In order to allow precise observation of a specimen at an observation point with a desired depth without changing the working distance of an objective optical system while employing a simple configuration, a laser scanning microscope according to the present invention includes an objective lens having a plurality of optical elements that are disposed with gaps therebetween in an optical-axis direction and that condense laser light emitted from a light source onto a specimen and also having an adjustment ring that allows changing of the focal point by moving the optical elements in the optical-axis direction; a scanner that has a galvanometer mirror capable of oscillating about a predetermined oscillation axis and that scans the laser light condensed onto the specimen by the objective lens in accordance with an oscillation angle of the galvanometer mirror; a light detecting unit that obtains image information of the specimen on the basis of return light returned from the specimen scanned with the laser light; and a scanner controlling unit that controls the oscillation angle of the galvanometer mirror so as to maintain an observation range of the specimen observed by the light detecting unit on the basis of the positions of the optical elements moved by the adjustment ring.

A super resolution technique, intended mainly for fluorescence microscopy, acquires the three-dimensional position of an emitter, through a hybrid method, including a number of steps.

In a first step the two-dimensional position of an emitter is acquired, using a technique, named in this application as an Abbe’s loophole technique., In this technique a doughnut, or a combination of distributions, having a zero intensity at the combined center of the distributions, is projected onto the sample containing the emitter, under conditions wherein the doughnut null is moved towards the emitter to reach a position in which the emitter does not emit light.

In a second step, an axial measurement is obtained using a 3D shaping method, characterized by the fact that the emitted light is shaped by an additional optical module creating a shape of the light emitted by the emitter, this shape being dependent of the axial position and means to retrieve the axial position from the shape.

High-speed optical targeting systems and methods are described, wherein a light source, e.g., a laser, is optically coupled with a spatial light modulator. Some embodiments include a device for two-dimensional light steering. In some embodiments, the device comprises a spatial light modulator, and a laser in optical communication with the spatial light modulator. In some exemplary methods, an area is scanned withing a microscope with millisecond revisit time, such as with at least 500 individually targeted points of light. In other exemplary methods, a beam of light is directed from a laser light source into an optical system, through which the light may be focused into a line on a spatial light modulator, wherein the light can be scanned across the spatial light modulator, and directed from the spatial light modulator onto a sample. Other exemplary methods are drawn to the construction and use of the embodiments escribed herein.

A method of performing imaging includes operating a light sheet projection module in a first state during a first measurement process and using a first primary objective for illumination of a specimen using a light sheet and detection of a first fluorescent emission. The method also includes operating the light sheet projection module in a second state during a second measurement process and using a second primary objective for illumination of the specimen using the light sheet and detection of a second fluorescent emission.

For checking the confocality of a scanning and descanning microscope assembly comprising a light source providing illumination light focused into a focal area in a focal plane, a detector detecting light coming out of the focal area and having a detection aperture to be arranged in a confocal fashion with respect to the focal area, and a scanner, an auxiliary detection aperture of an auxiliary detector arranged in the focal plane is scanned with the focal area of the illumination light to record a first comparison intensity distribution of the illumination light registered by the auxiliary detector, and the detection aperture of the detector is scanned with auxiliary light that exits out of an auxiliary emission aperture of an auxiliary light source concentrically arranged with respect to the auxiliary detection aperture in the focal plane to record a second comparison intensity distribution of the auxiliary light registered by the detector.

Beam deflection units in light-scanning microscopes are usually arranged in planes that are conjugate to the objective pupil. The scan optics, which is required for generating the conjugate pupil planes, is complicated and not very light efficient. The invention is intended to enable a higher image quality, simpler adjustment and a lower light loss microscope.

The optical system comprises a concave mirror (36) for imaging a respective point of the first and second beam deflection units (30A, 30B) onto one another. The concave mirror (36), the first beam deflection unit (30A), and the second beam deflection unit (30B) are arranged such that the illumination beam path is reflected exactly once at the concave mirror (36). A first distortion caused by the concave mirror (36) and a second distortion of the imaging caused by the first and second beam deflection units (30A, 30B) at least partly compensate for one another.

A microscope apparatus provided with: a disk unit obtained by integrally forming a pinhole array disk in which pinholes are arranged and a microlens array disk in which microlenses are arranged; a dichroic mirror focusing illumination light that has been transmitted through the microlenses of the disk unit, on the corresponding pinholes and splitting off fluorescence from a specimen that has passed through the pinholes in the reverse direction from the illumination light; an objective lens radiating the illumination light that has passed through the pinholes onto the specimen and focusing the fluorescence from the specimen on the pinholes; an illumination-light-axis adjustment mechanism adjusting the position and the angle of the optical axis of the illumination light; an installation-angle adjustment mechanism adjusting the installation angle of the disk unit; and a unit insertion/removal mechanism removably supporting the disk unit onto the optical axis of the illumination light.

The invention relates to a beam manipulation device for a scanning microscope, comprising a main colour splitter for coupling excitation light into an illumination beam path and for separating excitation light from detection light of a detection beam path, said device comprising a scanner, preferably positioned on a pupil plane, for scanning the excitation light. The device is characterised in that: an additional optical section is provided comprising optical elements which influence a beam path; at least one pupil plane and/or at least one intermediate image plane is formed in the additional optical section by the optical elements which influence the beam path; and an adjustable selection device is provided for activating either a first beam segment of the illumination and/or detection beam path, or the additional optical section, wherein the first beam segment of the illumination and/or detection beam path does not contain a pupil plane of the illumination and/or detection beam path.

A beam-scanning optical design is described for achieving up to kHz frame-rate optical imaging on multiple simultaneous data acquisition channels. In one embodiment, two fast-scan resonant mirrors direct the optical beam on a circuitous trajectory through the field of view, with the trajectory repeat-time given by the least common multiplier of the mirror periods. Dicing the raw time-domain data into sub-trajectories combined with model-based image reconstruction (MBIR) 3D in-painting algorithms allows for effective frame-rates much higher than the repeat time of the Lissajous trajectory. Because sub-trajectory and full-trajectory imaging are different methods of analyzing the same data, both high-frame rate images with relatively low resolution and low frame rate images with high resolution are simultaneously acquired.

A microscopy system that includes a first laser emitting a first laser pulse along a first beam line, the first laser pulse being converted into an annular Bessel pump beam; and a second laser emitting a second laser pulse along a second beam line, the second laser pulse being a probe beam, the annular Bessel pump beam and the probe beam being delivered to a sample at right angles to each other allowing the annular Bessel pump beam to shrink a focal axial diameter of the second beam line thereby enabling dipole-like backscatter stimulated emission along the second beam line.