022] For example, if the sample can move horizontally or is found to move, a vertical motion can be added to the horizontal motion, which can then be compensated for if desired.

[0023] The controller is further configured to determine the position of the component in the sample container for each imaging interval. As noted, over time, and thus between imaging intervals, the position for the same component may be shifted vertically or horizontally or otherwise moved in the sample.

[0024] The controller is configured to determine information associated with the component from the position. This information can be density, or relative density compared to the density of the sample liquid. The controller can also be configured to determine this information based on time differences in imaging information. From changes in position, such as depth, and the time period between detections at those positions, a velocity of movement can be determined. This velocity is indicative of the difference in density between the sample liquid and that of the component. Velocity can also or alternatively be indicative of the type of component, such as which type of white blood cell the component is.

[0025] In other situations, the controller may also or alternatively determine the size of the component: the component may sink (or float upward) at a rate determined by or indicative of its size, such as its cross-sectional area in a vertical plane.

[0026] In a preferred embodiment, the imaging information is a hologram. Accordingly, the imaging element is configured to generate a hologram or series of holograms. Such imaging elements typically include a radiation or light emitter and a detector, often in the form of a 2D detection array such as a CMOS sensor, a CCD array, or the like. Various devices can be used, including on-axis (in-line) and off-axis devices, the latter using a tilted reference beam to separate twin images from the real object in Fourier space. Additionally or alternatively, multiple cameras can be used to capture images or data at different wavelengths, thereby allowing more information to be determined from the sample. In one scenario, radiation is emitted through a pinhole and passes through the sample before hitting the detector.

[0027] When the imaging information is a hologram, methods involve obtaining sample information, often a 2D image or representation, from the hologram, where the representation indicates not only the component present at a particular distance / depth but also other components. From this information, it is possible to determine which components are present at a particular distance / depth. Such methods can be direct methods such as angular spectrum backpropagation or transport-of-intensity, iterative methods such as Gerchberg-Saxton or sparse regularization reconstruction, and / or machine learning techniques such as UNet, GAN, and Deep-Image-Prior methods, which have various levels of reconstruction accuracy.

[0028] In alternative embodiments, the imaging information is generated by a more standard imager, such as a camera with adaptive optics or multiple sets of fixed optics, which can provide images with various focal depths / distances.

[0029] In one embodiment, when the imaging information is a hologram, the controller: - configured to obtain, after the receiving step, from each hologram an image representation of the sample, each image representation being associated with a distinct focal length / depth at at least two different focal lengths / depths; - the identifying step is configured to identify each of a plurality of components in at least two image representations associated with two different holograms and two different focal depths / distances.

[0030] As mentioned, in general the same components are determined at different times and at different distances / depths, from which information can be obtained.

[0031] In one embodiment, the controller is configured to determine, as information associated with at least one of the plurality of components and from the location of that one component, the density of the component of interest, either absolute or relative to the sample liquid, from which the type of component can be determined.

[0032] In that embodiment, the controller can be configured to determine the density for the component of interest based on the difference in focal length / height of the position, which is indicative of vertical motion that may occur due to gravity and density differences between the liquid and the component within the sample.

[0033] In one embodiment, the component is a cell, such as a red blood cell, a white blood cell, a circulating tumor cell, or a bacterium. In that or other embodiment, the component can be a crystallized material, a parasite, or a bacterium. Alternatively, the component can be a parasite or a platelet. Cells are often sufficiently heavy and / or sized to allow them to move predictably, evenly, or generally downward, whereas platelets are small and tend to remain suspended. Because cells, and particularly parasites, can themselves be motile, their position within the sample cannot be simply determined vertically. A parasite can be determined when it moves horizontally or even upward far enough. Horizontal movement can be determined earliest when the sample is stationary within the sample container.

[0034] It should be noted that it is also possible to determine Brownian motion from imaging information, such as from the horizontal motion of platelets, which are lighter than cells and therefore do not tend to settle in the sample, or any impurities lighter than cells. This motion can be used, for example, to derive the temperature of the sample. Also, the motion of any such component, for example, when detected in unison for many or all such components, can be used to indicate that the sample is moving. This can be used to compensate for any motions determined for other components or to discard the determination.

[0035] Then, in one embodiment, the position over time of one of the components exhibits stochastic motion, and information associated with that one component indicates that the component is a platelet.

[0036] In that or another embodiment, the position of one of the components over at least a predetermined minimum time period indicates upward or sideways movement, and the information associated with that one component indicates that the component is a parasite or a bacterium. Essentially, the predetermined minimum time period is long enough to rule out stochastic movement, such as that caused by Brownian motion. The time period can be 1 second or longer, such as 2 seconds or longer.

[0037] In such an embodiment or another embodiment, the position over time of one of the plurality of components indicates generally downward movement at a decreasing rate within a first velocity interval, and information associated with that one component indicates that the component is a cell.

[0038] Additionally, or alternatively, the position over time of one of the components exhibits a generally downward movement at a rate of decline within the second rate interval, and the information associated with the one component indicates that the component is a bacterium. The rates of decline for cells and bacteria can be determined by testing.

[0039] It may be desirable to base the determination of the information on additional information, such as various densities and sizes, as well as any internal structure, edge structure or shape.

[0040] In one embodiment, the controller is configured to determine edge information associated with edges of components in one or more image representations, and to determine information associated with the components also based on the edge information. The edges of white blood cells are called cell walls and differ between white blood cell types, and this information can be used in the determination.

[0041] A second aspect of the invention relates to the method of claim 8.

[0042] All embodiments, considerations, circumstances, etc. of the first aspect of the invention are equally relevant to the second aspect of the invention.

[0043] Thus, the sample, bodily fluid, component, sample container, imaging element, imaging information, and information representing the image may be as described above.

[0044] Additionally, component identification and information determination may be as described above.

[0045] Also, as noted above, in a preferred embodiment, the generating step includes generating a series of holograms. In this context, the method may further include obtaining at least two image representations from each hologram, each image representation associated with a distinct focal length / depth of at least two different focal lengths / depths, and the identifying step includes identifying each of the plurality of components in the at least two image representations associated with the two different holograms and the two different focal depths / distances.

[0046] In one embodiment, the determining step includes determining the density of the associated component as information associated with at least one of the plurality of components, as described above. Accordingly, the determining step can include determining the density of the associated component based on a focal length / depth difference of the locations.

[0047] In one embodiment, the components of the sample are cells or bacteria, but platelets, parasites, crystallized material, and other types of components can also be looked for and analyzed.

[0048] As mentioned above, Brownian motion can be determined and used to confirm measurements and / or to correct the determined motion.

[0049] As indicated above, in one embodiment, the position over time of one of the components exhibits stochastic motion, and information associated with that one component indicates that the component is a platelet.

[0050] In that or another embodiment, the position of one of the plurality of components over at least a predetermined minimum time period indicates upward movement, and the information associated with that one component indicates that the component is a parasite or a bacterium, the time period being further described above.

[0051] In any of these or other embodiments, the position over time of one of the plurality of components indicates generally downward movement at a decreasing rate within a first velocity interval, and information associated with that one component indicates that the component is a cell.

[0052] Additionally or alternatively, the position over time of one of the plurality of components indicates generally downward movement at a decreasing rate within the second rate interval, and information associated with that one component indicates that the component is a bacterium.

[0053] In one embodiment, the determining step also includes determining edge information associated with edges of the component in the one or more image representations, and determining information associated with the component also based on the edge information.

[0054] Another aspect of the invention is based on the same relative motion and density, but is directed to determining parameters of fluids. This aspect relates to an apparatus for determining information related to bodily fluids, such as blood, serum, saliva, urine, etc., comprising: - a sample container configured to receive a sample comprising one or more particles having one or more predetermined parameters and a liquid; - an imaging element configured to generate a series of imaging information over time, each imaging information including information representing an image of a sample within a sample container at at least two different focal lengths / depths; a controller, - receive a set of imaging information; - identifying the same component in the different imaging information and determining the location of the component in the sample container with respect to each imaging information; - Determine fluid and associated information from its location With a controller configured as Includes.

[0055] Also in this context, the motion of particles is determined and used to generate information, where the unknown component is a liquid.

[0056] Obviously, the location of multiple components can be determined in various imaging information, as explained above.

[0057] The liquids, measuring devices, sample containers, controllers and all other parameters, embodiments, circumstances etc. may be as described above.

[0058] The sample may be filtered, if desired, or may otherwise be derived from a bodily fluid. Other fluids may also be analyzed in this manner. It is preferred that particles are visible in the fluid, and therefore that the fluid is not too turbid, such as being translucent to at least one or more wavelengths used in the measurement device. The liquid may contain other components, if desired.

[0059] The particles have one or more predetermined or predefined parameters, such as density and / or size. As discussed above, the relative density and, optionally, size of the particles aid in determining the vertical velocity of the particles. Thus, by knowing the composition and parameters of the particles, information can be obtained from the fluid.

[0060] A final aspect of the invention relates to a method for determining information associated with a fluid, the method comprising: - providing a sample in a sample container, the sample including a component and a fluid having predetermined parameters; - generating a series of imaging information over a period of time, each imaging information including information representing an image of the sample within the sample container at at least two different focal lengths / depths; - identifying the same component in the different imaging information and determining the location of the component within the sample container with respect to each imaging information; - determining information associated with the fluid from the location; Includes.

[0061] This therefore also relates to the determination of information associated with the fluid.In connection with this aspect of the invention, the same considerations as explained above are valid.

[0062] In the following, preferred embodiments will be described with reference to the drawings. [Brief explanation of the drawings]

[0063] [Figure 1] FIG. 1 shows a first preferred embodiment of an apparatus using a holographic imager. [Figure 2a] Figures 2a and 2b show the sedimentation of cell-like beads within the sample. [Figure 2b] Figures 2a and 2b show the sedimentation of cell-like beads within the sample. [Figure 3] Figure 3 shows the density of a typical blood cell. [Figure 4] FIG. 4 shows an alternative embodiment of the device that uses adaptive optics. [Figure 5] FIG. 5 shows an alternative embodiment of the device that uses replaceable optics. DETAILED DESCRIPTION OF THE INVENTION

[0064] 1, a preferred embodiment of the device 10 is shown, in which a sample holder 20 holds a sample, the sample being derived from a body fluid and including components of the body fluid such as cells, bacteria, platelets, parasites, etc. The sample may be a prepared body fluid, such as a sample in which components or constituents of the body fluid have been removed or altered, such as blood from which red blood cells have been lysed or milk from which fat has been broken down.

[0065] The image information provider 30 is provided in the form of a holographic imager configured to provide a hologram of the sample or a portion thereof. In this embodiment, the image provider includes a light source 24, such as an LED, and a pinhole 28 through which light is provided to the sample holder 20. A detector or imager 24, such as a detector array, such as a CMOS array, is provided to receive the light that has passed through the pinhole and the component-bearing sample. The LED and pinhole can be replaced by other configurations of light sources, such as lasers or concentric LEDs. It may also be advantageous to perform wavefront modulation at the source, for example, via an SLM or DOE.

[0066] A controller 40 is provided to receive the output of the imager 24 and obtain multiple images or information representative of the images of the sample at various focal planes or depths.

[0067] The generation of such images or information can be found in "Nonmechanical parfocal and autofocus features based on wave propagation distribution in lensfree holographic microscopy" by Dharmawan, Mariana, Scholz, Hormann, Schulze, Triyana, Garces-Schroder, Rustenbeck, Hiller, Wasisto, and Waag in Scientific Reports, (2021) 11:3213. Note that when the determination of images or information is based on holograms, all such images or information are obtained at the same time.

[0068] The image information provider outputs a series of holograms over time, and the controller then generates a series of images or sets of information from the series of holograms.

[0069] For the same sample, if holograms are provided over a period of time, preferably while the sample is generally stationary, several different types of information can be obtained from the sample.

[0070] When moving through various focal depths or distances in a generally vertical direction, the density of components of the sample can be determined from the determination of components, such as cells, at increasing or decreasing depths at increasing time points. If the density of the liquid portion of the sample is known, the rate of settling or flotation of the component can be used to determine the density of the component.

[0071] Components such as cells present at a certain depth in a particular image or image can be determined from the sharpness of the edges or components of the cells such as the nuclei.

[0072] In the applicant's co-pending application EP22182762.9, filed on 4 July 2022, a technique is exemplified which may be able to determine not only whether a cell or other component is in the focal plane of an image, but also whether the component is close to or far from the imager. This technique may therefore alternatively or additionally be used to determine the depth at which a component of an image is seen at the time of recording the associated hologram.

[0073] The location of components 25, such as cells, within the sample can be determined as coordinates in the horizontal plane and in depth, and thus cells can be identified in subsequent holograms and at other, typically larger, depths by at least substantially the same coordinates in the horizontal plane, but at other depths.

[0074] In FIG. 1, one particle 25 is above the depth-defining plane 27, one is in the plane, and two are below the plane.

[0075] Figures 2a and 2b show fluid measurements in which beads 10 μm in diameter and 1.055 g / ml in density are immersed in deionized water (1.0 g / ml). Because the beads have a density slightly higher than that of the liquid, they settle and therefore reside at greater depths over time. In Figure 2a, the depth over time of 25 beads is seen as detected over a 140-second period. In Figure 2b, the settling of individual beads is seen, along with a linear fit to the downward velocity of each bead.

[0076] Figure 3 shows the density of different types of blood cells. From Figure 3, it can be seen that different cell types sediment at different rates due to their different densities, which can be used to characterize or distinguish between cell types.

[0077] Additionally, other information can be used to characterize components, such as visual information such as the shape, contour, color, etc. of parts of a component, such as the cell membrane, cell nucleus, or other components of the cell. Different white blood cells have different sizes and shapes of membranes and nuclei, and different "fluffiness" thereof. These parameters can be determined from the image / information and used to characterize individual components.

[0078] Obviously, components with a density lower than that of the liquid will move upward within the sample, and the upward velocity can likewise be used to characterize the component and / or obtain information associated therewith.

[0079] Horizontal motion can also be determined where components are identified over time at at least substantially the same depth.

[0080] Horizontal motion can be due to, for example, Brownian motion, whereby components that are approximately the same density as the liquid and / or that are so small in size that they settle or float very slowly, also move somewhat stochastically and therefore vertically. From the motion of such components, the size of the Brownian motion can therefore be determined, which can be used to compensate for the determination of the density or velocity of heavier / lighter / larger components.

[0081] Such small components of the sample can be impurities such as platelets, large aggregates of platelets, bacteria, and unintended parts of the sample such as colored crystals from sample preparation.

[0082] It may also be desirable to determine non-Brownian motion of particles, such as convection from nearby bubbles. This, too, can be used as an indication that the sample is not stationary and the measurement is flawed. Furthermore, if some, e.g., all, of the determined components move in unison or have a moving component, this unison motion can be used as an indication that the sample in the sample container is not stationary. Thus, the motion of individual components can be compensated for this unison motion, and the analysis can be paused until a later time when the sample is assumed to be stationary.

[0083] Parasites may also be present in bodily fluids. Some parasites and some bacteria are capable of moving within bodily fluids. Thus, parasites / bacteria can be characterized or identified when determined at various depths, such as when moving in a horizontal plane or both upward and downward, beyond any movement that may be due to Brownian motion.

[0084] In FIG. 4, an alternative embodiment of apparatus 80 can be seen, in which images of a sample in a sample container are obtained using an imager 90, which has adaptive optics, such as a variable lens, to provide a series of images of the same sample at different focal lengths or depths. By rapidly changing the optics, the position of the sample components can be assumed to be substantially stationary, and a series of images with different focal depths can be provided. Note that the determination of the above information related to component depth / location and time can be performed, where images are obtained at different depths of the same sample at different time points. These time points can be logged and used in determining information related to the sample components.

[0085] 5, another alternative 100 can be seen, in which the imager 90' has replaceable lenses 92, each fixed at a particular focal depth. Thus, the lenses 92 can be sequentially introduced into the optical path between the sample container and the imager 90', thereby again providing a series of images of the same sample at different focal depths.

[0086] As indicated above, the figures and description relate to determining parameters of particles in a liquid, the relevant parameter(s) of which are preferably known. If the particle parameters are known, the same apparatus can be used to determine the liquid parameter(s). Thus, if a particle is known, its upward / downward movement, determined as described above, can be used to determine the liquid parameters.

[0087] In this environment, as also explained above, the liquid may be a bodily fluid, such as a filtered or otherwise prepared bodily fluid.

Claims

1. 1. An apparatus for determining information associated with a component of a bodily fluid, comprising: a sample container configured to receive a sample containing said component of said body fluid; an imaging element configured to generate a series of imaging information over time, each imaging information including information representing an image of a sample in said sample container at at least two different focal lengths / depths; a controller, receiving said set of imaging information; - identifying each of a plurality of components in different imaging information and determining, for each imaging information, the location of each one of the plurality of components in the sample container; - determining information associated with each of said plurality of components from said locations; With a controller configured as An apparatus comprising:

2. 10. The apparatus of claim 1, wherein the imaging information is a hologram.

3. 3. The apparatus of claim 2, wherein the controller: - after said receiving step, obtaining from each hologram an image representation of said sample, each image representation being associated with a distinct focal length / depth of at least two different focal lengths / depths, - in said identifying step, identifying each one of said plurality of components in at least two image representations associated with two different holograms and two different focal depths / distances; Consists of an apparatus.

4. 4. The apparatus of claim 1, wherein the controller is configured to determine the density of at least one of the plurality of components as information associated with the one component and from the location of the one component.

5. 5. The apparatus of claim 4, wherein the controller is configured to determine a density for the associated constituent based on a difference in focal length / height of the locations.

6. 6. The device of claim 1, wherein each one of the plurality of components is a cell, a platelet, a crystallized material, a parasite, or a bacterium.

7. 7. An apparatus according to claim 1, wherein the controller is configured to determine edge information associated with an edge of at least one of the plurality of components in one or more of the image representations, and to determine the information associated with the one component also based on the edge information.

8. 1. A method for determining information associated with a component of a bodily fluid, comprising: - providing a sample containing said component of said body fluid in a sample container; generating a series of imaging information over time, each imaging information including information representing an image of a sample within a sample container and at least two different focal lengths / depths; - identifying each one of a plurality of components in different imaging information and determining the position of each one of the plurality of components in the sample container with respect to each imaging information; - determining from said locations said information associated with each of said plurality of components; A method comprising:

9. 9. The method of claim 8, wherein the generating step comprises generating a series of holograms.

10. 10. The method of claim 9, further comprising obtaining at least two image representations from each hologram, each image representation associated with a respective focal length / depth of at least two different focal lengths / depths, and wherein the identifying step comprises identifying each of the plurality of components in at least two image representations associated with two different holograms and two different focal lengths / depths.

11. 11. The method of claim 8, wherein the determining step comprises determining, as information associated with at least one of the plurality of components, a density of the associated component.

12. 12. The method of claim 11, wherein the determining step includes determining the density of the associated component based on a difference in focal length / depth of the locations.

13. 13. The method of any one of claims 9 to 12, wherein each of the plurality of components is a cell, a platelet, a crystallized material, a parasite, or a bacterium.

14. 14. A method according to any one of claims 8 to 13, wherein the determining step also comprises determining edge information associated with edges of the component in one or more of the image representations, and determining the information associated with the component also based on the edge information.

Images