60 results about "Super-resolution microscopy" patented technology

The invention relates to large-field high-resolution microscopy and photolithography setups operating with polychromatic light. It includes the use of a plurality of focusing micromirrors.

The present invention is a method and apparatus that utilizes an inertia-free diffraction mechanism to control both phase and rotation of the standing wave pattern that results in super-resolution at unparalleled imaging speeds. In some embodiments of the present inventions, AODs are utilized to control period, phase, and rotation of the SW pattern in contrast to the commonly used mechano-optical principles. This allows 2D (and 3D) super-resolution imagining at high stability and speed not limited by mechanical constraints. The present invention can be utilized, for example, for real time observations of dynamic processes in living cells.

A microscopy method and apparatus includes placing a specimen to be observed adjacent to a reflective holographic optical element (RDOE). A beam of light that is at least partially coherent is focused on a region of the specimen. The beam forward propagates through the specimen and is at least partially reflected backward through the specimen. The backward reflected light interferes with the forward propagating light to provide a three dimensional interference pattern that is at least partially within the specimen. A specimen region illuminated by the interference pattern is imaged at an image detector. Computational reconstruction is used to generate a microscopic image in all three spatial dimensions (X,Y,Z), simultaneously with resolution greater than conventional microscopy.

The invention discloses a method for two-photon fluorescence stimulated emission differential super-resolution microscopy. The method includes the steps that (1) after being collimated, pulsed laser beams are converted into linear polarized light and polarization modulation is conducted on the linear polarized light, so that radial polarized light is obtained; (2) the radial polarized light is converted into circular polarized light and projected onto a sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore first signal light intensity I1 is obtained; (3) polarization modulation is conducted on the linear polarized light obtained in the step (1) and the linear polarized light is converted into tangential polarized light; (4) the tangential polarized light is converted into circular polarized light and projected onto the sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore second signal light intensity I2 is obtained; (5) effective signal light intensity I is calculated according to a formula I=I1-gammaI2 , so that super-resolution imaging is achieved. The invention further discloses a device for two-photon fluorescence stimulated emission differential super-resolution microscopy. The device is simple, free of light division, low in light power, capable of weakening the photobleaching effect, higher in resolution and larger in imaging depth.

The present disclosure provides improved microscopic imaging techniques, equipment and systems. More particularly, the present disclosure provides advantageous microscopy assemblies with illumination engineering (e.g., 3D microscopy assemblies with illumination engineering), and related methods of use. Disclosed herein is an imaging technique / assembly that uses a spatial light modulator (“SLM”) for 3D tomographic imaging with brightfield or fluorescence illumination that can also be utilized for bright-field, dark-field, phase-contrast, and super-resolution microscopy. Disclosed herein are methods and instrumentation / assemblies having preferred uses for 3D tomographic imaging, and phase-contrast and super-resolution imaging. The present disclosure advantageously provides for assemblies and methods configured to create 3D tomographic images by way of acquiring a series of images with varied angle illumination using a SLM and computational reconstruction that substantially eliminates the need to move the sample. The disclosed assemblies and methods are also able to acquire bright-field, dark-field, various contrast, and super-resolution images.

The invention discloses a structure illumination super-resolution microscopy imaging system and an imaging method thereof. The imaging system comprises an illumination light source, a rotating structure light generator, a first convergent lens, a light splitter, an objective lens, an object stage, a sample, a second convergent lens, a digital imaging device and a computer. An original image is processed through an imaging image reconstruction algorithm; super-resolution can be achieved only by rotating structured light stripes for four times without phase displacement; the imaging image reconstruction algorithm is based on frequency domain processing instead of non-spatial domain processing; a spectrum process of a super-resolution image of a sample is different from a traditional frequency domain method, structured light directions are not analyzed one by one and all directions are merged for analysis; in order to obtain traditional uniform 2-fold resolution improvement in various directions, the theoretical quantity of original images can be reduced to 4 from 9 of a traditional method; the quantity of the original images is further reduced to 3 and uniform 1.5fold resolution improvement in various direction can be theoretically obtained.

A method and apparatus for nanoscopy comprising a salt microlens. The microlens-based nanoscope comprises a conventional microscope, a microlens, and a XYZ piezoelectric stage is shown (SEE FIG. 1A). The microlens is mounted on a Z-stage and can be driven to accomplish the scanning. The specimen is placed above the microlens which is a plano-convex lens. The set up employed for the Salt Microlens for Ultra-high Resolution Imaging (SAMURI) can use a halogen-tungsten lamp with a dominant wavelength at 600 nm. A magnified virtual image of the specimen is obtained when the distance between microlens and specimen is less than the focal length of the microlens. The virtual image can then be magnified by the microscope and captured by eyes or a CCD camera.

Accurate localization of isolated particles is important in single particle based super-resolution microscopy. It allows the imaging of biological samples with nanometer-scale resolution using a simple fluorescence microscopy setup. Nevertheless, conventional techniques for localizing single particles can take minutes to hours of computation time because they require up to a million localizations to form an image. In contrast, the present particle localization techniques use wavelet-based image decomposition and image segmentation to achieve nanometer-scale resolution in two dimensions within seconds to minutes. This two-dimensional localization can be augmented with localization in a third dimension based on a fit to the imaging system's point-spread function (PSF), which may be asymmetric along the optical axis. For an astigmatic imaging system, the PSF is an ellipse whose eccentricity and orientation varies along the optical axis. When implemented with a mix of CPU / GPU processing, the present techniques are fast enough to localize single particles while imaging (in real-time).

Method for spatially high-resolution luminescence microscopy in which label molecules in a sample are activated to emit luminescence radiation comprising activating only a subset of the label molecules in the sample, wherein activated label molecules have a distance to the closest activated molecules that is greater or equal to a length which results from a predetermined optical resolution, detecting the luminescence radiation, generating a frame from the luminescence radiation, identifying the geometric locations of the label molecules with a spatial resolution increased above the predetermined optical resolution, repeating the steps and forming a combined image, and controlling the acquisition of the several frames by evaluating at least one of the frames or a group of the frames and modifying at least one variable for subsequent repetitions of the steps of generating frames for combining into an image.

This invention discloses a super-resolution microscopy method and device, of which the method comprises the following steps: converting laser beam into linearly polarized light after collimation; linearly polarized light is deflected and phase modulated by a spatial light modulator; the deflected beam is focused, collimated and then converted into circularly polarized light for projection on the sample to collect signal light from various scanning points on the sample, and obtaining the first signal light intensity; switching over modulation function to project linearly polarized light modulated by the second phase modulation on the sample to collect signal light from various scanning points on the sample, and obtaining the second signal light intensity; calculating valid signal light intensity to obtain the super-resolution image. This device features in a simple structure and easy operation, which can obtain a super-resolution beyond diffraction limit at a lower luminous power; it is quick in image formation with the frame frequency over 15 frames when the number of scanning points in each image is 512×512 .

The invention discloses a random positioning super-resolution microscopy method and device based on a fluorescence-emission kill mechanism. The method includes the following steps: coaxial and common-path exciting light and restraining light are simultaneously focused on a sample; the position on the sample, with the stimulated fluorescence-emission characteristic, randomly emits fluorescent light; fluorescent signals are collected to generated a sparse fluorescent distribution image; diffraction spots are positioned in a unimolecule manner, and the final product is obtained after different fluorescent light positioning images are synthesized. The device includes a first laser light source, a second laser light source, a reflector, a first dichroic mirror, a kohler mirror group, a second dichroic mirror, a microobjective, a sample, an optical filter, a field lens, an ocular, a wide field sensing element and a computer. The invention has the advantages that the resolution ratio and fineness are high, 20 nm crosswise super-resolution images can be obtained; the structure is simple, and the cost is low; irreversible damage of intensive laser or fluorescence photobleaching to samples is reduced, and repeating utilization ratio of samples is increased; the function extensibility is strong.

The invention discloses a real-time fluorescence radiation differential super-resolution microscopy method based on parallel spot scanning and device. In the method, a laser beam is divided to S-polarized light and P-polarized light, the S-polarized light is modulated to a circularly-polarized solid spot, and the P-polarized light is firstly modulated to eddy polarized light and is then modulatedto a circularly-polarized hollow spot; solid spot excitation light and hollow spot excitation light are staggered for at least 200 nm on an object plane; the solid spot excitation light and the hollowspot excitation light are used to perform two-dimensional scanning on a fluorescence sample simultaneously, and a positive confocal fluorescence intensity map obtained by solid spot modulation and anegative confocal fluorescence intensity map obtained by hollow spot modulation are obtained; and the two fluorescence intensity maps are subjected to shift matching. As two spots are adopted for simultaneous scanning, in comparison with the method of switching a modulation spot back and forth by the traditional fluorescence emission differential microscopy system, the sampling speed is more thantwice the traditional speed, the super-resolution dynamic microscopy effects under the confocal scanning speed can be realized, and the imaging speed can be improved significantly.

A method for high-resolution luminescence microscopy of a sample marked with marking molecules that can be activated to excite particular luminescent radiation, including: repeated activation of a subset of the marking molecules to emit luminescent radiation; repeated imaging of the sample along a depth direction and with a predetermined optical resolution; and producing images from the repeated imaging. Locations of the marking molecules are determined with a spatial resolution that is increased above the predetermined optical resolution. Activation of the marking molecules can be through radiation introduced into multiple regions, each extending along a plane substantially perpendicular to the depth direction. The regions can be arranged so that the regions are behind one another and overlap only partially. Separate images of the sample may be recorded for activation in each of the regions in order to obtain depth information relating to the marking molecules from the separate images.

The invention discloses a method for Fourier domain iterative splicing super-resolution microscopy based on surface wave illumination. The method comprises the following steps of (1) by changing the illumination angle of incident illumination light, stimulating the surface wave which is propagated along different directions at an interface of a sample and air; (2) enabling a surface wave illumination sample to generate frequency spectrum shifting along the corresponding transverse wave vector, and shifting a high-efficiency component of an object to the lens low objective low passband range; (3) enabling a CCD (charge coupled device) to shoot up an image corresponding to each illumination angle, substituting into a Fourier domain iterative splicing (FP (Fabry-Perot)) algorithm, and finally reconstructing the strength and phase distribution of the complicated sample. The invention also discloses a device for the Fourier domain iterative splicing super-resolution microscopy based on the surface wave illumination. The method has the advantages that the restored quantitative phase does not need to be obtained by interference; when the etching depth of the restored etching grid sample is calculated, the accuracy is verified through AFM (atomic force microscope) detection; the broad application prospect is realized in the material and life sciences.

Described are optical methods and reagents for sequencing polypeptides. A probe that exhibits different spectral properties when conjugated to different N-terminal amino acids is conjugated to the N-terminal amino acid of a polypeptide. Sequentially detecting one or more spectral properties of the probe conjugated to the N-terminal amino acid and cleaving the N-terminal amino acid produces sequence information of the polypeptide. The use of super-resolution microscopy allows for the massively parallel sequencing of individual polypeptide molecules in situ such as within a cell. Also described are probes comprising hydroxymethyl rhodamine green, an isothiocyanate group and a protecting group.

Methods and systems for the super-resolution imaging can make visible strongly subwavelength feature sizes (even below 100 nm) in the optical images of biomedical or any nanoscale structures. The main application of the proposed methods and systems is related to label-free imaging where biological or other objects are not stained with fluorescent dye molecules or with fluorophores. This label-free microscopy is more challenging as compared to fluorescent microscopy because of the poor optical contrast of images of objects with subwavelength dimensions. However, these methods and systems are also applicable to fluorescent imaging. Their use is extremely simple, and it is based on application of the microspheres or microcylinders or, alternatively, elastomeric slabs with embedded microspheres or microcylinders to the objects which are deposited on the surfaces covered with thin metallic layers or metallic nanostructures. The mechanism of imaging involved use of the plasmonic near-fields for illuminating the objects and virtual imaging of these objects through microspheres or microcylinders. These methods and systems do not require use of fragile probe tips and slow point-by-point scanning techniques. These methods and systems can be used in conjunction with any types of microscopes including upright, inverted, fluorescence, confocal, phase-contrast, total internal reflection and others. Scanning the samples can be performed using micromanipulation with individual spheres or cylinders or using translation of the slabs. These methods and systems are applicable to dry, wet and totally liquid-immersed samples and structures.

The invention discloses a high speed adaptive optical ring spot correction system and method based on machine learning. According to the system and the method, a learning model is established, a mapping relationship between distorted ring spot forms and phase reconstruction coefficients required for correcting deformation is established, a to-be-tested distorted ring spot is input into a trained model, the phase reconstruction coefficient for correcting the distortion can be solved, and further correction phase reconstructed by the phase reconstruction coefficient is loaded to a spatial lightmodulator, so the distortion can be corrected. According to the system and the method, rapid correction can be carried out on ring spot aberration in a stimulated emission depletion (STED) fluorescence microscopy, precision is high, the system is simple and is easy to operate, and the resolution reduction problem resulting from the aberration and scattering generated when sample deep layer imagingis carried out in an STED super-resolution microscopy imaging technology is solved.

The invention discloses a multiband fluorescence loss method, a multicolor super-resolution imaging method and a device. Multiband fluorescence refers to up-conversion fluorescence emitted in the process of electrons reaching a Tm<3+> high energy level and then sequentially migrating energy to the Gd<3+> ions and shell activated ions X<3+> in the Yb<3+> / Tm<3+> sensitization and up-conversion process of an other activated ions-doped shell wrapping a NaGdF4:Yb<3+> / Tm<3+> core. The up-conversion fluorescence can be different by changing the activated ions by means of the same sensitization, up-conversion and energy migration process. The fluorescence loss process is the process of loss of electrons of the Tm<3+> high energy level and the loss of multiband fluorescence emitted during the transfer of the Tm<3+> high energy level from Gd<3+> to X<3+>, caused by the stimulated emission transition of intermediate energy level electrons to a low energy level in the up-conversion process by using the stimulated emission of laser with the wavelength nearby 810 nm between the Tm<3+> matching energy levels. Nano-particles of different activated ions are synthesized based on the fluorescence loss method, and multicolor super-resolution microscopy imaging is realized using the same pair of excitation light and hollow loss light. An optical system is greatly simplified, and the cost of the system is reduced.

The invention discloses a multi-mode optical fiber super-resolution imaging device based on wavefront shaping and a light spot correction method thereof, and belongs to the field of super-resolution microscopy; after quenching light generated by a first laser and excitation light generated by a second laser are injected into a multi-mode optical fiber, the light spot at the exit end of the multi-mode optical fiber is imaged on a camera of a correction system, a modulation signal on a spatial light modulator is continuously converted, the light spot intensity information acquired by the camerais taken as the data base of a multi-mode optical fiber mode correlation correction method to correct the modulated signals of the spatial light modulator, at the exit end of the multi-mode optical fiber, Elie spot-shaped excitation light spots and bread ring-shaped quenching light spots are generated. The quenching light and the excitation light are moved to scan a sample, and the imaging of different depths in the biological tissue sample is achieved by moving the optical fiber, so that the imaging quality reduction caused by scattering in the biological tissue imaging process is overcome, high resolution and large imaging depth are achieved, so that the method is widely applied in biomedicine.

A super-resolution microscopy method includes forming an image of an emitting particle in a detection plane of a detector by a microscopy imaging system and correcting, by a wavefront-modulating device, at least some of the optical defects present between the emitting particle and the detection plane. The method further includes introducing, via the wavefront-modulating device, a deformation of the wavefront emitted by the emitting particle, of variable amplitude, allowing a bijective relationship to be formed between the shape of the image of the emitting particle in the detection plane and the axial position of the emitting particle relative to an object plane that is optically conjugated with the detection plane by the microscopy imaging system. The method further includes controlling the amplitude of the deformation of the wavefront by controlling the wavefront-modulating device, as a function of the given range of values of the axial position of the particle.

The invention discloses a multi-color super-resolution microscopy system and method based on a single channel. The system comprises a loss light source, a first collimating unit, a phase plate, a first excitation light source, a first dichroic mirror, a second collimating unit, a half wave plate, a polarizing beam splitter, a second excitation light source, a first detector, a second dichroic mirror, a lens-aperture-lens group, a third dichroic mirror, a scanning mirror, a second detector, a third collimating unit, a quarter wave plate, a microscopic objective and a sample stage. According to the system and method, two-color (or multi-color) super-resolution microscopy can be realized with one loss light source, and the system is simple in architecture and lower in cost. Stronger background noise caused by large energy consumption and crosstalk in a conventional single-channel multi-color super-resolution system can be overcome, the work precision is high, and the result is accurate. Therefore, compared with the prior art, the work efficiency and the experiment precision of the multi-color super-resolution microscopy system can be improved, and the cost is effectively reduced.

The invention discloses a super-resolution microscopy method based on speckle illumination. The method comprises the following steps: modulating laser beam, focusing the laser beam onto a sample to be tested, so as to form a speckle illumination pattern, and collecting fluorescence emitted by the sample to be tested, so as to obtain a fluorescence intensity image; changing the speckle illumination pattern, so as to obtain a plurality of fluorescence intensity images under different speckle illumination patterns; adding all the fluorescence intensity images together to obtain an image serving as a wide field image, and performing deconvolution on the wide field image, so as to obtain the initial estimation of the sample; calculating an initial illumination image through a gradient descent algorithm according to the obtained initial estimation; working out a sample image with higher resolution on the basis of an obtained object image and an initial illumination image through an FP algorithm; taking the worked out sample image as an estimated value of the sample, repeating iteration till iteration is accomplished, so as to obtain a super-resolution image. The invention further discloses a super-resolution microscopy device based on speckle illumination.

Methods and systems for the super-resolution imaging can make visible strongly subwavelength feature sizes (even below 100 nm) in the optical images of biomedical or any nanoscale structures. The main application of the proposed methods and systems is related to label-free imaging where biological or other objects are not stained with fluorescent dye molecules or with fluorophores. This label-free microscopy is more challenging as compared to fluorescent microscopy because of the poor optical contrast of images of objects with subwavelength dimensions. However, these methods and systems are also applicable to fluorescent imaging. Their use is extremely simple, and it is based on application of the microspheres or microcylinders or, alternatively, elastomeric slabs with embedded microspheres or microcylinders to the objects which are deposited on the surfaces covered with thin metallic layers or metallic nanostructures. The mechanism of imaging involved use of the plasmonic near-fields for illuminating the objects and virtual imaging of these objects through microspheres or microcylinders. These methods and systems do not require use of fragile probe tips and slow point-by-point scanning techniques. These methods and systems can be used in conjunction with any types of microscopes including upright, inverted, fluorescence, confocal, phase-contrast, total internal reflection and others. Scanning the samples can be performed using micromanipulation with individual spheres or cylinders or using translation of the slabs. These methods and systems are applicable to dry, wet and totally liquid-immersed samples and structures.

The invention discloses a multi-color super resolution microscope system with an automatic alignment function, belonging to the field of microscopic imaging. The system couples an excitation beam anda suppression beam into the same single mode polarization-maintaining fiber to form a mixed beam. Only the suppression beam is affected while the excitation beam is unaffected when the mixed beam is phase modulated via a phase modulation assembly. According to the multi-color super resolution microscope system with the automatic alignment function, the influence of beam drift on the performance ofthe super resolution microscope system can be avoided, so that the system structure is simplified, and the system stability is improved. Compared with the prior art, the system disclosed by the invention can conveniently realize multi-color imaging.

The present invention relates to a method of processing images captured following structured illumination of a sample, the method comprising the steps of: identifying emission spots within each captured image; verifying the emission spots; and reconstructing an enhanced image of the sample from the emission spots. The method may comprise identifying only in focus emission spots. By identifying and processing only in focus spots, whether or not they are centred on expected illumination positions, improvements in resolution can be achieved compared to known SIM methods. In particular, by suitable selection of in focus spots, significant improvements in lateral and axial resolution can be achieved.

The invention discloses a two-dimensional super-resolution microscopy method. The two-dimensional super-resolution microscopy method comprises the following steps of: (1) starting a first confocal imaging mode, and collecting signal light emitted by a sample to be tested, and thus obtaining I1 (x,y); (2) starting a second confocal imaging mode, collecting signal light emitted by the sample to be tested, and thus obtaining I2 (x,y); (3) starting a first negative confocal imaging mode, and collecting signal light emitted by the sample to be tested, and thus obtaining I3 (x,y); (4) starting a second negative confocal imaging mode, and collecting signal light emitted by the sample to be tested, and thus obtaining I4 (x,y); and (5) carrying out calculation according to a formula to obtain an effective signal light intensity I (x,y), and obtaining an super-resolution image by virtue of I (x,y). The invention further discloses a two-dimensional super-resolution microscopy apparatus. The two-dimensional super-resolution microscopy method and the two-dimensional super-resolution microscopy apparatus have the advantages of high imaging speed, simple apparatus and good signal to noise ratio.