WO2011072698A1

WO2011072698A1 - Stroboscopic counting of particles

Info

Publication number: WO2011072698A1
Application number: PCT/DK2010/050345
Authority: WO
Inventors: Martin Christian Valvik; Niels Agersnap Larsen
Original assignee: Unisensor A/S
Priority date: 2009-12-18
Filing date: 2010-12-17
Publication date: 2011-06-23

Abstract

The invention relates to a method for detecting particles in a liquid sample. A stroboscopic principle is applied to image the particle and to provide an optical sectioning of the particles. The method has a large variety of applications such as live monitoring of e.g. the amount of microorganisms, such as bacteria, in water samples from waterworks.

Description

STROBOSCOPIC COUNTING OF PARTICLES

The invention relates to a method for detecting particles in a liquid sample. Such a method can be applied for live monitoring of e.g. the amount of microorganisms, such as bacteria, in water samples from waterworks.

The requirement for the quality of the water that comes from the waterworks is very high, demanding a frequent analysis of the water. The quality is usually checked at least once a month in smaller waterworks, and for larger waterworks much more frequent. The quality check is performed manually by acquiring a water sample from the works or from somewhere along the water pipes. The sample is transported to a laboratory for analysis. A range of parameters is determined, such as the level of iron, level of different toxins and the pH-value. These parameters will usually not change significantly from test to test. One important parameter that must be kept very low in the water is the number of microorganisms or total bacterial count. If the number of bacteria, such as E. coli bacteria increases the health of the user of the water is at risk. In case of pollution the number of bacteria may rise to health threatening levels very rapidly, and there is therefore a need for live monitoring of the total bacterial count. If pollution occurs, the water supply should be stopped immediately. Microscopy may be used for monitoring the quality of the water.

When searching for particles such as microorganisms in samples with low or very low concentration using techniques such as optical sectioning and digital image analysis, it is necessary to search in a relatively large volume of a sample. The sample may e.g . be water at the waterworks that should be scanned for bacteria. Searching for small particles in a large volume requires a large number of high resolution images to be grabbed and processed. The high resolution is necessary to be able to determine if a detected particle is a microorganism or a particle comprised e.g. by ochre or lime scale.

The acquisition of a large number of images involves read-out of data from a camera to a storage location which for large amounts of high quality images may take unacceptable long time. Furthermore the acquired images must be processed which involves reading the images from the storage location and applying complex algorithms which expands the time required per image acquired . If the microorganisms are al ive, they may move around in the sample, and therefore have changed position from one image to the next, making optical sectioning difficult or impossible.

If the water has been polluted with a high level of microorganisms such as E. coli, it is desired to stop the supply immediately to avoid polluting the pipes and exposing the users of the water to the risk of becoming sick. It is therefore desired to have the measurement results ready immediately after acquisition of the images. Optical sectioning of large sample volumes for bacteria may therefore involve expensive electronics comprising fast processors and lots of RAM and disk space.

For decreasing the requirements to the processing electronics, the image size may be reduced. This unfortunately reduces the size of the sample that may be scanned in a given time, and therefore reduces the probability of detecting the microorganisms at an early stage.

A stroboscopic image is known which is produced by illuminating a scene multiple times in a single exposure with a stationary camera. It shows, as a single image (photograph), a manner of movement of the object. In the days of film cameras, such a stroboscopic image was taken by performing plural strobe light emissions on the moving object during a long-term exposure.

When searching for microorganisms in water the sample of water may be contained in a sample device. In order to be able to use optical sectioning, it is necessary that the microorganisms are substantially at stand still. Substantially at stand still may refer to the situation where there is no mass flow of the sample during the acquisition of the images.

If the microorganisms were moving, i.e. if the sample of water is moving within the sample device, the optical sectioning may be impossible to carry out. It will thus not be possible to determine if a particle found is a harmful microorganism or if it is a harmless solid particle. Such stroboscopic imaging of moving objects may therefore not be used, as the objects are not moving.

There is therefore a need for a reduction of the amount of data to be transferred (read out) and processed without going on compromise on the amount of sample scanned for microorganisms. Further the images acquired must be of a quality where the optical sectioning may be utilised to determine the type of particles detected.

It is one object of the present invention to provide a method for reducing the number of images of an optical sectioning to be acquired at a given size of sample without compromising the certainty of detecting microorganisms in the sample.

In one embodiment of the present invention an optical detection assembly is provided. The optical detection assembly comprises an illumination device, such as a laser or a LED. The optical detection assembly further comprises an image acquisition device, such as a CMOS camera or a CCD camera. The optical detection assembly optionally comprises optics for providing an image of an object to be created on the image acquisition device. The optics may comprise lenses, apertures, prisms, and other optical devices used to create an image of an object. The optics may be assembled to comprise a dark field type or oblique type of microscope, but also other types of microscope assemblies may be used. The optical detection assembly further comprises a shutter, such as an iris-type shutter or a mirror type shutter as in a standard reflex camera and/or an electronic shutter. The optical detection assembly may further comprise a sample device base for holding a sample device comprising a liquid sample.

The optical detection assembly may further comprise at least one translation unit for translating the sample device and the image acquisition device relative to each other.

The optical detection assembly may also comprise a stimulating device for stimulating the sample in the sample device. The optical detection assembly may also comprise a processing device for processing the images acquired from the image acquisition device. The processing device may be adapted to store at least one image, such as being adapted to store a plurality of images. The processing device may be adapted to process the images acquired using one or more image processing algorithms. The processing device may be adapted to control the operation of the optical detection assembly. The controlling may comprise controlling the image acquisition device, i.e. activating and deactivating the shutter, read-out of images, storing of images, controlling the stimulating device, controlling the translation unit(s) and their mutual timing and activation.

The optical detection assembly may be arranged in a housing supporting the optical detection unit, the sample device base, the illumination device, the stimulation device and the translation unit. The housing may further comprise the processing device as well as an output device for outputting data. The processing device may be an embedded processor running embedded software, or it may be a standard personal computer connected to the optical detection assembly. The output device may be a screen, a serial or parallel wired connection or it may be a wireless connection.

In one embodiment of the present invention at least a sub-volume of a liquid sample is prepared to be imaged / analysed. If the sample to be imaged / analysed is large, say many litres, and the concentration of the particles to be detected is very small, the number of scans to be made by the optical detection assembly may be very high. In this case it may be chosen to concentrate the particles in a smaller volume e.g. by filtering the sample. If the measurement to be made is a spot check of the sample, the measurement may be made without concentrating the sample.

The preparation of the liquid sample may also comprise staining, tagging and/or filtering the particles in the sample, or if the sample comprises blood, the preparation may comprise haemolysing. In one embodiment the method comprises providing said sample to said sample device. The sample device may be a one-time use only disposable e.g. made of plastic or it may be a re-useable device e.g. made of glass or other long- lasting material. In one embodiment, the sample comprised in the sample device may be replaced by a new sample. The amount of liquid sample in a sample device may be in the range of 0.1 micro litres to 100 micro litres.

In one embodiment, the sample device may be a part of a shunt to a main water pipe from the water work, said shunt going from the main water pipe though a valve to the sample device, and optionally further back to the main water pipe or through an outlet as wastewater. When a fresh sample of water is to be applied to the sample device, the valve is opened for a short period. When the water in the sample device has been completely exchanged with new water, the valve is closed again for the water to settle.

In one embod iment, the method comprises activating the shutter for acquisition of a long-time exposure image. The long-time exposure time may be as short as few milliseconds or even shorter, or, as long as hundreds of milliseconds or even many seconds. The shutter may be a physical shutter such as an Iris-type shutter or other aperture such as a mirror. When the shutter is activated, a physical barrier stopping light from exposing the image acquisition device is removed. When the shutter is deactivated, the physical barrier stops the light to fall upon the active area of the image acquisition device. The shutter may be an electronic shutter operated from the controlling device. The electronic shutter may be adapted to start and stop exposure of the image acquisition device.

Stimulation may be provided to the sample from the stimulating device. The stimulation is in one embodiment applying an electric field across the sample. The electric field may be DC or AC. The stimulation may be accomplished by applying a magnetic field, applying acoustic waves or electromagnetic waves, such as ultra violet light (UV). The purpose of applying stimulation may be to create a change in the sample that may be used to characterise particles in the sample. If e.g. an electric field is applied, electrically loaded particles may be observed moving towards the poles of the field. The stimulation may be provided when the shutter is activated and the image acquisition device is open for exposure to electromagnetic radiation from the sample device and/or when the shutter is deactivated.

In one embodiment, the method comprises providing illumination from the illumination device to the liquid sample comprised in the sample device. In one embodiment of the invention the illumination comprise providing a short pulse of electromagnetic radiation (such as a flash of light) to the sample for illuminating at least a portion of the sample that is imaged onto the image acquisition device. The duration of the flash may range between a few microseconds to tenths or hundreds of milliseconds depending on the properties of the light source and the sample. The illumination may be provided as pulsed electromagnetic radiation. The intensity may be varied between successive flashes. The illumination may be generated using a LED or a laser, or it may be generated using a light bulb or other electromagnetic emitting means. In one embod iment the ill um ination is provided as a continuous wave illumination, i.e. the l ight is on as long as the shutter is activated. In one embodiment the illumination comprises providing wavelength multiplexing of at least two different electronic waves. The illumination may be comprised of electromagnetic waves with different polarisation. I n one embodiment, said illumination comprises photoluminescence or said illumination triggers the emission of photoluminescence. The electromagnetic radiation may be intensity multiplexed.

In one embodiment, said illumination comprises electromagnetic radiation, said electromagnetic radiation preferably being in the range of about 200 nm to about 1 100 nm, such as in the range of about 300 nm to about 800 nm, in the range of about 400 nm to about 700 nm, in the range of about 450 nm to about 600 nm, in the range of about 495nm to about 570nm.

In one embodiment, the method of the present invention comprises translating the sample device and the image acquisition device relative to each other. The translation is in one embodiment of the invention to move the just illuminated part of the sample device partly or completely out of the view of the image acquisition device and a new part of the sample device partly or completely into the view of the image acquisition device. After movement of the sample device, a new illumination may be applied for further illuminating the sample and acquiring an image with the image acquisition device.

The translation may be a rotation of the sample device or it may be a linear translation along one or more axes, or a tilt, or a combination of these. The translation may be relative to the image acquisition device.

In one embodiment, the sample device is moved a distance corresponding to at least the field of view of the image acquisition device between each activation of the illumination. This ensures that a particle is only illuminated once, making it easier to count the particles in the images, as every particle will show up on the image only once.

In one embodiment, the sample device is moved a distance shorter than the field of view of the image acquisition device between each activation of the illumination. In this embodiment the particles are illuminated more than one time for each image, and each particle will therefore be seen as similar objects in the image with equidistant spacing, and when used for optical sectioning the image may also yield a focus stack for each object.

In one embodiment of the invention, the sample device is translated a distance corresponding to approximately 1 -10 times the diameter of the particles in the sample device between each activation of the illumination device, such as 2-8 times the diameter, such as 3-5 times the diameter. This embodiment may be used for determining the trace of particles in the sample. For such an application, the translation between the activations of the illumination could be selected to account for the movements of the particles between each illumination, in order to have the images of the particles separated in the combined image.

The method may comprise a first procedure comprising said illumination, optionally said stimulation and optionally said translation. The first procedure may also be referred to as the measurement procedure. In one embodiment, all parts, including the optional parts, of said first procedure are performed. The illumination, optionally the stimulation and optionally the translation of the first procedure may be carried out in different orders. E.g. the stimulation may be carried out before the illumination. If the stimulation of the sample generates a response from the sample that may comprise electromagnetic radiation, such as luminescence, it may not be necessary to carry out the illumination, as the illuminating is done in the stimulation process.

The first procedure may be repeated at least one time. The illumination of the sample, and optionally the stimulation of the sample using the stimulation device, and optionally the translation of the sample device relative to the image acquisition device may be repeated at least one time, preferably more than one time, such as more than 5 times, such as more than 20 times or more than 100 times.

When the illumination, stimulation and translation of the first procedure_have been repeated at least one time, the method may comprise deactivating the shutter for stopping the exposure of the active area of the image illumination device.

In one embodiment the shutter is deactivated only during the illumination and stimulation and activated during the translation, i.e. the shutter is deactivated and activated a number of times before the image is read out. When the exposure of the image illumination device has been stopped, the method may comprise reading the image from the image acquisition device and to store the image in the processing device. After the reading and storing process the image is ready for processing in the processing device using image processing algorithms. In one embodiment, the method of the present invention comprises processing the image to determine parameters characterising the particles in the liquid sample. The processing may comprise determining values of parameters relating to the concentration of particles, the relative concentration of different particles, the relative concentration of living and dead microorganisms, and/or to the movements of living microorganisms in said sample. The processing of the images may involve applying advanced algorithms to extract information regarding the particles. The extracted information may comprise the number of particles found in the image, the size and shape of the particles, the type of the particles, the movement pattern of the particles, the orientation of the particles, the particle focus stacks and the relative number of particles of each type of particles found.

The processing may comprise an optical sectioning if said sample and/or one or more particles in said sample.

When processing the image one or more parameters may be determined and the determined parameters may be written to the output device. The output device may be a display, an external device such as a USB device, as input to further procedures. In one embodiment, the parameters to be output are to be used in a later part of the method and said output of said value of said parameters comprises applying said value to the later part of the method. The parameters may be stored internally in the processing device such as in dedicated registers of the processing device.

After processing the image and determining parameters characterising the particles in the image, the parameters may be used to determine if a predetermined condition has been reached . The method may comprise repeating a second procedure comprising one or more of said activating the shutter, said providing stimulation, said illumination, said translation, said deactivation of said shutter, said reading an image, said processing to determine parameters, and said outputting said determined parameters. Said second procedure may be repeated until a predetermined first condition has been reached. The first condition may be reached in the first repetition and the procedure is therefore only activated once. The second procedure may thus comprise said first procedure of illumination, stimulation and translation. In one embodiment, all parts including the optional parts of said second procedure are performed.

The method may comprise a third procedure comprising providing a new sample into the sample device and re-initiating the second procedure from the beginning, i.e. said third procedure may comprise one or more of said preparing a liquid sample, said providing said sample to said sample device, said activating the shutter, said providing stimulation, said illumination, said translation, said deactivation of said shutter, said reading an image, said processing to determine parameters, said outputting, and said determining whether a first condition has been reached. The third procedure may thus comprise said first procedure and said second procedure. In one embodiment, all parts, including the optional parts, of said third procedure are performed. The starting over again with a new sample in the sample container may be repeated forever or at least a predetermined time or a predetermined number of times or to some predeterm ined cond ition has been reached . The predetermined stop condition could e.g. be the situation where an increased concentration of micro-organisms has been discovered. The measurement procedure may then be stopped and an alarm given. The alarm could e.g. be a sound signal given, a mail send to a predetermined receiver, or an electrical signal used to stop the pumps or close a valve at the waterworks.

The method of the present invention may comprise repeating said third procedure, until a predetermined second condition has been reached. The second condition may be reached in the first repetition and the third procedure therefore only be activated once. The first condition and the second condition may be equal.

The first condition and second condition determined from the determined parameters may be to stop imaging when detecting the end of the sample device. The first and/or second conditions may be to stop imaging when a desired volume of sample has been measured . The first and/or second conditions may be to stop imaging when detecting a particle in the sample, or a specific type a particle in the sample, or a specific number of particles in the sample. The first and/or second conditions may be to stop imaging when a predetermined number of translations has been made, or to stop when the translations has moved the sample a predetermined distance relative to the image acquisition device.

The number of times the sample is illuminated and the sample container is moved may in one embodiment depend on the number of particles found in the previous repetition of the fi rst procedure. I f th e re i s a ve ry l ow concentration of particles in the sample, the number of illuminations may be increased to a high level. This will reduce the number of times to invoke the advanced algorithms and thereby lowering the requirements to the hardware. In one embodiment, said processing comprises determining values of parameters relating to the relative concentration of different particles in said sample, such as the relative concentration of living and dead microorganisms in said sample.

I n one embod iment, said processing comprises determining values of parameters relating to the movements of living microorganisms in said sample.

In one embodiment of the invention, the sample device is moved a distance corresponding to approximately N times the diameter of the particles in the sample device, before the activation of the illumination device. N may be 1 ,2,3,4,5,6,7,8,10 or in principle any integer or non-integer number. When N is around 5 an image is exposed where a particle is imaged many times and may be used to extract information regarding the movement of the particle. Using a well-known translation of the sample device, it is possible to predict where a particle should be positioned in an image during next exposure. If the particle is not located at the expected position, the particle has moved, and it is possible to determine parameters of the movement, such as direction and speed.

In one embodiment of the invention the method for determining the value of at least one parameter characterising particles in a liquid sample, comprises: a) providing an optical detection assembly comprising an illuminating device, an image acquisition device and a shutter,

b) optionally providing a stimulating device for providing stimulation to said sample,

c) providing a sample device for holding said liquid sample,

d) optionally preparing at least a sub-volume of said liquid sample, e) providing said liquid sample to said sample device f) activating said shutter for exposing said image acquisition device to electromagnetic waves from said sample device,

g) optionally providing stimulation to said sample from said stimulating device

h) providing illumination from said illuminating device to said liquid sample comprised in said sample device

i) translating said sample device and said image acquisition device relative to each other, using a translation unit

j) repeating g), h) and i) at least one time

k) deactivating said shutter,

I) reading an image from said image acquisition device

m) processing sa id image to determ ine sa id value of at least one parameter characterising said particles in said liquid sample; and n) outputting said value of said parameter to an output device

The method may comprise repeating f) to n) until a predetermined first condition has been achieved.

The method may comprise repeating d) to o) until a predetermined second condition has been achieved. In one embod iment, i) comprises deactivating said shutter before said translation and activating said shutter after said translation.

In one embod iment, the predetermined first condition and said second condition is selected from the group of detecting the end of said sample device, sampling a desired volume of sample, detecting a particle, detecting a desired number of particles or achieving a predetermined number of translations.

Brief description of the drawings

Fig. 1 is a schematic view of an optical detection assembly (here an optical microscope) to be used when carrying out the method of the present invention, Fig. 2 shows a schematic view of the timing involved in optical sectioning,

Fig. 3 shows a schematic view of the timing involved in stroboscopic optical sectioning,

Fig. 4 shows a schematic diagram of an embodiment of the invention, Fig. 5 shows a schematic diagram of one embodiment of the invention, Fig. 6 shows a schematic diagram of one embodiment of the invention, Fig. 7 shows a theoretical example of a measurement Fig. 8 shows an image acquired using 20 illuminations of a sample, Fig. 9 shows a trace identification graph of the image shown as Fig. 8, Fig. 10 shows a slice of the image shown as Fig. 8, and

Fig. 1 1 shows a trace identification graph of the image shown as Fig. 10. The figures are schematic and may be simplified for clarity.

Further scope of applicability of the present invention will become apparent from the detailed description g iven hereinafter. However, it should be understood that the detailed description and specific examples, wh ile indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The invention is defined by the features of the independent claim(s). Preferred embodiments are defined in the dependent claims. Any reference numerals in the claims are intended to be non-limiting for their scope.

Some preferred embodiments have been shown in the foregoing, but it should be stressed that the invention is not limited to these, but may be embodied in other ways within the subject-matter defined in the following claims. Referring to Fig . 1 , an arrangement that may be applied in an optical microscope used as said optical detection assembly, will be described. A sample device 18 comprising a sample 12 is shown. Referring to the coordinate system 22 the sample device 18 has a first confinement 26 and a second confinement 28 confining the sample 12 in the Z-direction. The sample device 18 may extend beyond an image acquisition area 10 in the X-direction as well as in the Y-direction. Especially in the X-direction the sample device 18 may extend beyond the initial image acquisition area 10. The sample 12 may be confined in all three dimensions to make sure the sample 12 is at a non- moving state or steady state when performing the measurements.

An illuminating device 24 illuminates the sample 12 within the sample device 18. The first confinement 26 and the second confinement 28 are made of a material transparent for the electromagnetic waves from the illuminating device 24.

An optical detection assembly 15 comprises an image acquisition device 16 and an objective lens 14. The objective lens 14 comprises a first optical axis 13 and an object plane 17 perpendicular to the first optical axis 13. The image acquisition area 10 of the sample 12 is arranged to be coinciding with the object plane 17 of the objective lens 14. This enables a 2-dimensional image or 2-dimensional measurement of the image acquisition area 10 of the sample 12 to be imaged onto the image acquisition device 16.

In order to get the first confinement 26 and the second confinement 28 imaged onto the image acquisition device 16 and thereby comprised in the images, the image acquisition area 10 may intersect the first confinement 26 as well as the second confinement 28.

The sample device 18 may be moved relative to the optical detection assembly 15 using a translation unit 20 - in the figure symbolized by an arrow. The sample device 18 may be moved in the X direction in steps and for each step an image from the image acquisition device 16 is captured and stored in an image storing device for later use. The movement in the X d irection intersects the first optical axis 13. In one embodiment of the invention a translation unit for moving the sample device 18 in the Y-direction is used to enlarge the measurement volume. The size of the sample in the Y-direction may be sufficiently large to comprise the desired number of steps in that direction. The image acquisition area 10 may extend beyond the sample device 18, or at least extend beyond the first confinement 26 and the second confinement 28 of the sample device 18. The acquired images may comprise an image of the two confinements, and this information may be used to determine the height of the image acquisition area 10 and subsequently the distance between the two confinements.

In fig. 2 a schematic view of the timing involved in standard optical sectioning is shown. The figure comprises 4 synchronised timelines each indicating when there is activity of one of the parts in an optical microscope (the optical detection assembly) used for optical sectioning. Line no. 1 indicates when the processing device is active applying advanced algorithms to images acquired. Line no. 2 indicates activity in the camera (i.e. the image acquisition device). It is indicated using literal Έ' when the camera is open for Exposure, and indicated using literal 'RO' when the image is Read Out from the camera. In line 3 it is indicated using literal "T" when the translation unit is moving the sample device relative to the camera, while in line 4 it is indicated using literal "L" when the illumination is turned on.

As may be seen, the sequence is to turn the illumination on (i.e. said sample is illuminated) and to open the camera for exposure (i.e. to activate the shutter so that said image acquisition device is exposed to electromagnetic radiation from the sample device). After app. 30ms the illumination is turned off, and the camera exposure is ended. The camera may be open for exposure before or after the illumination is turned on and accordingly the camera may be closed for exposure before or after the illumination is turned off. If the optical microscope has been designed to eliminate external illumination, it will only be the period where both the camera is open for exposure and the illumination is turned on, that the camera will see an illuminated object. Then the image is read out from the camera and it is stored in the storing device. The read out time is app. 200ms. After the read out, the data is available to the processing device for applying advanced algorithms to determine parameters describing any particles found in the image. Depending on the number of particles and the complexity of the algorithms, the processing time is typically larger than 500ms. During the processing of the data, the translation device may be invoked to move the sample to the next image acquisition position . The translation time is typically app. 1 ms. After processing of the data, the sequence may be repeated starting at the opening of the camera for exposure. The overall sequence time for imaging one section of the sample device is at least 730ms and usually larger when more complex algorithms are applied.

The times indicated may of cause be varied, e.g. the illumination time may be varied depending on the type of illuminator used, the read-out time may be shorter or longer depending on the camera and other hardware in the setup, etc. but the overall picture of a long time for read out and calculation and a short time for exposure and translation will remain the same. In Fig. 3, a schematic view of the timing involved in utilising the method according to the present invention is shown. The figure comprises the same 4 synchronised timelines as shown in Fig. 2.

The sequence is initiated by opening a camera (an image acquisition device) for exposure (Line 2) by activating said shutter. During the exposure time, a sub-sequence is initiated. The sub-sequence comprises turning the illuminator on for app. 30ms and then off again and invoking the translation unit to translate the sample device and the camera relative to each other. The subsequence may be repeated a number of times, where after the camera is closed for exposure (i.e. the shutter is deactivated). After end of exposure, the image is read out to a storage device, and the processing device may begin processing the image. Immediately after end of reading out, the camera may be opened for exposure again and the sub-sequence of illuminating and translating may be continued. The processor may work simultaneously with the sub-sequence (said first procedure) of illuminating and translating.

The number of sub-sequences may be varied depending on the type of information required. When just counting particles in the sample, the number of sub-sequences may be high, such as 50 or more, and when there is a need of more information regarding the single particle detected, the number of subsequences may be reduced to less than 1 0. If for an example 1 0 subsequences are needed, the processing time may be calculated to 10 times (illumination + translation) + one time read out which equals app. 310ms + 200ms = 51 0ms (using the same times as for Fig . 2.). In th is case the processing time is app. 70% of the processing time as described in Fig. 2. What is more important, the scanned volume is 10 times higher! This gives an overall processing time for one section of a sample to app. 51 ms or 14 times faster than for Fig. 2.

If the number of sub-sequences is increased to 50, the processing time for one sequence is calculated to 50 sub-sequences + one read out time = 1750ms which equals 35ms for each section in the sample similar to 21 times faster than for Fig. 2. The best performance for getting parameters regarding the particles in the sample for as large a sample volume as possible is when the sub-sequence is stopped as soon as the calculations in the processing time has finished. Using the times indicated previously, the number of sub-sequences will therefore be 500ms divided by 31 ms which equals app. 16 illuminations per image. It should be emphasised that the indicated times for illumination, translation, read-out etc is for reference only - the times will vary depending on the hardware setup and the sample and illuminator used.

Fig. 4 shows a schematic diagram of an embodiment of the invention. The method comprises preparation of a sample for measurement. After the preparation, the integration time of the camera (image acquisition device) is started, and the sequence of illuminating and translating the sample may be executed a number of times, such as X times. Finally the integration is stopped, and the processing of data including reading out from the camera may begin. It is thus seen that the camera is only exposed once but using X illuminations. Fig. 5 shows a schematic diagram of one embodiment of the invention. The method is similar to the one shown in Fig. 4, but the sequence of exposing the camera (incl. illuminating and translating) is repeated Y times to get Y images each comprising X illuminations. The images are acquired from the same sample device, and may be acquired from separate parts of the sample device or from the same part.

Fig. 6 shows a schematic diagram of one embodiment. The method is similar to the one shown in Fig. 4, but the sequence of exposing the camera Y times is repeated Z times. For each of the Z times a new sample is prepared and the complete measurement method is repeated Y times each comprising X illuminations.

Figure 7 shows an example of a measurement using the method of the present invention. The figure shows four different points in time ti (100), t₂ (101 ), t₃ (102) and t (104) of a measurement. For each point in time the sample an imaging system is depicted together with the intermediate image formed on the camera (the image acquisition device) of the imaging system. The sample device 106 containing the sample, which in this example consists of two objects, a circle 1 10 and a triangle 1 1 1 , is imaged using an imaging system 104, and illuminated using a light source 109. The imaging system has a focus plane 107 intersecting the sample at an oblique angle. The two objects are depicted with their out of focus properties by to triangles with their main axis perpendicular to the focus plane of the imaging system 107. At time ti the light source is flashed, yielding an intermediate image to be formed on the camera of the imaging system 104. This intermediate image is showed in 108. The image depicts the two objects; the circle which will be in focus since the focus plane of the imaging system intersects the location of the circle in the sample, while the triangle will be out of focus since the focus plane does not intersect the triangle in the sample. A time t₂ the sample is moved along the direction 105 and the light source is flashed again. The resulting intermediate image is depicted in 1 12 - now the sample has moved so the circle is slightly out of focus, and the triangle is slightly more in focus than in 108. Notice that the objects from image 108 are still visible, since the imaging system is open for exposure during the whole period ti - t . In the following images 1 13 and 1 14 it is shown how the circle becomes more and more out of focus and eventually disappears from the image field, while the triangle is becoming in focus and out of focus again. At time t (103), the image may be read from the camera of the imaging system and analyzed. In Fig. 8 an image acquired using the method of the present invention is shown. The image is acquired using 18 illuminations of a sample comprising yeast. The translations between each illumination has been adjusted to fit to the size of the camera used as image acquisition device, i.e. a sample imaged in the lower part of the image in illumination no. 1 will be imaged in upper part of the image in illumination no. 18, and will be moved out of the image if an illumination (and translation) no. 19 had been used.

To reduce the accumulated background intensity, the image has been cleaned up. The translation of the sample between each illumination corresponds to the vertical dimension (columns) in the image. The image clearly shows a number of bright areas (dots) concentrated in a number of columns in the image. Each column of dots corresponds to a series of illumination of a single particle. For calculating the number of particles a vertical trace identification algorithm has been applied and the result is shown in Fig. 9. The vertical trace identification algorithm simply creates a sum of all the pixel intensity values for each column in the image. The upper trace in Fig. 9 shows the result of the identification algorithm for an un-cleaned image, and the lower trace shows the result of the identification algorithm for the cleaned-up image shown in Fig. 9.

By counting the spikes of the lower trace the number of particles in the part of the sample covered by the measurement may be determined. In this case 24 spikes are easily identified.

In Fig. 10 a slice of the image of Fig. 8 has been cut-out for further processing. The slice is made out of 40 columns of pixels centred on one of the particles identified in Fig. 9. A horizontal trace identification algorithm has been applied. The horizontal trace identification algorithm works similar to the vertical trace identification algorithm i.e. it creates the sum of pixel intensity values in the horizontal direction. The result of the horizontal trace identification algorithm is shown in Fig. 1 1 . The trace has a peak for each of the bright areas in the image, and the distance between the peaks are substantially equal . The distance between the peaks corresponds to the distance the translation of the sample relative to the camera. The column is further analysed for multiple particles by looking at the presence of multiple peaks within the travelled distance. The image of Fig . 10 may also be used for optical sectioning for determining details regarding the particle.

Claims

A m ethod for d eterm i n i ng th e va l u e of at l east on e pa ra m eter characterising particles in a liquid sample, comprising:

a) providing an optical detection assembly comprising an illuminating device, an image acquisition device and a shutter,

c) providing a sample device for holding said liquid sample,

d) optionally preparing at least a sub-volume of said liquid sample, e) providing said liquid sample to said sample device

f) activating said shutter for exposing said image acquisition device to electromagnetic waves from said sample device,

g) optionally providing stimulation to said sample from said stimulating device

j) repeating g), h) and i) at least one time

k) deactivating said shutter,

I) reading an image from said image acquisition device

m) processing sa id image to determ ine sa id value of at least one parameter characterising said particles in said liquid sample

n) outputting said value of said parameter to an output device

The method according to claim 1 , further comprising

o) Repeating f) to n) until a predetermined first condition has been achieved. 3. The method according to claim 2, further comprising

p) Repeating d) to o) until a predetermined second condition has been achieved.

4. The method according to any of claims 1 to 3, wherein said preparing of said l iqu id sample is selected from the group of tagging, staining, haemolysing, and/or filtering said particles. 5. The method according to any of claims 1 to 4, wherein said illumination comprises electromagnetic radiation, said electromagnetic radiation preferably being in the range of about 200 nm to about 1 100 nm, such as in the range of about 300 nm to about 800 nm, in the range of about 400 nm to about 700 nm, in the range of about 450 nm to about 600 nm, in the range of about 495nm to about 570nm.

6. The method according to claim 5, wherein said electromagnetic radiation is pulsed. 7. The method according to claim 5, wherein said electromagnetic radiation is intensity multiplexed.

8. The method according to any of claims 1 to 7, wherein said illumination comprises wavelength multiplexing of at least two different electromagnetic waves.

9. The method according to any of claims 1 to 8, wherein said illumination comprises photoluminescence or said illumination triggers the emission of photoluminescence.

10. The method according to any of claims 1 to 9, wherein said stimulation is selected from the group of applying an electric field, a magnetic field, an electromagnetic field or an acoustic wave to said sample. 1 1 .The method according to any of claims 1 to 10, wherein said translation comprises rotating said sample device relative to said image acquisition device.

12. The method according to any of claims 1 to 1 1 , wherein said translation comprises a linear translation of said sample device relative to said image acquisition device. 13. The method according to any of claims 1 to 12, wherein said translation comprises tilting said sample device relative to said image acquisition device.

14. The method according to any of claims 1 to 13, wherein said processing comprises optical sectioning of said sample.

15. The method according to any of claims 1 to 14, wherein said processing comprises determining values of parameters relating to the concentration of particles in said sample.

16. The method according to any of claims 1 to 15, wherein said processing comprises determ ining values of parameters relating to the relative concentration of different particles in said sample. 17. The method according to any of claims 1 to 16, wherein said processing comprises determ ining values of parameters relating to the relative concentration of living and dead microorganisms in said sample.

18. The method according to any of claims 1 to 17, wherein said processing comprises determining values of parameters relating to the movements of living microorganisms in said sample.

19. The method according to any of claims 1 to 18, wherein said output of said value of said parameters comprises writing said parameters to a display.

20. The method according to any of claims 1 to 19, wherein said output of said value of said parameters comprises writing said parameters to an external device.

21.The method according to any of claims 1 to 20, wherein said output of said value of said parameters comprises applying said value to a later part of the method. 22. The method according to any of claims 1 to 21, wherein said predetermined first condition and said second condition is selected from the group of detecting the end of said sample device, sampling a desired volume of sample, detecting a particle, detecting a desired number of particles or achieving a predetermined number of translations.

23. The method according to any of claims 1 to 22, wherein i) comprise deactivating said shutter before said translation and activating said shutter after said translation.