A fluorescence scanning microscope includes excitation and de-excitation light sources, which are designed to generate an excitation and a de-excitation light distribution, respectively. An illumination unit combines the light distributions to form a light distribution scanning over multiple illumination target points of a sample in such a way that an intensity maximum of the excitation light distribution and an intensity minimum of the de-excitation light distribution are spatially superimposed on one another. A detector detects fluorescence photons emitted from the respective illumination target point as a function of their arrival times. A processor evaluates the fluorescence photons with respect to the arrival times, generates a first pixel and a second pixel based thereon, assembles the first and second pixels to form first and second sample images, respectively, and, by means of the two sample images, determines a spatial offset between the intensity maximum and the intensity minimum.

A microscopic imaging method for creating a microscopic sample image (1A) of a sample (1) comprises the steps of arranging the sample (1) on a sampling crystal (10); irradiating the sample (1) with excitation laser pulses (2, 3) and generating sample response pulses (4) with a sample response field as a result of an interaction of the excitation laser pulses (2, 3) with the sample (1); irradiating the sampling crystal (10) with probe laser pulses (5) being temporally synchronized with the excitation laser pulses (2, 3) and spatially overlapped with the sample response pulses (4) in the sampling crystal (10), wherein the probe laser pulses (5) have a shorter wavelength than the excitation laser pulses (2, 3); detecting the sample response field by electric-field sampling with the sampling crystal (10), using the sample response pulses (4) and the probe laser pulses (5); and calculating the sample image (1A) based on the detected sample response field, wherein the excitation laser pulses (2, 3) have a wavelength in a range from mid-infrared to visible light and the sample response pulses (4) are created by a coherent interaction process induced in the sample (1) and with a fixed phase relationship relative to the excitation laser pulses (2, 3), the sampling crystal (10) is a non-centrosymmetric crystal, the irradiating step is repeated at multiple sample points (1A), wherein at each sample point (1A) the irradiating steps are successively repeated with multiple temporal probe delays of the probe laser pulses (5) relative to the excitation laser pulses (2, 3), at each probe delay, a sum or difference frequency pulse (6) of a sample response pulse (4) and a probe laser pulse (5) is generated, and at each probe delay, a spectral interference pulse (7) is created by a spectral interference of the sum or difference frequency pulse (6) and the current probe laser pulse, the detecting step includes sensing a polarization state of the spectral interference pulse (7) by an ellipsometer device (40) at each probe delay, wherein the local sample response field at the sample point (1A) is derived from the polarization states sensed at all probe delays, and the sample image (1A) is calculated based on the sample response field detected at the sample points (1A). Furthermore, a microscopic imaging apparatus is described.

A ptychography system is presented for imaging an object located in an object plane. The ptychography system comprises an optical system, and a detection device. The optical system comprises a single shot ptychography arrangement configured and operable to create light response patterns from the object in the object plane on a pixel matrix of the detection device during the same exposure session of the detection device, wherein the optical system further comprises at least one light coding device configured and operable to apply at least one predetermined coding function to at least one of illuminating light and the light response of the object being collected, and said detection device is configured and operable with a predetermined duration of the exposure session during which the pixel matrix detects the collected light, such that image data indicative of the detected light during a single exposure session is in the form of a coded light response of the object being illuminated.

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 microscope includes an illumination system configured to illuminate a sample chamber with laser pulses. The illumination system includes a control device with stored, modifiable illumination parameters, with trigger outputs, to which at least one externally triggerable laser system is connectable in each case, and with a trigger generator configured to produce temporally successive trigger signals for triggering the at least one laser system. The microscope is configured such that an assignment of the trigger signals to the trigger outputs and/or a time interval between successive ones of the trigger signals depends on the illumination parameters.

A multi-photon imaging system includes a laser module having a first channel for outputting a two-photon excitation laser pulse and a second channel for outputting a three-photon excitation laser pulse. The system further includes a first optical path for guiding the two-photon laser pulse from the first channel of the laser module and a second optical path for guiding the three-photon laser pulse from the second channel of the laser module. A microscope is also provided for simultaneously receiving the two-photon laser pulse from the first optical path and the three-photon laser pulse from the second optical path, and simultaneously, or with well controllable delays, delivering the two-photon laser pulse and the three-photon pulse to a target volume. The system further includes a photodetector configured to collect photons generated within the target volume in response to simultaneous excitation of the target volume by both the two-photon laser pulse and the three-photon laser pulse.

A laser scanning system for capturing an image of a specimen is described herein. The laser scanning system includes a light source configured to emit a light beam for illuminating the specimen, a scanning unit including a plurality of reflectors for scanning the light beam along first and second axes, and a data acquisition unit configured to control acquisition of the image. The laser scanning system can include a control circuit configured to receive a reference clock signal for the first reflector and generate a synchronization clock signal based on the reference clock signal. The laser scanning system can include a synchronization controller configured to control the scanning unit and the data acquisition unit. The synchronization controller can be configured to receive the synchronization clock signal, receive a plurality of imaging parameters, and generate a plurality of control signals based on the synchronization clock signal and the imaging parameters.

A SPIM-microscope (Selective Plane Imaging Microscope) having a y-direction illumination light source and a z-direction detection light camera. An x-scanner generates a sequential light sheet by scanning the illumination light beam in the x-direction. The SPIM-microscope has an illumination optics having a zoom optics provided in a beam path of the illumination light beam, the zoom optics being adapted to change the focal length of the illumination light beam and adapted to detect a larger area of the object by sequentially detecting sequences of images along the y-direction that have an increased resolution along the z-direction. An image processing unit combines these sequences of images by image stitching into one large overall image.

Among other things, motility of at least one individual microorganism or a change in motility of at least one individual microorganism or both is or are characterized. The characterized motility or change in motility is used to detect the presence or count of the at least one individual microorganism, or determine the identity of a species or strain of the at least one individual microorganism, or determine a susceptibility of the at least one individual microorganism to one or more antibiotics or other antimicrobials.

The invention relates to a method for correcting motion artifacts of in vivo fluorescence measurements using a 3D laser scanning microscope containing two pairs of orthogonally arranged acousto-optic deflectors, the method comprising: selecting a region of interest, selecting a plurality of guiding points along the region of interest, extending the guiding points to objects selected from scanning lines and/or surface elements and/or volume elements which, together, substantially cover the region of interest, repeatedly scanning the objects by generating continuous drifts to cover the objects, projecting the obtained scanning data to frames and obtaining a time series of the frames, correcting motion artifacts by shifting the data of the successive frames with respect to each other so as to maximize fluorescence cross correlation between the data of the frames.