Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B19/00—Condensers, e.g. light collectors or similar non-imaging optics
  • G02B19/0033—Condensers, e.g. light collectors or similar non-imaging optics characterised by the use
  • G02B19/0047—Condensers, e.g. light collectors or similar non-imaging optics characterised by the use for use with a light source
  • G02B19/0061—Condensers, e.g. light collectors or similar non-imaging optics characterised by the use for use with a light source the light source comprising a LED
  • G02B19/0066—Condensers, e.g. light collectors or similar non-imaging optics characterised by the use for use with a light source the light source comprising a LED in the form of an LED array
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/082—Condensers for incident illumination only
  • G02B21/084—Condensers for incident illumination only having annular illumination around the objective
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/12—Condensers affording bright-field illumination
  • G02B21/125—Condensers affording bright-field illumination affording both dark- and bright-field illumination

Definitions

• the present invention relates to an illumination system intended in particular to be included in a microscope, and the microscopes comprising such a system.
• Optical microscopy brings together a vast set of techniques used to observe and analyze objects at microscopic scales. These very varied techniques have been developed for several centuries. They advantageously provide an optical source of excitation of biological samples to acquire enriched data on these samples, such as morphological information, by allowing super-resolution, optical image processing and/or optical tomography.
• Known microscopes are based on an architecture comprising a stand, a microscope objective, a lighting system, and a photosensitive sensor, which can be a camera or the eye.
• This type of microscope is used for direct observation of biological tissues in the medical field, in particular for diagnostic or observation purposes. It only requires parts of limited complexity, allows interpretation of the images by its user, for example a doctor, an anatomopathologist or any other specialist, and poses few constraints as to the working environment. Nevertheless, the variability of biological tissues and the multiplicity of local know-how, in particular with regard to the preparation of samples specific to each laboratory, or the eye of the expert, make it delicate G universality of the adjustment parameters of digital tools that can be associated with this type of microscopes. In addition, such a type of microscope does not make it possible to observe a large sample surface with high resolution, so the more one seeks to obtain fine details on the sample, the smaller the surface observed. This does not allow a global and precise analysis of the tissues.
• Digital holography microscopy is a particularly rich and sensitive data recording technique.
• the objective of this technique is to record quantitative information on the optical properties of tissues.
• the physical characterization of the sample is here measured from images.
• Different mathematical treatments and physical models are then used to access different properties of the tissue, such as the optical thickness, the 3D morphology of the tissue, or its optical anisotropies.
• This quantitative information once isolated, in turn makes it possible to feed various optical and biological models from which the desired representations of the tissue will be formed, such as images, a cartography of the optical index, a representation in volume, for example in tomography.
• a simple registration allows a multiplicity of representations which mainly depends on the framework of abstraction or the formulation of the direct problem set up.
• Data acquisition is based on an interferometric system, which can incorporate microscope objectives or not, or, in the case of on-line holographic microscopes, not include any interferometer. To be usable, these microscopes must be installed in a well-controlled environment, providing thermal and mechanical stability.
• Microscopy by Ptychography in the Fourier domain is a very recent microscopy technique, initially developed in the field of electron microscopy nearly twenty years ago and transposed in 2013 in the optical regime, as described in the article by Zheng et al “Wide-field, high-resolution Fourier ptychography microscopy”, Nature Photonics, vol. 7 No. 9, pages 739-745, 2013.
• This technique is based on a traditional microscope architecture, except that the source of illumination, for the excitation of the sample, consists of a matrix of light-emitting diodes or LEDs.
• the microscope objective collects part of the scattered electromagnetic field, resulting from the interaction between the source and the sample, and whose inclination is low enough to be collected.
• This field is recorded by a photosensitive matrix detector, for example a charge transfer camera (CCD) or CMOS sensor (“complementary metal-oxide-semiconductor”).
• CCD charge transfer camera
• CMOS sensor complementary metal-oxide-semiconductor
• the Gershberg-Saxton algorithm allows for example to deduce the phase function c
• the error function is chosen according to the formulation of the direct problem chosen and associated with the measurement process.
• the microscope objective is modeled by a band-pass filter.
• its template is a disc of space pulsation radius
• j (x,y) satisfies the following constraint: the Fourrer transform T of the intensity as measured by the camera, must be included in a ray disc related to the numerical aperture of the microscope objective and centered at Cj.
• a sufficiently regular sampling in the Fourrer space must be performed, as explained in the article by Zheng et al mentioned above.
• the sampling used must be such that the overlap between the band-pass filters relating to each diode is of the order of 60%.
• the diodes are lit one by one, and for each diode, a snapshot of the sample is recorded.
• the images are then digitally combined in Fourrer space sequentially following a well-defined path, for example through the Gershberg-Saxton algorithm, described in particular in the article by JR Fienup, "Phase retrieval algorithms: a comparison » Applied Optics, vol. 21, No. 15, page 2758, 1982.
• the conventional limits such as the compensation of the chromatic aberrations of the objectives or the aberrations of the wavefront by digital adaptive optics, and compensation for focusing errors, can be exceeded in many respects.
• the reconstructed phase is quantitative, the inverse problem formulation of the complex electromagnetic field and its resolution allow tomographic reconstruction and access to the three-dimensional (3D) structure of the biological tissue and its various components.
• the greater the dynamic range sought the greater the effective time required for the acquisition of the images becomes, time which is added to the time necessary to carry out the calculations for the reconstruction of the phase.
• Known orders of magnitude exceed one minute for acquisition and a few minutes for calculations.
• one solution is to produce an excitation of the samples with several point-sources lit simultaneously, in particular several dark-field diodes as described in the articles by Tian et al "Computational illumination for high -speed in vitro Fourier ptychographic microscopy”, Optica, vol. 2, no. 10, pages 904-911, 2015 and “Multiplexed coded illumination for Fourier ptychography with a LED array microscope”, Biomed. Opt. Express, vol. 5, n° 7, pages 2376-2389, 2014.
• This technique provides phase reconstruction thanks to a modification of the calculation algorithm, and the excitation of the sample by several diodes simultaneously makes it possible to increase the power optics received by the sensor and therefore to reduce the exposure time used.
• the gain is significant and makes it possible to obtain complete FPM acquisitions at a rate of about ten per minute.
• the diodes are specifically placed on concentric circles to respect the symmetry of revolution of the properties of the objective and ensure the optimal meshing of the Fourier space. It significantly reduces the image acquisition time but at most doubles the resolution of the reconstructed images, the super-resolution factor being deliberately limited to 2.
• a single lens is also placed above the diodes to concentrate the exciting light coming from the different diodes on the sample.
• the mesh of the Fourier space cannot be chosen in a completely arbitrary way. Indeed, the overlap factor of the filters relating to two neighboring diodes cannot exceed a limit value (to ensure good convergence of the reconstruction algorithm).
• This covering makes it possible to have a redundancy of information on the spectrum of the sample. It is used to ensure proper phase referencing across the spectrum (rather than per chunk).
• This limit value can be reduced when several diodes are lit simultaneously, or even when variants in the data processing algorithm are made, such as described in the aforementioned article by Tian et al “Multiplexed coded illumination for Fourier ptychography with a LED array microscope”.
• the size of the diodes commercially available cannot be less than the minimum size of the cases used for their packaging (generally square in shape), their spacing is constrained.
• the minimum spacing between two diodes is of the order of a few mm for current low-cost technologies. This spacing imposes a condition on the minimum distance separating the matrix of LEDs from the sample. For a fixed numerical aperture, this distance can be easily calculated by geometric approach. It is imposed by the mesh conditions of the desired Fourier space.
• the theoretically accessible super-resolution factor g then depends solely on the area covered by the matrix of diodes. If the set of diodes used allows the complete tiling of a disk of Fourier space of radius SO :
• NAsynth represents the synthetic aperture associated with the arrangement of the diodes used, which is expressed, with Q the angle of incidence of the light beam from one of the peripheral diodes:
• This factor is, in a known manner, limited to approximately 6 or 7 for a numerical aperture NA equal to 0.1.
• the surface that must be covered by the diodes is then reasonable and the optimization of the design of the diode matrix is rather easy in the case of a single microscope objective.
• this optimization becomes more complicated when one seeks to use the same matrix of diodes for an FPM microscope with different microscope objectives with distinct numerical apertures.
• the surface of the matrix which must then be covered is all the more important as the difference between the numerical apertures of the various objectives is high.
• the known methods can use a cooled sensor.
• exposure times reduced to the best can be used, the power of the thermal noise being directly related to the statistics of thermal excitation of the carriers and therefore to the exposure time used to acquire each image, for a working temperature fixed.
• This reduction is particularly beneficial for acquiring dark field images, but is not easy to obtain.
• the sources are arranged in a plane, or on a spherical or semi-spherical surface, which makes it possible, in the second case, to have a uniform and identical sample excitation power for each source. . But these arrangements cannot overcome the constraint imposed by the distance between the sample and the sources in the bright field, as mentioned previously.
• the object of the present invention is precisely to meet this need.
• the subject of the present invention is thus an illumination system, intended in particular to be included in a microscope or a microscopic imaging device, comprising a plurality of light sources arranged according to plane regions defining a solid space intersecting an axis of reference passing through a system reference point.
• the invention is based on a lighting system consisting of several point-sources distributed in a volume, and makes it possible to reduce the noise of measurement in intensity and in phase thanks in particular to the optimization of the optical flows, and therefore to improve the quality of the images obtained compared to known methods using sources distributed over a flat or spherical surface.
• the lighting system according to the invention makes it possible to better manage the optical flow arriving at a sample under test, in particular in the case where the system is included in a microscope or a microscopic imaging device.
• the accessible super-resolution factor depends on two parameters: the inclination of the incident beams coming from the sources, and the density of the sampling in the Fourier space of the waves associated planes. In methods known, these two constraints lead to moving the light source a certain distance away.
• the optical flux arriving on the sample proportional to l/d 2 where d is the distance between the source and the sample, is therefore limited and leads to long exposure times.
• the volume arrangement according to the invention makes it possible to bring some of the source points closer and therefore to reduce the overall acquisition time while reducing the dark noise, proportional to the acquisition time, without bringing additional difficulties compared to the constraint relating to the minimum inter-source distance imposed by the size of the casing of each source.
• the invention makes it possible to finely and flexibly manage the sampling carried out in Fourier space. It is advantageously possible to finely choose the inclination of the beams by bringing the sources closer to a sample under test without reducing the sampling step for low inclinations, the sources being placed at a further distance for this.
• This fine mesh of Fourier space is advantageously produced for a fixed size, imposed on the one hand by the design of known microscopes but also by the minimum size of the sources used.
• the lighting system according to the invention can thus be designed in a very versatile way to be usable on all known microscopes and transform them, if necessary, into an FPM microscope.
• This flexible and fine sampling also makes it possible to perform image processing in a fully optical and flexible manner. This can be particularly beneficial for spotting objects of interest on a slide. Rather than performing this operation with specialized, inflexible, and expensive devices, such as phase contrast objectives, or the implementation of dark field imaging, the user can program the desired filtering function by choosing sources to light.
• a single illumination system according to the invention can be suitable for different microscope objectives at the same time, objectives which can range from high magnification to low magnification. It is thus not necessary to change the matrix of sources or to adjust them again to optimize the optical flows each time the objective is changed.
• solid space it is necessary to understand a three-dimensional figure, limited by a closed surface of measurable volume and of which all the points are at invariable distances, so that its shape and volume are thus determined.
• the system according to the invention advantageously comprises a multiplicity of layers of light sources.
• the invention thus offers a multilayer solution.
• the solid space can define an envelope surface with at least one non-planar face.
• the solid space defines an envelope surface having only planar faces, the solid space notably forming a cube.
• each of the faces of said cube preferably carries at least one light source, better still a plurality of light sources.
• the reference axis corresponds, in known manner, to the optical axis of said microscope or imaging device microscopic.
• the distance from a light source to said reference point is advantageously a function of the angle of incidence between the reference axis Z and the straight line connecting said light source to the reference point.
• the reference point is not included in the solid space defined by the light sources.
• the light sources can be arranged on regions of the plane, each of which intersects the reference axis perpendicularly.
• At least a portion of the plurality of light sources is disposed on at least one plane region obliquely intersecting the reference axis.
• each light source comprises one or more emissive elements, each emissive element of each of the light sources being point-like.
• the light sources located at a greater distance from said reference point are advantageously configured to acquire bright field images.
• the light sources located at a closer distance from said reference point are advantageously configured to acquire dark field images.
• a strong power is in fact obtained by approaching the sample.
• the system according to the invention may comprise flexible electronic circuits forming the plane regions and carrying the light sources. These circuits can be carried by a structure, in particular produced by 3D printing, which makes it possible to provide a more rigid structure with an easily adaptable angular orientation of the sources. Care should be taken to provide a vacuum for each light source to access the sample.
• the system according to the invention may include collimating microlenses associated with each light source.
• the system includes a global lens.
• the system according to the invention advantageously comprises a control module configured to select the light sources to be switched on, in particular for carrying out a function of filtering the spatial frequencies of a sample to be studied placed centrally at the level of said point of reference.
• the size of the filtering function to be achieved in an all-optical way can be adjusted flexibly by adjusting the intensity of each light source. This makes it possible to reveal the frequencies sought, which is particularly interesting for replacing certain stages of image processing by an entirely optical processing, for example to identify objects of interest.
• the intensity can be the same for each light source.
• the intensity of one or more light sources is adjustable. Thanks to the invention, it is not necessary to adjust the intensity of the light sources, the same intensity for each source is used, whereas a different power reaches the sample because of the variable distance of the sources of light to the sample.
• the light sources are diodes.
• Another subject of the invention is a microscope comprising the lighting system according to the invention, as defined above.
• Another subject of the invention is a microscopic imaging device comprising the lighting system according to the invention, as defined above.
• the microscopic imaging device may comprise an additional light source, being in particular a laser source or a Kohler source.
• This additional light source brings additional functionalities, in particular a measurement holographic through the laser source, and traditional lighting through the Kohler source.
• Another subject of the invention is a method for analyzing a sample using a lighting system according to the invention, comprising at least the following steps:
• the compensation of the powers arriving on the sample is due to the variable distance between the sample and each light source.
• the invention Insofar as the angles of illumination possible by the sources are very varied, and that the mesh of the Fourier space is fine, it is possible to use the invention to reveal selected details of the sample, for simple source programming. For example, it can be used to obtain dark-field images and reveal the high spatial frequencies of the sample.
• Another subject of the invention is a computer program product for implementing the method for analyzing a sample according to the invention, the computer program product comprising a support and recorded on this support of the instructions readable by a processor so that, when executed,
• the powers of each light source are analyzed, in the frequency domain, in an overlap zone created between two adjacent sources, in order to compensate for the powers arriving on the sample, and - the spectrum is initialized so as to be able to remove any light source placed on the reference axis in order to carry out the reconstruction of the sample.
• FIG 1 figure 1, previously described, illustrates the result of a ptychographic reconstruction in Fourier space carried out in a prior art FPM microscopy installation with identical exposure times and powers for each of the diodes used and arranged on a single plane,
• Figure 2 illustrates an example of an illumination system according to the invention included in a microscopic imaging device
• FIG. 2 An example of a lighting system 1 according to the invention.
• the lighting system 1 is included in a microscopic imaging device 10 intended to study a sample E, in particular a biological sample, placed at a reference point P r of the system 1.
• the lighting system 1 comprises a plurality of light sources 2 arranged in plane regions defining a solid space S perpendicularly intersecting a reference axis Z passing through said reference point P r of the system.
• the solid space S defines an envelope surface having only planar faces, and forms a cube having six faces. Each of the six faces of said cube carries a plurality of light sources.
• the system according to the invention thus comprises a multiplicity of layers of light sources.
• the microscopic imaging device 10 comprises a microscopic lens 6 and a camera 7.
• the distance from a light source 2 to said reference point P r is advantageously a function of the angle of incidence between the reference axis Z and the straight line connecting said light source to the reference point.
• the reference point P r is also not included in the solid space S defined by the light sources 2.
• the intensity is the same for each light source 2, and the light sources are diodes.
• the system 1 can also comprise a control module, not illustrated, configured to select the light sources 2 to be turned on, in particular for the realization of a function of filtering the spatial frequencies of the sample E to be studied placed at said reference point P r .
• a man-machine interface makes it possible to make the link between the system and the study of the sample.
• the solid space S defined by the plane regions on which the light sources are arranged defines an envelope surface having non-planar faces, the light sources 2 being arranged on plane regions obliquely intersecting the reference axis Z.
• the microscopic imaging device 10 may comprise an additional light source 8, being in particular a laser source, an optical condenser, or a Kohler source.
• the invention is not limited to the examples which have just been described.
• other light sources can be used, as well as other arrangements for the latter.
• the invention can be used in conventional microscopy, digital holography, in particular online, optical tomography, lensless microscopy, or microscopy by Fourier ptychography.
• the lighting system according to the invention can be used for medical diagnosis, through the observation of biological tissues, in particular for the observation of malaria or the various abnormal blood cell lines, such as schistocytes, clusters platelets, or abnormal white blood cells.
• the invention can be advantageously used for the production of virtual biological slides, or digital clones, for automated diagnosis, and associated for example with artificial intelligence algorithms.
• the data reconstructed with the system of the invention are largely digitally manipulable, and are rich, both in intensity and in phase, and fine.
• the lighting system according to the invention can in particular be used instead of known lighting systems for microscopes with increased flexibility and advantageously, since it offers a simpler system, in particular not requiring any mechanical element and optics to adjust the inclination of the beams. If all the sources are switched on at the same time, it is possible to form images quite similar to those obtained by a Kohler source. To be adapted to the various traditional microscopy applications, the adjustment of the Kohler source is replaced by the programming of the sources to be switched on, which can be much more varied and fine in the invention than in a Kohler source. Such a setting also allows an excitation of the sample inaccessible to Kohler sources.
• the invention can also be used to perform differential phase contrast microscopy, described in the article by Tian et al “Quantitative differential phase contrast imaging in an LED array microscope”, Optics Express, vol. 23n no. 9, pages 11394-11403, 2015.
• the invention can be used for all imaging applications where it is useful to be able to control the incidence of the beams and the power, in particular as a function of the inclination, for example in imaging applications frequency for the detection of objects of interest.

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• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Microscoopes, Condenser (AREA)

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• 2021
  • 2021-07-29 FR FR2108256A patent/FR3125893A1/fr active Pending
• 2022
  • 2022-07-28 WO PCT/EP2022/071270 patent/WO2023006915A1/fr active Application Filing
  • 2022-07-28 EP EP22744787.7A patent/EP4377733A1/de active Pending
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