Classifications

• • 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
  • G01N15/1429—Signal processing
  • G01N15/1433—Signal processing using image recognition
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
  • G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample
• • 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
  • G01N2015/1481—Optical analysis of particles within droplets

Definitions

• the field of the invention is that of devices and methods of detection in contact imaging for diagnosis and biological analysis.
• optical imaging for biological diagnosis if we restrict our to specific detection, excluding contact imaging, two types of methods are mainly used. These are "flow cytometry" and fluorescence molecular imaging.
• Flow cytometry is a powerful technique that can count, characterize and classify the different cells that cut the light beam of a laser. By analyzing the diffraction patterns, it is possible to determine the dimensions of the cell. Fluorescence measurements also make it possible to distinguish the different families of bacteria.
• the disadvantage of this technique is that it requires expensive and complex instrumentation.
• Another disadvantage is a relatively weak solid scanning angle, limiting the field of investigation.
• Fluorescence molecular imaging is a widely used method in biological diagnosis because of its very high efficiency.
• the sensitivity of the fluorescence measurements is that of the discrete events: using fluorescent markers, it is possible to detect individual molecules in microscopy. To obtain good results, it is necessary to perfectly separate the energy that excites the fluorescent molecules called “excitation” energy from that emitted by them, called “emission” energy. If excellent filters exist today, they impose a conditioning of the light beams, in particular a low opening of the beams. Consequently, the optical systems for implementing this method are complex and bulky. Fluorescence imaging also requires the prior addition of a fluorophore in the medium to be analyzed, which makes the process invasive.
• a contact imaging device is shown schematically in FIG.
• This device comprises a lighting source 1, which can be a source of small size, for example a light-emitting diode, a diaphragm 2 for limiting the opening of the source and an imager or sensor 3 which can be a matrix of photosites, "CCD” type matrix, meaning "Charge-Coupled Device” or "C-MOS”, meaning "Complementary Metal Oxide Semiconductor".
• Such imagers generally include microlenses associated with each photosite.
• the diaphragm 2 is not essential, but its use is advantageous. It is inserted between this matrix 3 and the light source 1, a transparent slide 4 of the microscope slide type carrying the object of the study 5.
• This object is a solution comprising micrometric particles, these particles possibly being biological particles such as cells or bacteria or other particles such as micro-beads.
• the analyzed drop rests on the transparent blade 4 and its meniscus is in contact with the ambient gas, this gas can be air.
• the matrix 3 is connected to a display and / or image processing system, not shown in FIG. 1.
• the distance between the diode 1 of the slide object 4 is preferably greater than 1 centimeter, and may be, for example, a few cm, typically between 2 and 5 centimeters.
• the distance separating the object from the surface of the sensor is between 0, 1 and 2 millimeters.
• the blade is made of a transparent material such as silica, quartz, and its thickness varies between a few tens of micrometers and 1 millimeter.
• This very simple device, without magnification optics can be, in some cases, an alternative to conventional methods of optical counting such as flow cytometry, high resolution optical microscopy or fluorescence molecular imaging techniques.
• FIG. 2 thus represents the signal S obtained on an axis x of the sensor passing through the center of a particle P.
• the signal S represents here the gray level measured by different pixels along the ordinate axis, the pixels constituting the X axis: This is a profile. We see that the signal-to-noise ratio is very low, barely enough to allow the detection of the particle.
• the method according to the invention does not have these disadvantages. It makes it possible, in fact, in a contact imaging device, to detect particles of micrometric size without using high sensitivity sensors.
• the main characteristic of the process according to the invention is to carry out measurement or measurements during or after the evaporation phase of the drop of liquid in which the micrometric particles (bacteria, cells, beads, etc.) to be detected are located.
• the drop is not arranged between two plates.
• the drop must be in contact with a gas, for example air, so that evaporation can take place. It has been found that the detection was very effective during or following evaporation.
• the subject of the invention is a method for the optical detection of particles or micrometric or sub-micrometric size organisms by means of a contact imaging device, said particles or organisms being immersed in a drop of liquid, the detection being carried out by means of a matrix of photosensitive cells or photosites, characterized in that said method comprises at least a first detection step carried out during the evaporation of the drop of liquid.
• the drop may be placed either on a blade, located in contact or at a short distance from the imager. The drop can also be placed directly in contact with the imager.
• said method comprises at least a second detection step carried out after the evaporation of the drop of liquid.
• the detection is carried out at the periphery of the drop, at the interface separating the drop from its evaporated portion.
• said method comprises a succession of detection steps performed at regular time intervals during and / or after the evaporation of the drop, each detection step making it possible to measure the distribution of particles or organisms in a plane. given, said plane being at a distance from the photosite matrix depending on the evaporation time, all of said distributions of particles or organisms obtained to reconstruct a three-dimensional distribution of particles or organisms in the initial drop before evaporation.
• the liquid is water or, when the particles to be detected are bacteria, a biological buffer, for example "TRIS", an abbreviation for trishydroxymethylaminomethane.
• the liquid comprises a wetting agent, for example TWEEN 20, abbreviation of polyoxyethylene (20) sorbitan monolaurate.
• the support which is either a transparent plate or the surface of the sensor carrying the drop of liquid can be functionalized. It can also be made hydrophilic. It can also be cooled below room temperature.
• Figure 1 shows a device in contact imaging
• Figure 2 shows the detection signal obtained with a low sensitivity photosensitive matrix
• FIG. 3 represents the principle of the detection method according to the invention.
• Fig. 4 shows a detection signal obtained with a low sensitivity photosensitive matrix using the method according to the invention
• FIG. 5 represents successive detection steps implementing the method according to the invention.
• Figures 6 represent representations of observations of bacteria in a drop of saline buffer, as well as in a film resulting from the evaporation of the drop.
• the profiles (6a and 6d) represent the intensity detected as a function of the position along a horizontal axis x;
• FIGS. 7 represent representations of observations of bacteria in a drop of saline buffer, before and after evaporation, the drop being deposited on a hydrophilic or hydrophobic slide.
• the curves 7a and 7c represent the intensity detected as a function of time, at a point situated at the level of the drop;
• FIG. 8 represents the image of a 500 nm diameter polystyrene particle, as well as a horizontal profile along an X axis of this image.
• the method according to the invention makes it possible to considerably increase the signal-to-noise ratio and consequently the detection efficiency.
• the detection systems according to the prior art detect microparticles or microorganisms within a drop of liquid. However, as the particles are immersed, they are hardly discernible, except to use a sensor with high sensitivity.
• the left-hand view of FIG. 3 represents, at time T, the image resulting from a partial view of a drop G comprising particles P. This image is derived from a sensor of an imaging device of contact as previously described. The particles are not discernible if the sensor has a sensitivity too low.
• FIG. 3 represents, at the time ⁇ + ⁇ , the image of a partial view of the preceding drop G partially partially evaporated.
• the particles P appear clearly in the zone Z of the drop which has evaporated.
• FIG. 4 thus represents the profile of the signal S obtained on an axis x of the sensor passing through the center of a particle P. It can be seen that the signal-to-noise ratio is now sufficient to allow the detection of the particle. The signal to noise ratio shown is sufficient to consider detection of a large part of the immersed cells. It is of the order of a few tens of%. This method is reproducible.
• the method according to the invention is therefore very simple to implement. It essentially consists of taking measurements during the evaporation of the drop of liquid. A measurement or a detection step essentially consists in recording the signals detected by the photosites of the imager of the device in contact imaging and analyzing their amplitude. The physical explanation of this contrast enhancement of illuminated particles during evaporation is complex.
• this increase can be attributed to the formation of a thin residual film covering the particle, this film remaining some time after the evaporation of the drop.
• this film is ephemeral and disappears in a few seconds, or even less, or this film is durable and remains for several seconds, or even a few tens of seconds or minutes. It seems that the presence of this residual film, covering the particle, acts as microlens and thus allows a detection of bacteria with a surprisingly high signal-to-noise ratio. Indeed, the intensity of the signal is maximum when the particle is at the level of the evaporation line and triple that at the meniscus of the drop.
• Figure 5 illustrates this principle. This figure shows at three different times the evaporation of a drop comprising particles, on the one hand a three-dimensional view of a portion of the drop and on the other hand the corresponding image from the matrix detector. of the contact imaging device. It should be noted that the three-dimensional views are not representative of the actual dimensions of the drops and their distribution. At time To, only two particles emerge from the drop and are identifiable on the image coming from the detector. They are represented by white circles. At time T1, after 30 seconds of evaporation, a larger number of particles emerge and are identifiable on the image resulting from the detector.
• the evaporation time of a drop lasts from a few seconds to a few tens of seconds, the volume of the drop being between 1 microliter and 20 microliter, or even beyond.
• TRIS Trishydroxymethylaminomethane
• purified water can be used.
• TRIS has the advantage of being a saline solution for preserving bacteria for a few days. It is therefore commonly used as a biological buffer. It is preferable that the slide carrying the drop is not too hydrophobic, or even hydrophilic, as will be shown later.
• the process works with different types of particles. Examples are Silanol beads 1 micrometer in diameter, Latex beads 1 micrometer in diameter and bacteria. Conclusive tests have been carried out on bacteria of the Escherichia Coli or Bacillus Subtilus type. It works in a wide range of concentrations, from one particle per drop to one hundred thousand particles per drop.
• the lighting from the source is as homogeneous as possible.
• the surface of the meniscus is illuminated at a substantially equal intensity at each point.
• the illumination has a certain spatial coherence, that is to say that the diaphragm disposed in front of the source is of small dimensions. For example, a diaphragm of 100 micrometers in diameter can be used.
• the acquisition device is simple and inexpensive since it only includes an electronic card for acquiring digital images from photosites of a low cost "CCD” type sensor or "CMOS " for a “webcam” type camera. ", A light emitting diode, a diaphragm and a glass slide.
• the pixels or photosites of the sensor may have average dimensions, of the order of two to ten microns. These sensors have a price much lower than that of high sensitivity sensors where the pixel size does not exceed two microns.
• the light source 1 is a light emitting diode of power 1 .7 W emitting at a wavelength centered around 555 nm (Model KI Luxeon I I I brand Luxeon, registered trademark). It is placed at a distance of 10 cm above the substrate 4.
• the latter is a microscope slide made of glass with a surface area of 70 mm ⁇ 25 mm and a thickness of 0.1 mm.
• the sensor 3 is a CMOS sensor comprising 800 x 600 pixels, the dynamic range being 8 bits. The dimension of each pixel is 3 ⁇ x 3 ⁇ . This sensor has been removed from a webcam (model Talkcam 200 brand V gear). In order to place the substrate 4 as close as possible to the photodetector 3, the plastic membrane covering this detector has been removed.
• a sample, with a volume of approximately 1 ⁇ l, of a liquid sample is deposited on the substrate 4 facing the sensor 3. drop evaporate, lasting a few minutes.
• the solution used is a 10 mM TRIS HCI saline buffer with a pH of 8.
• FIGS. 6a, 6b and 6c respectively show a horizontal profile P1, an image and a three-dimensional representation of said image obtained when the sample is illuminated by a spatially coherent light beam by placing a diaphragm, with a diameter of 100 ⁇ , between the light source and the liquid sample. In this way, we try to observe a diffraction task produced by the bacteria present in the drop.
• the volume of the drop is of the order of ⁇ .
• Figures 6d, 6e and 6f respectively show a horizontal profile PI, an image and a three-dimensional representation of the said image obtained when the sample is illuminated by the same source, without the diaphragm.
• the substrate being sufficiently hydrophilic, when the drop evaporates, there remains a wetting film covering the substrate and the bacteria deposited on the latter. This film remains all the longer as the substrate is hydrophilic, and the solution of the sample is wetting and has a low surface tension: it is called the formation of an ultra-wetting film.
• the configuration implemented during the test is favorable, since a hydrophilic blade is used, in this case an ultrasonically washed glass and an ethanol rinsing and the previously described biological buffer made wetting by the addition of a wetting agent (0.1% TWEEN). It can be seen that according to such a configuration, as illustrated in FIGS. 7, the ultra-wetting film formed following the evaporation of the gout persists for a long time: a few minutes or even hours.
• Figures 6d, 6e and 6f are compared respectively to Figures 6a, 6b and 6c. It can be seen that the signal-to-noise ratio obtained by producing a contact image of the bacterium covered with a wetting film is increased by a factor of 20 with respect to a contact image of the bacteria bathing in the drop. This allows unambiguous detection of this bacterium.
• a film resulting from the evaporation of the solution, is one of the key points of the process according to the invention.
• Such a film plays the role of one or more microlenses formed above the bacterium. This explains that it can detect the latter with a signal to noise ratio as high, of the order of 45.
• the signal-to-noise ratio is defined as follows:
• I amplitude of each pixel of the image
• hydrophobic and hydrophilic means that the angle of a drop in contact with this substrate is respectively less than and greater than 90 °, this definition being commonly accepted.
• Figures 7a and 7b illustrate the results obtained when the substrate is hydrophobic.
• Figure 7a shows the temporal evolution of a horizontal profile of the image of the bacterium shown in Figure 7b, the profile passing through the center of the image of the bacterium.
• Figure 7b-1 the drop evaporates and no significant signal is detected.
• An increase in the signal is observed, corresponding to the effect of the formation of this film on the bacterium.
• the film disappears rapidly, which explains the rapid decrease in signal intensity observed for t> 48.5s (FIG. 7b-3).
• Figures 7c and 7d illustrate the results obtained when the substrate supporting the drop is hydrophilic.
• Figure 7c shows the temporal evolution of a horizontal profile P of the image of the bacterium shown in Figure 7d, the profile passing through the center of the image of the bacterium.
• the substrate being hydrophilic the film remains for a long time on the surface of the substrate and the bacterium (FIG. 7d-3).
• a signal of significant intensity is observed over a much longer duration than in the previous case, the duration being here of a few tens of seconds.
• the method according to the invention makes it possible to detect a bacterium in solution at the moment when the drop containing the bacterium evaporates, and in particular when the drop / external environment interface is at the level of the bacterium. that is, when the bacterium is covered only with a thin film.
• the formation of the film is either ephemeral (duration less than 1 or 2s), or durable. The more the solution is wetting on the substrate considered, the more the formation of the film is durable. By durable, one hears a few tens of seconds, even a few minutes or several hours.
• a wetting agent in the solution.
• An agent of this type is, for example, the previously defined TWEEN 20, the concentration (by volume) of which is 0.1%.
• Tris HCl PH 8 diluted to 10 mM in distilled water, particularly satisfactory results having been obtained for this type of buffer. Tris is abbreviation for trishydroxymethylaminomethane, or 2-amino-2-hydroxymethyl-1,3-propanediol.
• buffers can be used. When it is desired to identify bacteria, biological buffers keeping alive bacteria will be preferred.
• a wetting agent such as tween, is generally very useful and allows the formation of a more durable microfilm.
• the volume concentration of such an agent is for example of the order of a few hundredths of a% to a few%, preferably a few hundredths of a percentage to a few tenths of%.
• PBS Phosphate Buffer Saline
• distilled water apart from biological use, distilled water.
• FIG. 8 represents an image (FIG. 8a) and a profile PI (FIG. 8b) of the observation of a polystyrene ball, covered with an ultra-wetting film formed consecutively to evaporation of a drop of the same saline buffer as that described in the previous examples.
• the diameter of this ball is 500 nm.
• the signal-to-noise ratio of the detection remains high (of the order of 20).
• the support of the drop below ambient temperature, the evaporation of the drop is slowed down, and the residual film is more durable. For example, when the ambient temperature is 20 °, the support can be brought to a temperature of between 5 to 10 °. It is also possible to use a cooled imager, which makes it possible to increase the signal-to-noise ratio of the detected signal.
• Another example is the bacteriological diagnosis of urinary tract infections.
• the bacteriological examination of the urine makes it possible to affirm the urinary infection when it shows the presence of a monomicrobial bacteriuria (a single species of bacteria) with a number of colonies greater than 100 bacteria / ⁇ , associated with a leucocyturia (presence of white blood cells in urine) greater than 10 leukocytes / ⁇ or pyuria.

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