39 results about "Structured illumination microscopy" patented technology

A multimode waveguide illuminator and imager relies on a wave front shaping system that acts to compensate for modal scrambling and light dispersion by the multimode waveguide. A first step consists of calibrating the multimode waveguide and a second step consists in projecting a specific pattern on the waveguide proximal end in order to produce the desire light pattern at its distal end. The illumination pattern can be scanned or changed dynamically only by changing the phase pattern projected at the proximal end of the waveguide. The third and last step consists in collecting the optical information, generated by the sample, through the same waveguide in order to form an image. Known free space microscopy technique can be adapted to endoscopy with multimode waveguide, such as, but not limited to, fluorescence imaging or Raman spectroscopy or imaging, 3D linear scattering imaging or two-photon imaging. Super-resolution, i.e., resolution below the diffraction limit, is achieved for example but not limited to, using the STimulated Emission Depletion microscopy (STED) technique or the Structured Illumination Microscopy (SIM) technique or a stochastic illumination based method (PALM, STORM) in combination with the multimode waveguide imaging method.

According to a first aspect, the invention relates to a multimodal optical sectioning microscope (200, 400, 600) for full-field imaging of a volumic and scattering sample comprising:—a full-field OCT system for providing an image of a first section in depth of the sample comprising an illumination sub-system (201, 401, 601) and a full-field imaging interferometer with a detection sub system (208, 408, 608) and an optical conjugation device for optically conjugating the sample and said detection sub system, wherein said optical conjugation device comprises a microscope objective (203, 403, 603),—a supplementary full-field optical sectioning imaging system for providing a fluorescent image of a second section in depth of said sample comprising a structured illumination microscope with an illumination sub system (623), means (421, 422) for generating at the focal plane of said microscope objective of said full-field imaging interferometer a variable spatial pattern illumination and a detection sub system (624), optically conjugated with said focal plane of the microscope objective.

An acquiring unit of a structured illuminating microscopy apparatus acquires at least two modulated images having the same wave number vector and the different phases; and a calculating unit of the structured illuminating microscopy apparatus, in a spatial frequency spectrum of each of at least the two modulated images acquired by the acquiring unit, separates a 0th-order modulating component and ±first-order modulating components of observational light fluxes superimposed on arbitrary two observation points based on at least four observation values regarding the two observation points which are mutually displaced by an amount of the wave number vector.

In order to realize a high-speed switching of structured pattern, a structured illuminating microscopy includes a driving unit generating a sonic standing wave in a sonic wave propagation path by giving a driving signal for vibrating a medium of the sonic wave propagation path to a light modulator, an illuminating optical system making at least three diffracted components of the exit light flux passed through the sonic wave propagation path to be interfered with one another, and forming interference fringes on an observational object, an image-forming optical system forming an image by an observational light flux from the observational object on a detector, and a controlling unit controlling a contrast of an image by modulating at least one of an intensity of the exit light flux, an intensity of the observational light flux, and the detector with a modulating signal of 1 / N frequency of the driving signal.

A multimode waveguide illuminator and imager relies on a wave front shaping system that acts to compensate for modal scrambling and light dispersion by the multimode waveguide. A first step consists of calibrating the multimode waveguide and a second step consists in projecting a specific pattern on the wave guide proximal end in order to produce the desire light pattern at its distal end. The illumination pattern can be scanned or changed dynamically only by changing the phase pattern projected at the proximal end of the waveguide. The third and last step consists in collecting the optical information, generated by the sample, through the same waveguide in order to form an image. Known free space microscopy technique can be adapted to endoscopy with multimode waveguide, such as, but not limited to, fluorescence imaging or Raman spectros copy or imaging, 3D linear scattering imaging or two-photon imaging. Super-resolution, i.e., resolution below the diffraction limit, is achieved for example but not limited to, using the STimulated Emission Depletion microscopy (STED) technique or the Structured Illumination Microscopy (SIM) technique or a stochastic illumination based method (PALM, STORM) in combination with the multimode waveguide imaging method.

Illumination phase controls that provide precise and fast phase control of structured illumination patterns used in structure illumination microscopy are described. A coherent light source is used to generate a beam of coherent light that is split into at least three coherent beams of light. In one aspect, an illumination phase control is composed of at least one pair of rotatable windows to apply at least one phase shift to at least one of the beams. An objective lens is to receive the beams and focus the at least three beams to form an interference pattern. The phase control can be used to change the position of the interference pattern by changing the at least one phase shift applied to the at least one beam.

The invention discloses a three-dimensional outline measuring method based on Structured Illumination Microscopy SIM. The method specifically comprises the following steps: I, acquiring three-dimensional profile distribution spot cloud data of a measured object according to a three-dimensional profile measuring method; II, acquiring a two-dimensional planar image of the measured object according to a structured illumination method; III, performing edge extraction processing on the two-dimensional planar image obtained in the step II, so as to obtain a super-resolution edge outline of the measured object; IV, fusing the three-dimensional profile distribution spot cloud data obtained in the step I with the edge outline obtained in the step III, thereby obtaining three-dimensional profile spot cloud images with accurate edge outlines. According to the method, a three-dimensional profile measurement method is combined with an SIM technique, three-dimensional edge information of the measured object can be accurately obtained, and defects of a conventional 3D profile measurement technique can be effectively overcome.

Methods, systems, and devices described herein enable single-image optical sectioning, even at depth within turbid media, such as human skin or other tissue. Embodiments can eliminate the need for multiple image samples or raster scans, making in-vivo or live biological imaging easier and faster than multi-image sectioning techniques. Better contrast and resolution than traditional three-phase structured illumination microscopy (SIM) is possible in turbid media. Embodiments enable imaging of cell nuclei. Resolution and contrast resulting from disclosed embodiments are less sensitive to motion of or within patients or other targets than confocal microscopy and three-phase SIM techniques. Three-dimensional images of target specimens can be provided based on a group of single-image optical sections. Real-time imaging can also be provided.

Presented are a structured illumination microscopy system using a digital micromirror device and a time-complex structured illumination, and an operation method therefor. A structured illumination microscopy system using a digital micromirror device and a time-complex structured illumination according to an embodiment may comprise: a light source; a digital micromirror device (DMD) for receiving light irradiated from the light source, implementing a time-complex structured illumination, and causing a controlled structured illumination to enter a sample; and a fluorescence image measurement unit for extracting a high-resolution 3D fluorescence image of the sample.

Variable orientation illumination-pattern rotators ("IPRs") that can be incorporated into structured illumination microscopy instruments to rapidly rotate an interference pattern are disclosed. An IPR includes a rotation selector and at least one mirror cluster. The rotation selector directs beams of light into each one of the mirror clusters for a brief period of time. Each mirror cluster imparts a particular predetermined angle of rotation on the beams. As a result, the beams output from the IPR are rotated through each of the rotation angles imparted by each of the mirror clusters. The rotation selector enables the IPR to rotate the beams through each predetermined rotation angle on the order of 5 milliseconds or faster.

A method for imaging an object using a microscope includes obtaining axial response data, the axial response data representative of a relationship between a separation between a top surface of the object and an objective lens of the microscope and an intensity of light reflected by the top surface of the object; positioning the object at a distance from the objective lens that is within a linear region of the axial response data; sequentially illuminating the object with a plurality of periodic patterns; obtaining a plurality of images of the object, each image resulting from the illumination of the object with a corresponding one of the plurality of periodic patterns; determining a reconstructed image of the object based on the plurality of images of the object; and, based on variations in the intensity of the reconstructed image, determining a topographic profile of the top surface of the object.

A fluorescence microscope for obtaining super-resolution images of a sample labeled with at least one fluorescent label by combining localization microscopy and structured illumination microscopy is provided. The fluorescence microscope includes one or more light sources, an illumination system having a structured illumination path for illuminating the sample with structured illumination light and a localization illumination path for illuminating the sample with localization illumination light.

A structured illumination apparatus includes a light modulator being disposed in a light path of an exit light flux from a light source, and in which a sonic wave propagation path is arranged in a direction traversing the exit light flux; a driving unit generating a sonic standing wave in the sonic wave propagation path by giving a driving signal for vibrating a medium of the sonic wave propagation path to the light modulator; an illuminating optical system making at least three diffracted lights of the exit light flux passed through the sonic wave propagation path to be interfered with one another, and forming interference fringes of the diffracted lights on an observational object; and a controlling unit controlling a contrast of the interference fringes by modulating a phase of at least one diffracted light among the diffracted lights in a predetermined pitch.

In order to realize a high-speed switching of structured pattern, a structured illuminating microscopy includes a driving unit generating a sonic standing wave in a sonic wave propagation path by giving a driving signal for vibrating a medium of the sonic wave propagation path to a light modulator, an illuminating optical system making at least three diffracted components of the exit light flux passed through the sonic wave propagation path to be interfered with one another, and forming interference fringes on an observational object, an image-forming optical system forming an image by an observational light flux from the observational object on a detector, and a controlling unit controlling a contrast of an image by modulating at least one of an intensity of the exit light flux, an intensity of the observational light flux, and the detector with a modulating signal of 1 / N frequency of the driving signal.

A structured illumination apparatus includes a light modulator being disposed in a light path of an exit light flux from a light source, and in which a sonic wave propagation path is arranged in a direction traversing the exit light flux; a driving unit generating a sonic standing wave in the sonic wave propagation path by giving a driving signal for vibrating a medium of the sonic wave propagation path to the light modulator; an illuminating optical system making at least three diffracted lights of the exit light flux passed through the sonic wave propagation path to be interfered with one another, and forming interference fringes of the diffracted lights on an observational object; and a controlling unit controlling a contrast of the interference fringes by modulating a phase of at least one diffracted light among the diffracted lights in a predetermined pitch.

Methods and apparatuses are disclosed whereby structured illumination microscopy (SIM) is applied to a scanning microscope, such as a confocal laser scanning microscope or sample scanning microscope, in order to improve spatial resolution. Particular aspects of the disclosure relate to the discovery of important advances in the ability to (i) increase light throughput to the sample, thereby increasing the signal / noise ratio and / or decreasing exposure time, as well as (ii) decrease the number of raw images to be processed, thereby decreasing image acquisition time. Both effects give rise to significant improvements in overall performance, to the benefit of users of scanning microscopy.

Various embodiments (300, 400, 500) for a multi-focal selective illumination microscopy (SIM) system for generating multi-focal patterns of a sample are disclosed. The embodiments (300, 400, 500) of the multi-focal SIM system perform a focusing, scaling and summing operation on each generated multi-focal pattern in a sequence of multi-focal patterns that completely scan the sample to produce a high resolution composite image.

An ultra-high-speed 3D refractive index tomography and structured illumination microscopy system using a wavefront shaper and a method using the same are provided. A method of using an ultra-high-speed 3D refractive index tomography and structured illumination microscopy system that utilizes a wavefront shaper includes adjusting an irradiation angle of a plane wave incident on a sample by using the wavefront shaper, measuring a 2D optical field, which passes through the sample, based on the irradiation angle of the plane wave, and obtaining a 3D refractive index image from information of the measured 2D optical field by using an optical diffraction tomography or a filtered back projection algorithm.

In a method and apparatus for determining the height of a plurality of spatial positions on a surface of a specimen, a light beam is projected on the surface. The light beam has a sinusoidal spatial pattern in at least two directions perpendicular to an optical axis of the light beam, and which is moved to different spatial pattern positions. The surface is scanned along said optical axis in different scanning positions. A fixed relationship between a moving distance between subsequent spatial pattern positions, and a scanning distance between subsequent scanning positions exists. The light reflected by the surface is detected in scanning positions with the spatial pattern having corresponding spatial pattern positions. From the detected light for each spatial position of the surface, an envelope curve of intensity values corresponding to scanning positions is determined. A maximum of the envelope curve and its corresponding scanning position being representative of the height of the spatial position of the surface is selected. The spatial pattern is moved in a sequence of 2n steps (n > 2) in a first and a second spatial direction over a distance of 1 / 4 and 1 / n pattern wavelength, respectively.

An apparatus (10) for structured illumination microscopy (SIM) comprises a pulsed femtosecond MiTai laser (11) operable to generate a pulsed beam. The beam pulses are directed on to a specimen (12) via an optical arrangement including a beam steering apparatus comprising a pair of acousto-optic deflectors AODx, AODy, each operable to vary the deflection angle of the beam in response to variation in the frequency of an applied acoustic deflection signal,and an acousto-optic modulator AOM. The frequency of the acoustic deflection signals applied to the AODs and / or the frequency of the acoustic compensation signal applied to the AOM in the present invention is dynamically modulated. This dynamic modulation can increase the effective AODxy scan angle in each direction by about 4 mrads (equivalent to a 15% increase in the area of the field of view). Furthermore, dynamic modulation of the compensation frequency also improves the evenness of the illumination over the field of view.

In structured illumination microscopy, the multiple recording of images with different phase positions of the structuring requires a high stability in the optical arrangement and sample throughout the entire measuring process. Also, the structuring must be projected into the sample in a highly homogeneous manner. The current invention optimizes recording of individual images in order to achieve the best possible resolution in the result image even in problematic samples. An optimization of this kind can be carried out in different ways, for example, by determining an optimal adjustment for at least one illumination parameter or recording parameter or by pulsed illumination such that an excitation from a triplet state of the fluorescent dye to a higher triplet state is reduced, or by illuminating the sample with depletion light for depopulating a triplet state of the fluorescent dye, which reduces bleaching.

An ultra-high-speed 3D refractive index tomography and structured illumination microscopy system using a wavefront shaper and a method using the same are provided. A method of using an ultra-high-speed 3D refractive index tomography and structured illumination microscopy system that utilizes a wavefront shaper includes adjusting an irradiation angle of a plane wave incident on a sample by using the wavefront shaper, measuring a 2D optical field, which passes through the sample, based on the irradiation angle of the plane wave, and obtaining a 3D refractive index image from information of the measured 2D optical field by using an optical diffraction tomography or a filtered back projection algorithm.

Methods and apparatuses are disclosed whereby structured illumination microscopy (SIM) is applied to a scanning microscope, such as a confocal laser scanning microscope or sample scanning microscope, in order to improve spatial resolution. Particular aspects of the disclosure relate to the discovery of important advances in the ability to (i) increase light throughput to the sample, thereby increasing the signal / noise ratio and / or decreasing exposure time, as well as (ii) decrease the number of raw images to be processed, thereby decreasing image acquisition time. Both effects give rise to significant improvements in overall performance, to the benefit of users of scanning microscopy.

Presented are a structured illumination microscopy system using a digital micromirror device and a time-complex structured illumination, and an operation method therefor. A structured illumination microscopy system using a digital micromirror device and a time-complex structured illumination according to an embodiment may comprise: a light source; a digital micromirror device (DMD) for receiving light irradiated from the light source, implementing a time-complex structured illumination, and causing a controlled structured illumination to enter a sample; and a fluorescence image measurement unit for extracting a high-resolution 3D fluorescence image of the sample.

Techniques are described for reducing the number of angles needed in structured illumination imaging of biological samples through the use of patterned flowcells, where nanowells of the patterned flowcells are arranged in, e.g., a square array, or an asymmetrical array. Accordingly, the number of images needed to resolve details of the biological samples is reduced. Techniques are also described for combining structured illumination imaging with line scanning using the patterned flowcells.

Techniques are described for reducing the number of angles needed in structured illumination imaging of biological samples through the use of patterned flowcells, where nanowells of the patterned flowcells are arranged in, e.g., a square array, or an asymmetrical array. Accordingly, the number of images needed to resolve details of the biological samples is reduced. Techniques are also described for combining structured illumination imaging with line scanning using the patterned flowcells.