175 results about "Laser scanning microscope" patented technology

A laser scanning microscope produces molecular excitation in a target material by simultaneous absorption of three or more photons to thereby provide intrinsic three-dimensional resolution. Fluorophores having single photon absorption in the short (ultraviolet or visible) wavelength range are excited by a beam of strongly focused subpicosecond pulses of laser light of relatively long (red or infrared) wavelength range. The fluorophores absorb at about one third, one fourth or even smaller fraction of the laser wavelength to produce fluorescent images of living cells and other microscopic objects. The fluorescent emission from the fluorophores increases cubicly, quarticly or even higher power law with the excitation intensity so that by focusing the laser light, fluorescence as well as photobleaching are confined to the vicinity of the focal plane. This feature provides depth of field resolution comparable to that produced by confocal laser scanning microscopes, and in addition reduces photobleaching and phototoxicity. Scanning of the laser beam by a laser scanning microscope, allows construction of images by collecting multi-photon excited fluorescence from each point in the scanned object while still satisfying the requirement for very high excitation intensity obtained by focusing the laser beam and by pulse time compressing the beam. The focused pulses also provide three-dimensional spatially resolved photochemistry which is particularly useful in photolytic release of caged effector molecules, marking a recording medium or in laser ablation or microsurgery. This invention refers explicitly to extensions of two-photon excitation where more than two photons are absorbed per excitation in this nonlinear microscopy.

Apparatus and methods for fluorescence imaging using radiofrequency multiplexed excitation. One apparatus splits an excitation laser beam into two arms of a Mach-Zehnder interferometer. The light in the first beam is frequency shifted by an acousto-optic deflector, which is driven by a phase-engineered radiofrequency comb designed to minimize peak-to-average power ratio. This RF comb generates multiple deflected optical beams possessing a range of output angles and frequency shifts. The second beam is shifted in frequency using an acousto-optic frequency shifter. After combining at a second beam splitter, the two beams are focused to a line on the sample using a conventional laser scanning microscope lens system. The acousto-optic deflectors frequency-encode the simultaneous excitation of an entire row of pixels, which enables detection and de-multiplexing of fluorescence images using a single photomultiplier tube and digital phase-coherent signal recovery techniques.

A method for investigating specimens, wherein a spectral splitting of the radiation coming from the specimen is carried out for specimen points or point distributions, for the operation of a laser scanning microscope or a fluorescence screening arrangement or a flow cylinder comprising the steps of generating a λ-stack so that the spectral distribution is measured by individual detection channels and storing the signals so as to be correlated to the detection signals with at least one of the spatial coordinates x, y and z and / or so as to be correlated to the measurement time t.

Systems and methods according to embodiments of the present invention facilitate imaging and sectioning of a thick specimen that allow for 3D image reconstruction. An example embodiment employs a laser scanning microscope and sectioning device, where the specimen, and optionally, the sectioning device are affixed to respective programmable stages. The stage normally used for aligning the specimen with the microscope objective is used as an integral component for sectioning the specimen. A specimen is imaged such that the imaging depth is less than the sectioning depth to produce overlap in contiguous sets of images; both acts are repeated until the imaging is completed. A substantially or completely seamless 3D image of the specimen is reconstructed by collecting sets of 2D images and aligning imaged features of structures in overlapping images or portions thereof. Specimen may be from a human, animal, or plant.

A method and / or arrangement for the analysis of fluorescing samples in an image-generating microscope system, preferably a laser scanning microscope, wherein the sample is scanned point-by-point or line-by-line in at least one surface section and a dispersive splitting of the radiation coming from the sample is carried out during the scanning, wherein the split radiation is detected by at least one line of detector elements in a wavelength-dependent manner, a selection of two-dimensional or three-dimensional sample parts which correspond to pre-stored two-dimensional or three-dimensional geometric objects or the like is carried out based on the recorded and stored intensity distribution of at least one of these detection elements and / or at least one other detection element for the radiation reflected from the sample by image processing, and an analysis of the spectral signature and / or spatial spectral sequence is carried out for at least a portion of these sample regions with respect to the fluorescence markers arranged thereon.

The invention describes a highly compact laser scanning microscope with integrated short-pulse laser. Direct coupling or a fiber coupling of the short-pulse laser with the laser scanning microscope is advantageously circumvented with this arrangement. This compact arrangement of the laser in the scan module of the laser scanning microscope can be used in a particularly advantageous manner, for example, in multiphoton microscopy for three-dimensionally resolved microscopic analysis, e.g., of biological specimens. Because of the inherent depth discrimination of the multiphoton technique, confocal pinholes can be entirely omitted in the detection beam path. Accordingly, the microscope system can be realized in a very simple manner with respect to engineering and is particularly simple to handle with respect to application. Through the use of a short-pulse laser system in which a plurality of wavelengths are available simultaneously, diverse applications can be realized in one and the same compact microscope system.

The invention relates to a method and apparatus for determining the polarization properties of light emitted, reflected or transmitted by a material using a laser scanning microscope with the tested material being illuminated point by point with a laser beam of known polarization state. According to the invention the light beam with a polarization state modified by the material or the light emitted by the material is being examined by measuring the intensity of two different polarization components of a selected light beam received from each point of said material essentially at the same time and assigning a signal obtained by processing the two intensity signals to a respective point of an image of said material. The apparatus has a polarization state generator between the laser light source and the material being tested, and a detector in a light beam for determining the intensity of light with a polarization state modified by the material or the intensity of light emitted by the material, the improvement of which is that a means for dividing the polarization components in space or time is used in front of the detector.

A method for generating an image of a sample by a microscopy method including varying local resolution, wherein at least two of the following microscopy methods are combined: laser scanning microscopy, a microscopy method wherein the sample is excited to luminescence by structured line or wide area illumination, and a first microscopy image is generated from the images thus obtained, having increased local resolution greater than the optical resolution of the image, a further microscopy method according to the PAL principle, by which a second microscopy image is generated, indicating geometric locations of marker molecules emitting luminescent radiation at an increased local resolution relative to the optical resolution, and a further microscopy method, wherein the sample is marked using marking molecules suitable for the STED, ESA, or RESOLFT technique, and a third microscopy image is generated of STED, ESA, or RESOLFT, wherein the obtained images are superimposed.

Apparatus and methods for fluorescence imaging using radiofrequency multiplexed excitation. One apparatus splits an excitation laser beam into two arms of a Mach-Zehnder interferometer. The light in the first beam is frequency shifted by an acousto-optic deflector, which is driven by a phase-engineered radiofrequency comb designed to minimize peak-to-average power ratio. This RF comb generates multiple deflected optical beams possessing a range of output angles and frequency shifts. The second beam is shifted in frequency using an acousto-optic frequency shifter. After combining at a second beam splitter, the two beams are focused to a line on the sample using a conventional laser scanning microscope lens system. The acousto-optic deflectors frequency-encode the simultaneous excitation of an entire row of pixels, which enables detection and de-multiplexing of fluorescence images using a single photomultiplier tube and digital phase-coherent signal recovery techniques.

A method for investigating specimens, wherein a spectral splitting of the radiation coming from the specimen is carried out for specimen points or point distributions, for the operation of a laser scanning microscope or a fluorescence screening arrangement or a flow cylinder comprising the steps of generating a lambd-stack so that the spectral distribution is measured by individual detection channels and storing the signals so as to be correlated to the detection signals with at least one of the spatial coordinates x, y and z and / or so as to be correlated to the measurement time t.

A laser scanning microscope includes an acousto-optic element or an electro-optic element in an illumination optical system which introduces a laser beam from a laser beam source of the laser scanning microscope into a microscope. The laser scanning microscope further includes a beam expander disposed between the laser beam source and the acousto-optic element or the electro-optic element.

A technique is described for performing critical parameter analysis (CPA) of a semiconductor device (DUT) by combining the capabilities of conventional automated test equipment (ATE) with a focused optical beam scanning device such as a laser scanning microscope (LSM). The DUT is provided with a fixture such that it can be simultaneously scanned by the LSM or a similar device and exercised by the ATE. The ATE is used to determine pass / fail boundaries of operation of the DUT. Repeatable pass / fail limits (for timing, levels, etc.) are determined utilizing standard test patterns and methodologies. The ATE vector pattern(s) can then be programmed to “loop” the test under a known passing or failing state. When light energy from the LSM scanning beam sufficiently disturbs the DUT to produce a transition (i.e., to push the device outside of its critical parameter limits), this transition is indicated on the displayed image of the DUT, indicating to the user which elements of the DUT were implicated in the transition.

An arrangement for coupling at least one beam of optical tweezers for trapping particles and / or a treatment beam into a microscope beam path, preferably in a laser scanning microscope, wherein means are provided for changing the position of the beam focus of the optical tweezers and / or of the treatment beam in a freely adjustable manner for purposes of changing the focal position of the microscope.

For the purpose of calibration which is simple and can also be carried out as often as desired, an arrangement and a method for calibrating a preferably confocal laser scanning microscope, it being possible for an object (1) to be scanned by a scanning beam (2), are defined by calibration means (12) which are arranged in the plane of an intermediate image (11) and can likewise be scanned by the scanning beam (2).

A method of finding, recording and optionally evaluating object structures, especially on slides, preferably of fluorescent object structures such as gene spots. A microscope with a CCD camera, a scanning microscope or a preferably confocal laser scanning microscope can be used for recording and for the rapid and reliable detection of the object structures, with the image data being recorded using an illumination pattern that is projected into the object plane.

Provided is a laser scanning microscope having a first focusing-position control unit; and a confocal detecting unit. The first focusing-position control unit shifts a first focusing position of a laser beam on a sample in a direction of an optical axis of an objective lens. The confocal detecting unit has a confocal aperture for a confocal detection of a light emitted from the first focusing position. The microscope may include a second focusing-position control unit shifts a second focusing position of the light emitted from the first focusing position focused by the confocal detecting unit in a direction of an optical path thereof.

In a confocal laser scanning microscope with an illuminating configuration (2), which provides an illuminating beam for illuminating a specimen region (23), with a scanning configuration (3, 4), which guides the illuminating beam over the specimen while scanning, and with a detector configuration (5), which via the scanning configuration (3, 4) images the illuminated specimen region (23) by means of a confocal aperture (26) on to at least one detector unit (28), it is provided that the illuminating configuration (2) of the scanning configuration (3, 4) provides a line-shaped illuminating beam, that the scanning configuration (3, 4) guides the line-shaped illuminating beam over the specimen f while scanning and that the confocal aperture is designed as a slit aperture (26) or as a slit-shaped region (28, 48) of the detector unit (28) acting as a confocal aperture.

A Device for coupling a short pulse laser into a microscope beam path, wherein the spectral components of the laser radiation are spatially separated by means of a dispersive element, the individual spectral components are manipulated and are then spatially superimposed again by means of another dispersive element.

The method for splitting and reflecting beams in a laser scanning microscope which produces a laser beam (1) comprises the steps of: reflecting a beam from a partially reflecting mirror (2); splitting the laser beam (1) with a splitting device into partial beams (5); causing the partial beams to travel in a plane; directing the partial beams to a sample under investigation; providing the partially reflecting mirror (2) with a constant transmission; placing the partially reflecting mirror between two high reflectivity mirrors (3,4); transmitting the laser beam (1) to one of the high reflectivity mirrors (3); reflecting said beam (1) to the other one of the high reflectivity mirrors (4) with basically equal attenuation; repeatedly transmitting the beam to one of the mirrors; and repeatedly reflecting the beam to the other one of said mirrors (4); arranging the partially reflecting mirror and the high reflectivity mirrors (3,4) relative to each other to cause beams (5) reflected by the partially reflecting mirror (2) not to coincide with beams reflected by said high-reflectivity mirrors (3,4); shifting forwardly, at an exit region of the splitting device, an edge of the partially reflecting mirror (2) along an edge of one of the high-reflective mirrors (4); and shifting rearwardly an edge of the other high-reflective mirror (3).

A scanning optical microscope using a wavefront converting element suffers minimum off-axis performance degradation and allows the wavefront converting element to be controlled by a simple method. Further, a pupil relay optical system is simple in arrangement or unnecessary. A laser scanning microscope includes a laser oscillator and a wavefront converting element for applying a desired wavefront conversion to a laserbeam emitted from the laser oscillator. An objective collects a wavefront-converted approximately parallel laser beam emerging from the wavefront converting element onto a sample. A detector detects signal light emitted from the sample. An actuator scans the objective along a direction perpendicular to the optical axis.

An immersion microscope objective formed of thirteen or fewer lens elements includes, in order from the object side, first and second lens groups of positive refractive power, a third lens group, a fourth lens group having negative refractive power with its image-side surface being concave, and a fifth lens group having positive refractive power with its object-side surface being concave. The first lens group includes, in order from the object side, a lens component that consists of a lens element of positive refractive power (when computed as being in air) and a meniscus lens element having its concave surface on the object side. Various conditions are satisfied to ensure that images of fluorescence, obtained when the immersion microscope objective is used in a laser scanning microscope that employs multiphoton excitation to observe a specimen, are bright and of high resolution. Various laser scanning microscopes are also disclosed.

A method for controlling laser scanning microscopy of a probe comprising at least one cell is disclosed. The method comprises the steps of acquiring at least one initial image of the probe and identifying at least one cell within an initial probe image. Using a pre-defined grammar, a first set of scanning mode parameters for monitoring the cell(s); a first set of trigger parameters including at least one physiological parameter defining an event in the cell(s); and a second set of scanning mode parameters for monitoring at least one cell of the probe after an occurrence of the event is defined. A successive set of probe images acquired according to the first set of scanning mode parameters is provided and processed to determine if the event has occurred. Responsive to the event occurring, microscope modality is changed to the second set of scanning mode parameters.

A method is disclosed for operation of an image-generating optical system for detection of characteristic quantities of the wavelength-dependent behavior of an illuminated specimen, such as the emission behavior and / or absorption behavior and, in particular, the fluorescence and / or luminescence and / or phosphorescence and / or enzyme-active light emission and / or enzyme-active fluorescence, for the operation of a laser scanning microscope, comprising the steps of splitting the image point information of the specimen into spectral components in a spatially resolved manner on the detection side in dependence on wavelength and carrying out at least one summing for different spectral components.

Laser-scanning microscope with at least one detection radiation input, in which an aperture plate is installed in front of the detector, whereby optics with variable transmission lengths and a fixed focal distance is provided for focusing varying wavelengths of the detected light onto the aperture plate level at the detection radiation path, which realizes the imaging from the infinite space into an image level with a finite conjugate distance,and / orwith at least one light source launched via an optical fiber, whereby collimator optics with a fixed focal distance, and a variable conjugate distance are down-streamed from the fiber output, which transfers the point source at the fiber output with a numerical aperture into a parallel beam in the infinite space in front of the scan-objective lens, whereby a wavelength-dependent, at least partial compensation of the chromatic distortion of the micro-objective lenses occurs by means of turning the chromatic curve for the illumination wavelength used.

Apparatus for and methods of inspection using laser beam induced alteration are provided. In one aspect, an apparatus is provided that includes a laser scanning microscope for directing a laser beam at a circuit structure and a source for biasing and thereby establishing a power condition in the circuit structure. A detection circuit is provided for detecting a change in the power condition in response to illumination of the circuit structure by the laser beam and generating a first output signal based on the detected change. A signal processor is provided for processing the first output signal and generating a second output signal based thereon. A control system is operable to scan the laser beam according to a pattern that has a plurality of pixel locations, whereby the laser beam may be moved to a given pixel location and allowed to dwell there for a selected time before being moved to another pixel location.

A calibration device for managing a variety of performance tests and / or calibration tasks in a laser scanning microscope. The calibration device, which has focusing optics and a test structure arranged in the focal plane of the focusing optics, with structural elements detectable in reflected and / or transmitted light aligned to each other in a common mounting, can be switched into the microscope beam path in a laser scanning microscope, so that the pupil of the focusing optics coincides with the objective pupil of the laser scanning microscope or lies in a plane conjugated to it.

A laser scanning microscope according to the present invention is a laser scanning microscope which scans a laser beam on a sample by a scanning optical system in a scanning optical system main body to detect a fluorescence or reflected light from the sample, and a light source section comprising a light source manufactured by a semiconductor process and an optical fiber provided on a radiation side of the light source is incorporated in the scanning optical system main body.

A laser scanning microscope comprises at least one selectively switchable micro-mirror arrangement (DMD) in the illumination beam path and / or detection beam path which is used for the wavelength selection of dispersively divided illumination and / or object light such as reflection, fluorescence.