Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
  • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• • H—ELECTRICITY
  • H04—ELECTRIC COMMUNICATION TECHNIQUE
  • H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
  • H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
  • H04N23/70—Circuitry for compensating brightness variation in the scene
  • H04N23/72—Combination of two or more compensation controls
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T5/00—Image enhancement or restoration
  • G06T5/40—Image enhancement or restoration using histogram techniques
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T5/00—Image enhancement or restoration
  • G06T5/90—Dynamic range modification of images or parts thereof
  • G06T5/92—Dynamic range modification of images or parts thereof based on global image properties
• • 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
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N2015/0038—Investigating nanoparticles
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/10—Image acquisition modality
  • G06T2207/10056—Microscopic image

Definitions

• This presentation relates to the field of microscopy applied to the visualization of natural mixtures, in the sense of unfiltered, of particles, these natural mixtures comprising in an unpredictable way, particles resolved by an optical microscope or microparticles, and unresolved particles. by an optical microscope or nanoparticles.
• the present disclosure relates to the visualization, via a microscope and a camera, of such mixtures, composed in particular of biological particles, constituting phase objects, in particular in Brownian motion in a liquid medium, in particular aqueous.
• This presentation also concerns the visualization of mixtures containing micro-objects or nano-objects of intensity, attenuating the light which they transmit or reflect or absorb, such as for example gold microparticles and gold nanoparticles, especially in Brownian motion in a liquid medium.
• This presentation thus relates, in general, to the visualization of mixtures of phase or intensity objects, micro-objects or nano-objects, in particular subjected to Brownian motion, but also the visualization of particles of all sizes which may be stationary. .
• the prior art knows the resolved optical microscopy, in bright field ("brigthfield microscopy" in English) which makes it possible to form an image in intensity of an object resolved by the microscope, that is to say whose lateral dimension is greater than the lateral resolution limit of the microscope, for an illumination light source (in particular thermal source, light-emitting diode, laser, etc.) collected by the microscope.
• an illumination light source in particular thermal source, light-emitting diode, laser, etc.
• unresolved particles are in general invisible and therefore cannot be visualized at the same time as resolved particles.
• the prior art also knows, for example from the document published under the number FR3027107 (BOCCARA), a method and a device for optical interference detection of nanoparticles in an unnatural fluid sample, because filtered beforehand to contain only unresolved particles. .
• the prior art also knows operations for increasing the contrast of images and in particular images of phase objects, resolved, whether they are in Brownian motion or not.
• these operations of increasing the contrast of an image consist of transformations of a histogram of the image.
• histogram transformations modify images by processing each pixel independently. These transformations appear in almost all image processing and analysis processes.
• these transformations are common for digital images taken by a camera recording the images in the form of pixels each assigned a gray level or several gray levels associated with colors: in pre-processing to normalize the image, before recording, or in post-processing to improve visualization, after recording.
• Normalization of an image Extension of the histogram of an image, before or after its recording, so that this histogram presents gray levels extending over the entire range of gray levels of the sensor used for the recording or screen used for viewing, or the eye of an observer, to maximize contrast for a human observer.
• Brown motion Spontaneous movement of particles in a liquid or viscous medium, caused by thermal agitation and preventing the sedimentation of particles under the effect of gravity.
• Robotscope Device for limiting the duration of an image acquisition by a camera and allowing movement to be frozen; a strobe image is heard as an image short enough in duration to freeze a particular movement.
• Digital contrast enhancement Set of digital methods making it possible to increase the number of details visible in an image, by a human observer or a system capable of implementing such methods, in particular by normalization of the image and making it possible to correct overexposure in a digital image.
• the present disclosure relates to a method for obtaining an image, using a microscope, of a set of particles, conjugated from a digital camera via the microscope, the set of particles comprising nanoparticles not resolved by the microscope and microparticles resolved by the microscope, in which the particles are illuminated by a light source, in which the light source illuminates the digital camera through the microscope.
• This process includes the following steps:
• the method may include the following characteristics, considered alone or combined with one another (except for major technical incompatibility):
• nanoparticles include viruses and microparticles include aggregates of viruses
• the digital correction of the overexposure is obtained by transforming the histogram of the image
• the transformation of the image histogram is an extension of the image histogram
• the transformation of the histogram of the image is a translation of the histogram of the image
• the transformation of the image histogram includes a translation of the image histogram and an extension of the image histogram
• the extension of the histogram of the image is non-linear.
• the present disclosure also relates to a device for the implementation of the above method, in which the light source is of sufficient light intensity to overexpose an image recorded by the digital camera via the microscope, showing variations light intensity for a nanoparticle conjugated from the digital camera through the microscope.
• the device includes digital means for correcting the exposure of an image recorded by the camera.
• FIG 1 represents an example of a device for the observation of particles.
• the device of FIG. 1 comprises a microscope 1; a light source 2 illuminating the field of the microscope; a camera 3.
• the camera is arranged in a conjugate image plane of the object focal plane of the microscope 1 and collects direct light from the illumination source 2 through the microscope to form a bright field image.
• a strobe (not shown in this figure) or any other means to limit the acquisition time and digital image processing means for improving the contrast of the acquired image can be provided.
• the bright field configuration is carried out so as to collect on the one hand the variations in intensity, due to the resolved phase objects located in the field of the microscope and on the other hand, to collect the light scattered by the non-phase objects. solved also located in the field of the microscope.
• Modulation of direct light by resolved objects is used to visualize resolved objects and modulation due to scattered light interference from unresolved objects in the vicinity of the conjugate object focal plane of the camera is used to visualize unresolved objects .
• the device shown therefore makes it possible to visualize on the same image and in the same frame of reference resolved and unresolved objects located in a section extending over the depth of field of the microscope, from the object focal plane of the microscope, or more generally from a section extending through the depth of field of the microscope from the object plane conjugated to the plane of the camera by the microscope.
• the camera is assumed to be planar, although a camera conforming to the field curvature of the microscope can be envisioned without departing from the teaching of this paper.
• the device comprises a microscope 1 or optical system, a light source 2 or lighting source or source and a camera 3.
• the invention thus comprises the illumination source 2 collected by the microscope 1 and the camera 3.
• An object to be observed is placed in the object space of the microscope 1 and is thus illuminated and imaged on the camera 3 in a bright field.
• a collimated illumination of the source 2 is preferred to maximize the contrast of the interference signals observed for conjugated nanoparticles of the camera 3.
• a source 2 of the highest possible light power or in any case sufficiently powerful to fill the capacity or well depth of the camera during the exposure time is preferred. This may be a source 2 with the lowest possible bandwidth to wavelength.
• source 2 a visible light-emitting diode (for example an imperial blue LED from the Thorlabs brand marketed under the reference M405LP1) with a wavelength centered on 405nm.
• a visible light-emitting diode for example an imperial blue LED from the Thorlabs brand marketed under the reference M405LP1
• a white light-emitting diode or white LED for example an LED of the Thorlabs brand sold under the reference MWWHLP1
• NPs nanoparticles
• NA numerical aperture
• the lighting conditions can either be defined by the propagation in free space of the source, or defined by a condenser making it possible to change the lighting conditions (Kohler lighting, critical lighting or any other type of lighting, in particular collimated) .
• the magnification of the optical system, the size of the pixels of the camera 3 and the acquisition rate of the camera 3, in number of images per second, are chosen so that the displacement, between two images acquired or recorded, a nanoparticle is quantifiable.
• a bright field image is, in all cases, collected via the microscope 1, on a camera 3 of great dynamic range, ie of great depth of well, by example a CMOS camera of the Photon Focus brand marketed under the reference PHF-MV-D1024E-160-CL-12.
• CMOS camera of the Photon Focus brand marketed under the reference PHF-MV-D1024E-160-CL-12.
• Another camera having more pixels and a lesser well depth can also be used by grouping (in English “binning”) pixels.
• the camera 3 is preferably arranged in a conjugate image plane of the object focal plane of the microscope 1, according to an optical configuration for which the optical correction of the aberrations is optimized and the limit imposed by the diffraction is generally reached.
• the microscope 1 forms a bright field image of a sample containing resolved particles and laterally unresolved particles, for example an unfiltered natural sample.
• a sample containing resolved particles and laterally unresolved particles for example an unfiltered natural sample.
• the resolution limit is in a known manner close to 200nm.
• a test sample can be used in which both resolved and unresolved immobile particles are present.
• a sample consisting of natural unfiltered water and containing a priori viruses and virus aggregates, forming a population of phase objects, with dimensions between 10 nm and 10 ⁇ m can in particular be visualized with the invention.
• Any liquid sample containing biological particles can also be observed with the device and the method of the invention.
• the invention is particularly useful in general for observing natural media containing phase objects in Brownian motion.
• an acquisition rate of the camera of the order of approximately 130 images per second or more and on the other hand, on each image or strobe image, taken by the camera. camera, apply a contrast increase operation suitable for overexposed images (whose exposure was maximized without saturating the image) obtained with a bright field microscope. Any increase in the rate of the camera compatible with an imposed signal-to-noise ratio is therefore favorable to the visualization of increasingly small unresolved objects, in the presence of resolved objects.
• the acquisition rate is in all cases chosen as high as possible to allow the Brownian motion of the particles to be followed as well as possible, while completely filling the wells of the camera in order to minimize shot noise, which is a major source of noise in certain embodiments.
• the operation of increasing the contrast can be applied in real time if the image processing means allow it or a posteriori on a sequence of stroboscopic images, recorded in an image memory.
• An advantage of the invention is that the representations of the two types of particles by interferometric imaging and intensity imaging share the same spatial reference, having been obtained strictly with the same device in bright field. Subject to mechanically stabilizing the distance between the microscope and the object plane conjugated with the camera by the microscope, there is therefore a quantitative means of imaging on particles of dimensions smaller or greater than the resolution limit of the microscope, of sectioned way.
• An advantage of the invention is to be able to obtain the two types of imaging with the same instrument and therefore to naturally share the same spatial reference for the two types of imaging, interferometry between scattered and direct light and conventional intensity imaging, carried out automatically by the microscope according to the particle size. It is therefore possible not only to make distance measurements between two resolved particles or between two unresolved particles but also to make distance measurements between a resolved particle and an unresolved particle, reliably, thanks to the method of embodiment described above.
• This embodiment therefore makes it possible to obtain a sectioned imaging of resolved and unresolved natural particles, in a section of thickness equal to the depth of field of the microscope.
• the duration can be chosen to freeze the Brownian motion of the fastest moving particles, i.e. to ensure that the movement of the smallest particles to be imaged is not resolved by the microscope in the duration of acquisition of the strobe image.
• a minimum size of the particles that it is possible to image with the device and the method of this embodiment, can also be calculated, that is to say the power of interferometric resolution. of the device of the invention.
• the adjustment of the lighting in the image satisfies the condition of maximizing or overexposing to the maximum, the brightfield under the constraints of not saturating the interferometric image nor the intensity image, which is a classic adjustment in microscopy .
• This adjustment can be made on the strobe image viewed on a screen by a human observer.
• the illumination source can be a source of any type such as a heat source of the lamp type, a light-emitting diode (LED) or a laser.
• the resulting illumination for the microscope can be spatially consistent or inconsistent, temporally consistent or inconsistent.
• the device of this embodiment therefore makes it possible to carry out with the same microscope in a bright field, a common interferometric (in diffused and direct light) and intensity (in direct light attenuation) imaging.
• a histogram extension or expansion also known as image normalization
• the stroboscopic image being due to its bright field particularly saturated towards the whites and not revealing to the naked eye, in general no visible detail.
• the distribution over the entire range of the image levels of its histogram is thus particularly effective in increasing its contrast. It should be noted that this situation corresponds in classical or resolved microscopy on phase objects to a defective microscopy experiment, in which the lighting conditions have not been optimized, and in which a wide range of gray levels them. lower camera is unnecessary.
• non-linear normalization operations of the histogram of the stroboscopic image, depending on the type of particles observed.
• intensities of the resolved / unresolved particles generally change from sample to sample
• non-linear normalization may be sample-specific. Any other method making it possible to remove a background from the stroboscopic image and to improve its contrast can also be used with the invention.
• the invention is particularly suitable for the visualization, by an observer, of populations of particles, in the case where the size of each particle of the population is between 10nm and 10 microns or even comprises both unresolved particles and particles. particles resolved by an optical microscope, operating in the visible.
• a “histogram” refers to the distribution of light intensities or "gray levels" in a digital image.
• the invention is capable of industrial application or usable in the field of microscopy.

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• Optics & Photonics (AREA)
• Computer Vision & Pattern Recognition (AREA)
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• Signal Processing (AREA)
• Microscoopes, Condenser (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

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• 2019
  • 2019-03-13 FR FR1902589A patent/FR3093807B1/fr active Active
• 2020
  • 2020-03-10 WO PCT/EP2020/056382 patent/WO2020182828A1/fr unknown
  • 2020-03-10 CN CN202080020662.9A patent/CN113692545B/zh active Active
  • 2020-03-10 US US17/432,048 patent/US20220247909A1/en not_active Abandoned
  • 2020-03-10 JP JP2021555079A patent/JP2022524464A/ja active Pending
  • 2020-03-10 EP EP20714876.8A patent/EP3938831A1/de not_active Withdrawn

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