A scanning element routes a sheet or beam of excitation light through a first set of optical components and into a sample at an oblique angle. The position of the excitation light within the sample varies depending on the orientation of the scanning element. Fluorophores within the sample emit fluorescent detection light. The first set of optical components route the detection light back to the scanning element, and the scanning element routes the detection light through a second set of optical components and into a camera. The second set of optical components includes a phase modulating element that induces a controlled aberration so as to homogenize point spread functions of points at, above, and below a focal plane when measured at the camera. In some embodiments, the images captured by the camera are processed to correct for aberration by performing deconvolution of a point spread function.

A microscope system including: a source of light; a sample objective configured for focusing the light at a focal plane within a sample; a remote focus unit configured for changing a position of the focal plane along an axis perpendicular to the focal plane; one or more optical element configured for directing the focused light to a location within the focal plane; and a detector configured for detecting light emitted from the focal plane within the sample; wherein the one or more optical element is located after the remote focus unit along a beam path of the light from the source to the sample objective, such that the changing the position of the focal plane along the axis is performed before the directing the focused light to the location within the focal plane.

An optical scanning microscope includes an illumination system (160) and an objective lens (134) operable together to provide the excitation radiation (152, 154) in a focal volume (124) at sufficient intensity to cause emission of emission radiation from a sample in the focal volume. The objective lens (134) is scanned by an objective scanner (130, 132). In this example an x-y transducer (xyXD) (130) is connected to a kinematic flexure mechanism (132) which acts as a scanning lens mount. The kinematic flexure mechanism is operable to scan the objective in two dimensions transverse with respect to the objective's optical axis so as to scan the emitting focal volume in corresponding dimensions. The kinematic flexure mechanism may be a unitary 3D-printed member.

An observation apparatus includes: alight source that emits pulsed light; and an objective that includes a first optical element serving as a light guide part and irradiates a sample with the pulsed light. The first optical element consists of a medium that satisfies the following conditional expression for 1 and 2:
0.75<2/1<1.33 where 1=(n2n1)/(21) and 2=(n3n2)/(32) are satisfied (1 indicates a light wavelength of 706.52 nm; 2 indicates a light wavelength of 1529.6 nm; 3 indicates a light wavelength of 2325.4 nm; and n1, n2, and n3 respectively indicate refractive indexes that the medium has for 1, 2, and 3).

The present invention relates to an apparatus for simultaneous imaging and execution of contact-free directed hydrodynamic flow in a specimen with at least one light source, in particular a laser, adapted to dynamically heat the interior and/or a surface of the specimen, a microscope with an objective adapted to image at least a part of the specimen and to guide, in particular focus, a light beam of the light source, in particular a laser beam, into and/or onto the specimen to heat at least one specified location of the specimen, means for manipulating the specified location, and a sample chamber for the specimen that is accessible for imaging radiation and the light beam to allow simultaneous imaging and manipulation of the sample via the objective. Furthermore the present invention is directed to a method for simultaneous imaging and executing contact-free directed hydrodynamic flow in a specimen wherein, at least one light source, in particular a laser, dynamically heats the interior and/or a surface of the specimen via a light beam, in particular via a laser beam, the beam of the at least one light source is directed to the specimen through an objective of a microscope, the light beam is variably guided, in particular focused, to specified locations of the specimen inducing a hydrodynamic flow in the specimen, and imaging the specimen via the same objective as used for introduction of the light beam.

Techniques are described for imaging a sample where the techniques include acquiring a raster scan image of the sample, providing light from a light source, directing the light into a plurality of different light beam paths at different times, providing light in each of the plurality of light beam paths through an objective lens to the sample, and providing light in each of the plurality of beams to different locations within the sample. Fluorescence emission light from the sample is detected in response to excitation by light in each of the plurality of light beam paths, where the detected fluorescence emission light corresponds to fluorescence intensity projections of the sample with low mutual coherence, and an image of the sample is generated based on the detected fluorescence emission light and based on the raster scan image.

Systems and methods herein provide improved, high-throughput multiphoton imaging of thick samples with reduced emission scattering. The systems and methods use structured illumination to modify the excitation light. A reconstruction process can be applied to the resulting images to recover image information free of scattering. The disclosed systems and methods provide high throughput, high signal-to-noise ratio, and high resolution images that are depth selective.

An image acquisition device includes a spatial light modulator modulating irradiation light, a control unit controlling a modulating pattern so that first and second light converging points are formed in an observation object, a light converging optical system converging the irradiation light, a scanning unit scanning positions of the first and second light converging points in the observation object in a scanning direction intersecting an optical axis of the light converging optical system, and a photodetector detecting first observation light generated from the first light converging point and second observation light generated from the second light converging point. The photodetector has a first detection area for detecting the first observation light and a second detection area for detecting the second observation light. The positions of the first and second light converging points are different from each other in a direction of the optical axis.

A system includes a light source to generate an optical signal having a set of pulses at a first repetition rate. The system also includes a multiplexer circuit to generate a multiplexed optical signal from the optical signal n sets of pulses at a second repetition rate, where the n sets of pulses have different polarization states and are at the first repetition rate. The system also includes a focusing unit to split the multiplexed optical signal into n excitation signals to excite a sample. The system also includes an objective to receive the n excitation signals and to illuminate the sample. The objective and the focusing unit collectively focus each excitation signal of the n excitation signals on a different focal plane of the sample to generate a response signal. The system also includes a demultiplexer circuit to generate n emission signals based on the response signal.

A scanning element routes a sheet or beam of excitation light through a first set of optical components and into a sample at an oblique angle. The position of the excitation light within the sample varies depending on the orientation of the scanning element. Fluorophores within the sample emit fluorescent detection light. The first set of optical components route the detection light back to the scanning element, and the scanning element routes the detection light through a second set of optical components and into a camera. The second set of optical components includes a phase modulating element that induces a controlled aberration so as to homogenize point spread functions of points at, above, and below a focal plane when measured at the camera. In some embodiments, the images captured by the camera are processed to correct for aberration by performing deconvolution of a point spread function.