阅读说明：本技术 用于观察微米颗粒和纳米颗粒的设备及方法 (Apparatus and method for observing microparticles and nanoparticles ) 是由 M·格飞 L·塔里尼 于 2020-03-10 设计创作，主要内容包括：本发明涉及一种使用显微镜(1)获得颗粒集合的图像的方法,颗粒集合经由显微镜(1)与数码相机(3)共轭,颗粒集合包括未由显微镜(1)分辨出的纳米颗粒以及由显微镜(1)分辨出的微米颗粒,其中颗粒由光源(2)照射,其中光源(2)经由显微镜(1)照射数码相机(3)。该方法包括以下步骤：-通过光源(2)使由数码相机(3)记录的图像过度曝光,以使得对于观察者而言,在所记录的图像中显示关于纳米颗粒的光强度变化；-数字校正所记录的图像的过度曝光,以使得对于观察者而言,在所记录的图像中显示关于纳米颗粒的光强度变化的同时显示关于微米颗粒的光强度变化。(The invention relates to a method for obtaining an image of a collection of particles using a microscope (1), the collection of particles being conjugated to a digital camera (3) via the microscope (1), the collection of particles comprising nanoparticles not resolved by the microscope (1) and microparticles resolved by the microscope (1), wherein the particles are illuminated by a light source (2), wherein the light source (2) illuminates the digital camera (3) via the microscope (1). The method comprises the following steps: -overexposing the image recorded by the digital camera (3) by means of the light source (2) such that the light intensity variations with respect to the nanoparticles are displayed in the recorded image for the observer; -digitally correcting the overexposure of the recorded image so that, for the observer, the light intensity variations with respect to the nanoparticles are displayed at the same time as the light intensity variations with respect to the microparticles in the recorded image.)

1. A method of obtaining an image of a collection of particles using a microscope (1), the collection of particles being conjugated with a digital camera (3) via the microscope (1), the collection of particles comprising nanoparticles not resolved by the microscope (1) and microparticles resolved by the microscope (1), wherein the particles are illuminated by a light source (2), and wherein the light source (2) illuminates the digital camera (3) via the microscope (1), the method comprising the steps of:

overexposing, by means of the light source (2), the image recorded by the digital camera (3) to display to the observer, in the recorded image, the light intensity variations with respect to the nanoparticles; and is

Digitally correcting the overexposure of the recorded image to show the light intensity variation with respect to the microparticles at the same time as the light intensity variation with respect to the nanoparticles is shown to the observer in the recorded image.

2. The method of claim 1, wherein the nanoparticles comprise viruses, and wherein the microparticles comprise viral aggregates.

3. The method of claim 1, wherein the digital correction of overexposure is obtained via a transformation of a histogram of the image.

4. The method of claim 3, wherein the transformation of the histogram of the image is a stretching of the histogram of the image.

5. The method of claim 3, wherein the transformation of the histogram of the image is a translation of the histogram of the image.

6. The method of claim 4, wherein the stretching of the histogram of the image is linear.

7. The method of claim 4, wherein the stretching of the histogram of the image is non-linear.

8. The method of claim 3, wherein the transformation of the histogram of the image comprises a stretching of the histogram of the image and a translation of the histogram of the image.

9. An imaging device comprising a microscope (1), a light source (2) and a digital camera (3), the imaging device being configured to obtain an image of a set of particles using the microscope (1), the set of particles being conjugated with the digital camera (3) via the microscope (1), the set of particles comprising nanoparticles not resolved by the microscope (1) and microparticles resolved by the microscope (1),

wherein the particles are illuminated by the light source (2),

wherein the light source (2) illuminates the digital camera (3) via the microscope (1) and

wherein the light source (2) has a light intensity sufficient to overexpose an image recorded by the digital camera (3) via the microscope (1) and to cause a display of light intensity variations with respect to nanoparticles conjugated with the digital camera (3) via the microscope (1) to an observer in the recorded image,

the device further comprises digital means for correcting overexposure of the image recorded by the camera (3) to display the light intensity variations with respect to the microparticles at the same time as the light intensity variations with respect to the nanoparticles are displayed to the observer in the recorded image.

Technical Field

The present disclosure relates to the field of microscopy applied to observe natural mixtures of particles (in its unfiltered sense) that contain, in an unpredictable manner, particles (or microparticles) that are resolved by optical microscopy as well as particles (or nanoparticles) that are not resolved by optical microscopy.

Background

More precisely, the present disclosure relates to the observation of such mixtures via a microscope and a camera, in particular to the observation of mixtures, for example, composed of phase object-forming biological particles, which undergo brownian motion, in particular in liquid media (in particular aqueous media).

The present disclosure also relates to the observation of mixtures containing amplitude micro-or nano-objects, i.e. micro-or nano-objects that attenuate the light transmitted or reflected or absorbed, especially in case of brownian motion in a liquid medium, like for example gold micro-and gold nano-particles.

Thus, the present disclosure generally relates to the observation of mixtures of phase or amplitude objects, especially micro or nano objects when undergoing brownian motion, but the present disclosure also relates to the observation of particles of any size and possibly not moving.

Bright field resolving optical microscopes are known in the art, which make it possible to use a light source (in particular a thermal light source, a light emitting diode, a laser, etc.) of the illuminating light collected by the microscope to form an intensity image of an object (i.e. an object whose lateral dimensions are greater than the lateral resolution limit of the microscope) resolved by the microscope. In this system, the unrecognized particles are generally not visible and therefore cannot be observed simultaneously with the recognized particles.

An optical method and device for interference-based detection of nanoparticles in a non-natural fluid sample (containing only non-resolved particles because they have been pre-filtered) is also known in the prior art, for example from the document published under number FR3027107 (BOCCARA). Specifically, this document mentions an interferometric method of observing bionanoparticles that undergo brownian motion in an aqueous medium via a microscope and a camera. Furthermore, what is presented in the bright field interferometry method disclosed in this document, in particular on page 6 of this document, is a sample which in all cases needs to be filtered beforehand and contains only particles smaller than a few hundred nanometers. Given that the visible light resolution of the optical microscope used in this document is only a few hundred nanometers, this document teaches that the observed sample must contain only unidentified particles. Therefore, in this system, the recognized particles previously removed from the object to be observed by physical filtering are in principle not present in any image of the object to be observed and therefore cannot be observed simultaneously with the undistinguished particles.

Therefore, in the prior art, especially in the case where the particles are phase objects that undergo brownian motion in a liquid matrix, a technical problem of observing a mixture of particles recognized by a microscope and particles not recognized by the microscope (referred to as a natural mixture) in an image by a microscope and a camera is a difficult problem.

Operations for increasing the contrast of an image, in particular of a resolved phase object, whether or not the phase object is in brownian motion, are also known in the art. In particular, these operations for increasing the contrast of an image include transforming the histogram of the image. In image processing, histogram transformation modifies an image by processing each pixel independently. These transformations occur almost in any image processing and analysis process. In particular, these transformations are generally applied to digital images taken by cameras that record images in the form of pixels, each pixel being assigned a grey level or a plurality of grey levels related to the color, this process taking place: before the image has been recorded, during a pre-processing in order to normalize the image; or after recording the image, during post-processing to improve viewing.

In this document, the following definitions apply:

"normalization of images": stretching of the histogram of the image before or after it is recorded so that it includes gray levels stretched over the entire range of gray levels of the sensor used for recording, the display used for viewing or the eyes of the viewer to maximize the contrast seen by the human viewer.

"brownian motion": spontaneous movement of the particles in a liquid or viscous medium, which is caused by thermal disturbances and prevents the particles from settling under the influence of gravity.

"stroboscope": a device for limiting the exposure time of the image taken by the camera and allowing the motion to be frozen; a "stroboscopic image" is understood to be an image with an exposure time short enough to freeze a certain motion.

"digital contrast enhancement": a set of digital methods that increase the amount of visible detail in an image and correct overexposure in a digital image, in particular via normalization of the image, by a human observer or a system capable of implementing the digital methods.

Disclosure of Invention

The present disclosure relates to a method of obtaining an image of a collection of particles using a microscope, the collection of particles being conjugated to a digital camera via the microscope, the collection of particles comprising nanoparticles not resolved by the microscope and microparticles resolved by the microscope, wherein the particles are illuminated by a light source, and wherein the light source illuminates the digital camera via the microscope. The method comprises the following steps:

-overexposing, by means of the light source, the image recorded by the digital camera to display to the viewer, in the recorded image, the light intensity variations with respect to the nanoparticles;

-digitally correcting the overexposure of the recorded image to show the variation of light intensity with respect to the microparticles at the same time as the variation of light intensity with respect to the nanoparticles in the recorded image is shown to the viewer.

As a variant, the method may comprise the following features, which may be implemented alone or in combination with one another (except in cases that would lead to significant technical incompatibilities):

the nanoparticles comprise a virus and the microparticles comprise aggregates of the virus;

the digital correction of the overexposure is obtained via a transformation of a histogram of the image;

the transformation of the histogram of the image is a stretching of the histogram of the image;

the transformation of the histogram of the image is a translation of the histogram of the image;

the transformation of the histogram of the image comprises a translation of the histogram of the image and a stretching of the histogram of the image;

the stretching of the histogram of the image is linear;

the stretching of the histogram of the image is non-linear.

The present disclosure also relates to an apparatus for carrying out the above method, wherein the light source has a light intensity sufficient to overexpose an image recorded by the digital camera via the microscope, thereby presenting a light intensity variation with respect to nanoparticles conjugated with the digital camera via the microscope. The apparatus includes a digital device for correcting exposure of an image recorded by the camera. The teachings of the present disclosure apply to any combination of the above-described methods, variations, and apparatus.

The above features and advantages, and other features and advantages, will be apparent from a reading of the following detailed description of the apparatus and embodiments of the proposed method. The detailed description is made with reference to the accompanying drawings.

Drawings

The figures are schematic and not necessarily drawn to scale. The drawings are first intended to illustrate the principles of the invention.

Fig. 1 shows an example of an apparatus for observing particles.

Detailed Description

Examples of embodiments of the proposed invention are described in detail below with reference to the accompanying drawings. These examples illustrate the features and advantages of the present invention. It should be remembered, however, that the invention is not limited to these examples.

The apparatus of fig. 1 comprises: a microscope 1; a light source 2 for illuminating the field of view of the microscope; and a camera 3. The camera is placed in an image plane conjugate to the object focal plane of the microscope 1 and collects light directly emanating from the illumination source 2 via the microscope, forming a bright field image. A stroboscope (not shown in this figure) or any other means for limiting the exposure time and a digital image processing means for improving the contrast of the acquired image may be provided. The bright field configuration is arranged to collect, on the one hand, intensity variations caused by resolved phase objects located in the field of view of the microscope and, on the other hand, light scattered by non-resolved phase objects also located in the field of view of the microscope.

The modulation of direct light by the resolved object is used to observe the resolved object, and the modulation caused by interference of light scattered by an undistinguished object near the object focal plane conjugate to the camera is used to observe the undistinguished object.

The illustrated apparatus thus makes it possible to observe, in the same image and in the same reference frame, both resolved and non-resolved objects located in slices extending within the depth of field of the microscope from the object focal plane of the microscope, or more generally, both resolved and non-resolved objects located in slices extending within the depth of field of the microscope from the object plane conjugated to the camera plane by the microscope. The camera is assumed to be planar, but it is contemplated that the camera conforms to the curvature of field of the microscope without departing from the teachings of the present disclosure.

In a first embodiment, shown by way of example in fig. 1, the apparatus comprises a microscope 1 or optical system, a light source 2 or illumination source or source, and a camera 3. Thus, in all embodiments, the invention comprises a light source 2 of illumination collected by a microscope 1 and a camera 3. The object to be observed is placed in the object space of the microscope 1 and is thus illuminated and imaged in bright field mode on the camera 3.

Preferably, the illumination provided by the light source 2 is collimated to maximize the contrast of the interference signal observed for the nanoparticles conjugated to the camera 3.

The light source 2 with the highest possible luminous power is preferred, or in any case a light source with a power sufficient to fill the well volume or well depth of the camera during the exposure time. Which may be a light source 2 having a wavelength bandwidth as narrow as possible.

Thus, a light emitting diode (e.g. a royal blue Thorlabs LED with reference number M405LP 1) emitting visible light centered at 405nm may be used as the light source 2.

However, other light sources with a broader spectrum may also be used, as long as they allow detection of nanoparticles of a given size due to the signal-to-noise ratio that it allows. Thus, in one exemplary embodiment, a white light emitting diode (e.g., Thorlabs LED, reference MWWHLP 1) has been used to provide collimated illumination. Although not as suitable as the spectrally narrower blue LEDs described above, such white LEDs still allow Nanoparticles (NP) of 100nm to be observed (compared to 10nm for blue LEDs). Such nanoparticles are not distinguishable in visible light.

High Numerical Aperture (NA) objectives are preferred to maximize the collection of light scattered by the Nanoparticles (NPs).

Therefore, a microscope 1 equipped with an oil immersion objective (e.g. Olympus x100 objective) may be used. The illumination conditions may be defined by the propagation of the light source in free space or by a condenser allowing the illumination conditions to be changed (kohler illumination, critical illumination or any other type of illumination, in particular collimated illumination).

In all embodiments, the magnification of the optical system, the pixel size of the camera 3 and the acquisition rate of the camera 3 in units of images per second are chosen such that the motion between the two nanoparticle images acquired or recorded is quantifiable.

In all cases, the bright field image is imaged via a microscope 1 onto a high dynamic range (i.e. with a large well depth) camera 3, such as a CMOS camera manufactured by Photon Focus under reference PHF-MV-D1024E-160-CL-12. Another camera with more pixels and shallower well depths may also be used if the pixels are combined.

Preferably, the camera 3 is placed in an image plane conjugate to the object focal plane of the microscope 1 in an optical configuration in which the optical correction of aberrations is optimized and which typically reaches the diffraction limit.

Regarding the illumination adjustment of the light source 2, it is ensured that the microscope 1 forms bright field images of a sample (e.g. an unfiltered natural sample) comprising both resolved particles and laterally non-resolved particles. For an objective lens with a minimum visible wavelength (i.e. 400nm) and a numerical aperture equal to 1, the resolution limit is close to 200nm, as is known. It is also possible to use samples in which both non-moving and non-resolved particles are present.

With the present invention it is possible in particular to observe samples consisting of unfiltered natural water which theoretically contains viruses and virus aggregates forming phase populations, the size of which is between 10nm and 10 μm. Any liquid sample containing biological particles can be observed using the apparatus and method of the present invention. In general, the invention is particularly applicable to observing natural media that include phase objects that undergo brownian motion.

In certain embodiments, on the one hand, a camera acquisition rate of about 130 images per second or more may be defined, and on the other hand, a contrast enhancement operation may be applied to each image or strobe image taken by the camera, which is suitable for overexposure images obtained using bright field microscopy (i.e., to maximize their exposure without saturating the image). Thus, in the presence of resolved objects, any increase in the camera acquisition rate that is compatible with a given signal-to-noise ratio facilitates viewing smaller and smaller undisresolved objects. In all cases, the acquisition rate is chosen to be as high as possible so as to track the brownian motion of the particles as successfully as possible while ensuring that the wells of the camera are completely filled to minimize shot noise, which is the dominant noise source in some embodiments.

The contrast enhancement operation may be applied in real time if allowed by the image processing means, or subsequently to a sequence of strobed images stored in the image memory.

Thus, it is possible to cause a contrast-enhanced interference pattern due to interference between direct light and light scattered by particles not recognized by a microscope and an optical image of particles recognized by a microscope, which has high contrast and includes details visible to a human observer without saturation, to be displayed to human eyes in an image obtained with a single device.

One advantage of the present invention is that the representations of the two types of particles (generated by interference imaging and intensity imaging) share the same spatial frame of reference, since the two types of particles are obtained with exactly the same bright field device. If the distance between the microscope and the object plane conjugated to the camera by the microscope is mechanically stable, there is therefore a quantitative means of imaging particles with a size smaller or larger than the resolution limit of the microscope in a slice-wise manner.

This makes it possible in particular to observe not only spatially evolved resolved particles or sets of non-resolved particles (i.e. sets of different particles in which some particles are resolved and some are not), but also temporally evolved resolved particles or sets of non-resolved particles (i.e. sets of different sizes of the same particles, for example in the case of bubbles of various sizes that successively increase in size from a non-resolved diameter to a resolved diameter). In the present application, in all the embodiments of the invention, the expression "set of particles" covers at least the set that can undergo these two evolutions.

An advantage of the invention is that it is possible to obtain both types of imaging with the same instrument, thus naturally combining both types of imaging (interferometry between scattered light and direct light and conventional intensity imaging) performed automatically by the microscope according to the particle size in the same spatial reference system.

Thus, by the method of the above embodiment, not only the distance between two resolved particles or between two unrecognized particles can be reliably measured, but also the distance between the resolved particles and the unrecognized particles can be measured.

This embodiment thus makes it possible to obtain slice images of both naturally resolved and non-resolved particles in a slice whose thickness is equal to the depth of field of the microscope.

Furthermore, the steps of acquiring and increasing the optical contrast of the image may be applied simultaneously to achieve real-time imaging, or non-real-time imaging may be obtained after temporal processing of the image for noise reduction in a time-shifted manner.

For particles larger than 10nm in size, an exposure time shorter than or equal to 1/130 seconds may be selected. In general, this time can be chosen to freeze the brownian motion of the fastest moving particles, i.e., to ensure that the motion of the smallest particles to be imaged is not discernable by the microscope in the exposure time of the stroboscopic image.

Thus, for a minimum exposure time, the minimum size of the particles that can be imaged using the apparatus and method of this embodiment, i.e., the interference resolving power of the apparatus of the present invention, can be calculated.

Under the constraint that neither the interference image nor the intensity image is saturated, the adjustment of the image brightness satisfies the condition that the bright background is maximized or overexposed to a maximum, which is a conventional adjustment in microscopes. This adjustment may allow a human observer to observe the stroboscopic image on the display.

In the bright field device, the illumination source may be any type of light source, such as a lamp-type thermal light source, a Light Emitting Diode (LED) or a laser. With respect to microscopy, the resulting illumination may be spatially coherent or incoherent, and/or temporally coherent or incoherent.

Thus, the apparatus of this embodiment makes it possible to jointly achieve interference imaging (via scattered light and direct light) and intensity imaging (via attenuated direct light) with the same bright field microscope.

In order to increase the contrast of the stroboscopic image, or of its time average, which tends to be saturated, in particular white, due to its bright background, and which does not generally show any visible details to the naked eye, a stretching or expansion of the histogram (also referred to as a normalization of the image) can be applied in particular. Thus, the distribution of the histogram of the image across the entire level of the image is a particularly effective way to increase the contrast. It should be noted that in conventional or resolving microscopes of phase objects, this situation corresponds to a defective microscope setup, where the illumination conditions are not optimized, and where a wide range of lowest grey levels of the camera is not required.

With a system comprising only a bright field microscope, a system (in particular a camera) which makes it possible to acquire images in a stroboscopic manner, and image processing means for improving the contrast of the images, a device is thus obtained which, when applied to the observation of natural samples, is capable of generating simultaneous images of phase objects which undergo brownian motion, without the need for size-dependent physical filtering of the particles.

It should be noted that the normalization operation improves the contrast of the interference pattern of the unrecognized particles and the intensity pattern of the recognized particles.

Many variant embodiments are possible, in particular by implementing a shift of the histogram of the stroboscopic image, although this would result in a stroboscopic image of lower contrast after processing.

Depending on the type of particles observed, a linear or non-linear normalization operation may also be applied to the histogram of the stroboscopic image. In particular, each non-linear normalization may be sample-specific, since the intensity of the resolved/undisresolved particles typically varies from sample to sample.

Any other method that can remove the background from the strobe image and improve its contrast can also be used with the present invention.

Thus, in all embodiments of the invention, the process will start with an overexposure of the image recorded by the camera via the microscope by the light source used at its maximum power, in the hope of subsequently digitally correcting this overexposure. This method makes it possible to obtain in a single image the intensity variations (visible to the human eye) with respect to the nanoparticles not resolved by the microscope, and at the same time the intensity variations (visible to the human eye) with respect to the microparticles resolved by the microscope. In other words, the method makes it possible to obtain, in a single image, the variation of light intensity correlated to the interference of scattered light caused by the presence of non-resolved nanoparticles in the observed medium, while obtaining the variation of light intensity correlated to the presence of resolved microparticles in the same medium.

The invention is particularly suitable for observing a population of particles by an observer, in the case where the size of each particle of the population is comprised between 10nm and 10 microns, or in the case where the population of particles comprises particles not resolved by an optical microscope operating under visible light and particles resolved by the microscope.

In the context of the present invention, "histogram" is understood as the distribution of light intensities or "grey levels" in a digital image.

The invention can be used industrially or in the field of microscopes.

The embodiments or examples of embodiments described in this disclosure have been presented by way of illustration and not limitation; in light of the present disclosure, those skilled in the art will be readily able to modify these embodiments or examples of embodiments or to devise other embodiments while still remaining within the scope of the present invention.

In particular, if some features of the above-described embodiments or examples of the above-described embodiments alone are sufficient to achieve one of the advantages of the present invention, a person skilled in the art can easily conceive of variations including only these features. Furthermore, the different features of these embodiments or examples of embodiments can be used alone or in combination with each other. When combined, these features may be combined as described above or otherwise, and the invention is not limited to the specific combinations described in this specification. In particular, features described in relation to one embodiment or example of an embodiment may be applied in a similar manner to another embodiment or example of an embodiment, unless stated otherwise.