• 专利附录
  • 专利说明
  • 权利要求
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    • 专利摘要:
    The invention describes a procedure for correcting the scattering effect of light in a flat laser beam microscope comprising: calculating, by means of Monte Carlo simulations, the transfer function of a medium for different scattering coefficients and scanning depths, creating a transfer functions database: experimentally estimate the dispersion coefficient of a sample assuming that it is uniform throughout the sample: scan the sample using the flat laser beam microscope to obtain a set of images corresponding to different sections of the sample; and deconvolve each image of the sample obtained in the scan using the transfer function of the database that corresponds to the dispersion coefficient of the sample and the scanning depth of each image, thus correcting in each image the effect of the dispersion of the light. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2853354A1
    申请号:ES202030209
    申请日:2020-03-12
    公开日:2021-09-15
    发明作者:Vidal Asier Marcos；Lorenzo Jorge Ripoll；Fernandez Roberto Fernandez
    申请人:Universidad Carlos III de Madrid；
    IPC主号:
    专利说明:

    [0002] Procedure to correct the effect of light scattering in flat laser beam microscope measurements
    [0004] OBJECT OF THE INVENTION
    [0006] The invention belongs in general to the field of optics, and more particularly to flat laser beam microscopy.
    [0008] The object of the present invention is a method to reverse the effects caused by the scattering of light as it passes through the sample in flat laser beam microscopy images.
    [0010] BACKGROUND OF THE INVENTION
    [0012] Studies of embryos and large biological samples through light microscope present, unlike what happens with small samples, particular problems related to the optical properties of the tissue, absorption and scattering of light, which cause a marked loss in the resolution of the images. To solve these problems, significant improvements have been made in recent years over flat laser beam microscopes, the invention of which dates back to 1903.
    [0014] A flat laser beam microscope is made up of an illumination medium capable of emitting a thin sheet of light in one direction, called the "illumination direction" (x), and a detection means consisting of a camera coupled to a large objective numerical apertures arranged according to a direction called "detection direction" (z), which is perpendicular to the illumination direction, following the original configuration of Siedentopf and Zsigmondy.
    [0016] With this configuration, the camera can obtain a 2D image of the fluorescence emitted by the section of the sample excited by the illumination plane. If the sample is also moved in the direction of the detection axis and images are taken at different positions, a set or stack of 2D images is generated where each of the 2D images corresponds to the fluorescence emitted by the section excited by the plane of illumination in each position with respect to the sample. This stack of 2D images contains information about the position in z (depth of the sample according to the detection direction) obtained by moving the sample, and of the x and y positions, present in each 2D image. The 2D image stack can then be merged to generate a 3D image of the sample, as described in US 7,554,725 to Stelzer et al. Subsequently, it was proposed to rotate the sample around its own axis, usually vertical, to capture several stacks of 2D images (commonly called "angular measurements") and then merge them, which allows improving the anisotropy and quality of the images (S Preibisch et al, Nature Methods 7 (2010)).
    [0018] For a clearer understanding of this technique, Figs. 1a and 1b showing a first example of a flat laser beam microscope (100). The sample (107) is placed in a holder (101) within a cuvette (102) filled with a liquid. A linear Gaussian, Bessel, Airy or similar illumination beam (103) falls on a cylindrical lens (104) that focuses it thanks to an illumination objective (105) to generate the vertical flat illumination sheet (106). This vertical flat illumination sheet (106) falls on the sample (107) according to the illumination direction (DI), and the fluorescent light (108) emitted by that specific plane of the sample (107) is collected by an objective (109 ) of detection oriented according to the detection direction (DD), which is perpendicular to the illumination direction (DI). An image corresponding to the sample portion (107) illuminated by the illumination sheet (106) is thus obtained. To obtain a complete image of the sample (107), it is possible to move the support (101) according to the detection direction (DD) to take measurements corresponding to various sections of the sample (107), or the support (101 ) can rotate around its vertical axis to allow the taking of various angular measurements according to the technique proposed by Preibisch.
    [0020] Since 2015, the inventors of the present application have filed several patent applications aimed at various improvements in this type of microscopes. These patent applications are as follows:
    [0022] PCT / ES2015 / 070455 entitled "Microscope and procedure for the generation of 3D images of a collection of samples" that describes a new microscope that combines the SPIM (Selective Plane Illumination Microscope) flat laser beam technique with the tomography technique Optical Projection Tomography (OPT).
    [0024] PCT / ES2016 / 070714, entitled “Multiple loading device for beam microscope flat laser '' describing a multi-loading device for feeding a continuous, sequential flow of samples to a flat laser beam microscope.
    [0026] PCT / ES2017 / 070028, entitled "Automatic objective change device for flat laser beam microscope", which describes a device that allows to automatically change the image acquisition objective of a flat laser beam microscope depending on the magnification desired at all times.
    [0028] PCT / ES2017 / 070028, entitled "Rotary objective change device for flat laser beam microscope", which describes a device where the objective change is carried out through rotations of the cuvette itself.
    [0030] PCT / ES2017 / 070184 entitled "Microscope specimen clamping device", which describes a device to be able to mount samples and measure them on flat laser beam equipment.
    [0032] PCT / ES2019 / 070629 entitled "Microscope and flat laser beam procedure for large samples", which describes a system to allow obtaining large 3D images without the need to move the sample.
    [0034] In flat laser beam microscopy systems, the size of the sample determines the maximum image quality that can be obtained. As images of planes are obtained at greater depth, their resolution is affected, among other factors, by the scattering suffered by the light on its way to the detection target.
    [0036] Indeed, the fluorescence emitted by an excited plane of the sample must propagate through it a certain distance. Therefore, the further from the detection objective said plane is, the greater the distance that the light travels through the sample, and therefore the greater the deterioration of the images. For this reason, currently the use of this technique is limited to the study of samples that are very transparent (previously cleared) or of a size small enough so that the loss of resolution due to scattering is almost negligible.
    [0038] DESCRIPTION OF THE INVENTION
    [0040] The present invention describes a procedure that makes it possible to reverse the effect of dispersion of light in flat beam microscopy images in order to improve their quality. To do this, first the degree of light scattering in the sample is estimated and, later, a post-processing is applied to the images obtained to recover part of the lost resolution. This improvement allows the use of flat laser beam techniques to study samples whose size or limited transparency precludes their visualization with current flat laser beam techniques.
    [0042] In this document an x, y, z coordinate system is defined where: the " x direction" refers to the illumination direction; the " z direction" refers to the detection direction, or depth direction of the sample; and the " y direction" is perpendicular to the x and z directions, that is, it is the vertical direction in the natural position of use of a flat laser beam microscope as shown in the figures.
    [0044] In this document, the term "depth" referring to an image or plane of the sample refers to the distance between the plane of the sample excited by the illumination sheet and the detection chamber. The greater this distance, the greater the depth of said image or plane. The depth therefore corresponds to the z direction.
    [0046] In this document, the term "Gaussian beam " refers to a monochromatic beam whose intensity profile transverse to the axis of propagation follows a Gaussian function. This type of beam is the most common output of any laser light source, corresponding to the TEM 00 fundamental mode.
    [0048] The present invention describes a method for correcting the scattering effect of light in a flat laser beam microscope that basically comprises the following steps:
    [0050] to. Generating a database of transfer functions for semi-transparent media
    [0052] The objective of this step is to characterize the loss of resolution of an image when propagating in a semi-transparent medium with low dispersion. For this, simulations are carried out in order to estimate a transfer function H (kx, ky; z) for the medium that quantifies the attenuation suffered by each spectral component (kx, ky) of an image for a depth z.
    [0053] In this context, the parameters kx and ky refer to spatial frequencies in an image. These are expressed as cycles or line pairs per millimeter. In diffraction limited microscopy techniques, the maximum frequency in the image corresponds to the theoretical Abbe limit.
    [0055] The Monte Carlo method allows the simulation of the propagation of light in scattered media with great precision. In addition to estimating the spatial distribution of light intensity, this technique can be used to calculate the frequency response of the medium to a unit impulse. To do this, a simulation of the propagation of a collimated 'pencil' type source is carried out, in which the photons are generated in the z direction at a point (x, y) = (0, 0) in a three-dimensional volume. Once the simulation is complete, the Fourier transform is calculated for each z-plane to obtain the frequency response of the system.
    [0057] This process will be repeated for different values of the dispersion coefficient in order to create a database of transfer functions corresponding to each dispersion coefficient.
    [0059] Ultimately, in this step, the transfer function of a medium is calculated through simulations using the Monte Carlo method for different scattering coefficients and scanning depths, creating a database of transfer functions corresponding to each scattering coefficient. and scan depth.
    [0061] b. Experimental estimation of the sample dispersion coefficient using a flat beam microscope
    [0063] In order to carry out deconvolution correctly, it is necessary to use the transfer function of a medium with a dispersion coefficient equivalent to that of the sample. Since its value is unknown a priori, an experimental in situ estimation of it must be carried out. It is important to emphasize that the measurement will be carried out in the flat laser beam microscope with the sample mounted as a procedure prior to image acquisition.
    [0065] Thus, assuming that the sample has a dispersion coefficient relatively homogeneous, before scanning the sample and once mounted on the flat beam microscope, it is illuminated with a Gaussian beam at different depths (z) and positions (y) with respect to the camera. This can be done either simultaneously or sequentially, depending on whether the projection frequency is higher than the camera measurement frequency (frames per second, fps).
    [0067] The camera (without filter) captures the distribution of the light scattered by the sample as a consequence of the propagation of each of the beams as shown in Fig. 2. The divergence of the beams in each of the images due to the dispersion allows estimating the dispersion coefficient. To do this, during post-processing intensity profiles are measured in the image and the widening of the beam is quantified at various points of the x-axis (the beam propagation), as shown in Fig. 3. Minimizing the measurement From the error between the experimental curves and those of the data from the Monte Carlo simulations from the previous step, it is determined which is the dispersion coefficient that best fits.
    [0069] To increase the precision of the procedure, it is repeated for each of the measurement positions (y, z) in order to estimate an average dispersion coefficient of the sample.
    [0071] Ultimately, this step involves estimating experimentally using the flat laser beam microscope the dispersion coefficient of a sample assuming that it is uniform throughout the sample, where the estimation comprises the following steps:
    [0072] b1) mounting the sample on the flat laser beam microscope;
    [0073] b2) illuminating each plane of the sample with a Gaussian beam; b3) capture images of the propagation of the Gaussian beam in the sample and repeat the process in different positions; and
    [0074] b4) estimating the scattering coefficient of the sample by measuring the divergence of the beams caused by scattering;
    [0076] c. Sample scan
    [0078] The sample is now normally scanned by means of the flat laser beam microscope, obtaining a set of images corresponding to different sections of the sample. That is, the imaging of the sample is performed in a flat laser beam microscope following a conventional acquisition protocol, Either illuminating with a flat beam or with digital scanning.
    [0080] The images obtained will show artifacts caused by scattering, especially those corresponding to scanning planes located deeper than the detection target.
    [0082] d. Correcting the effect of scattering on scan images
    [0084] In order to reverse and reduce the effects of dispersion in the images obtained, the deconvolution of each image must be calculated with its corresponding transfer function of the medium, calculated in step a. To do this, the simulation database will be searched for the volume that corresponds to the dispersion coefficient estimated in step b, and the convolution with the transfer function for said volume will be calculated for each image.
    [0086] That is, each image of the sample obtained in the scan is deconvolved with the transfer function of the database that corresponds to the dispersion coefficient of the sample and the scanning depth of each image, thus reversing the effect of the scattering of light.
    [0088] Deconvolution can be performed with multiple methods, preferably Wiener deconvolution.
    [0090] BRIEF DESCRIPTION OF THE FIGURES
    [0092] Figs. 1a and 1b respectively show a perspective view and a top view of the main elements of a conventional flat laser beam microscope.
    [0094] Fig. 2 experimental measurement scheme to estimate the dispersion coefficient of the sample.
    [0096] Fig. 3 shows the procedure for estimating the dispersion coefficient from the experimental measurements.
    [0098] Fig. 4 shows a simplified flow diagram of the method of the present invention.
    [0099] PREFERRED EMBODIMENT OF THE INVENTION
    [0101] A complete procedure is described below. In any study carried out with the proposed method, it is assumed that the Monte Carlo simulations have been carried out previously, therefore there is a database with the spatial information to estimate the dispersion coefficient and a database with the functions transfer for a wide range of media and sample sizes.
    [0103] Thus, Fig. 2 shows a diagram of the assembly of the invention where the illumination objective (OI), the measurement positions (PM), the detection objective (OD), the sample (M), the measurements of beam spread (MD), an image analysis block (AI), a Monte Carlo simulation bank (MC), and the transfer function for the estimated scattering coefficient (TF (^ s)), where is the dispersion coefficient.
    [0105] Fig. 3 shows a diagram of the dispersion coefficient estimation procedure where the experimental dispersion measure (MDE) and the dispersion measure in Monte Carlo simulations (MD-MC) can be seen for different values of ^ s. The graphs show the value of the parameter y versus the intensity (I)
    [0107] Fig. 4 thus shows a complete flow chart of the process of the present invention. The following symbols and parameters are shown in Fig. 4:
    [0109] LS - Flat Beam Microscope
    [0110] H - Data transfer function
    [0111] I mc - Simulated intensity
    [0112] lo - Raw raw image
    [0113] I - Image after deconvolution
    [0115] Under these conditions, the steps for the procedure will be as follows:
    [0117] 1. Mounting the sample in the microscope
    [0119] 2. Image capture without filter on the detection objective and illumination of the sample with a Gaussian beam. Images are captured at different z depths and positions on the y axis to estimate the mean dispersion coefficient of the sample.
    [0120] 3. Image acquisition of the sample using the conventional flat beam protocol preferred by the user.
    [0122] 4. Calculation of the deconvolution of the images obtained in step 3 with the transfer function of the medium for the dispersion coefficient estimated experimentally in step 2.
    权利要求:
    Claims (2)
    [1]
    1. Procedure to correct the effect of light scattering in measurements of a flat laser beam microscope, characterized in that it comprises the following steps: a) calculate, through simulations using the Monte Carlo method, the transfer function of a semi-transparent medium for different scattering coefficients and scanning depths, creating a database of transfer functions corresponding to each scattering coefficient and scanning depth;
    b) estimate experimentally using the flat laser beam microscope the dispersion coefficient of a sample assuming that it is uniform throughout the sample, where the estimation comprises the following steps:
    b1) mounting the sample on the flat laser beam microscope;
    b2) illuminating each plane of the sample with a Gaussian beam;
    b3) capture images of the propagation of the Gaussian beam in the sample and repeat the process in different positions; and
    b4) estimating the scattering coefficient of the sample by measuring the divergence of the beams caused by scattering;
    c) normally scanning the sample by means of the flat laser beam microscope, obtaining a set of images corresponding to different sections of the sample; and
    d) deconvolve each image of the sample obtained in the scan with the transfer function of the database that corresponds to the dispersion coefficient of the sample and the scanning depth of each image, thus reversing the effect of dispersion in each image of the light.
    [2]
    2. Process according to claim 1, wherein the deconvolution is carried out using the Wiener method.
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    同族专利:
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