Devices and methods for super-resolution optical microscopy are described. Devices include an optical multiplexer to develop an excitation/illumination optical beam that includes alternating pulses of different profiles. Devices also include a signal processing unit to process a sample response to excitation/illumination beam and to subtract the neighboring pulses of the different profiles from one another on a pulse-to-pulse basis. Devices can be incorporated in existing confocal microscopy designs. As the subtraction effectively reduces the volume of the response signal, the spatial resolution of the systems can be markedly improved as compared to previously known optical microscopy approaches.

A method for imaging a sample using a microscope having an illumination unit, an imaging lens system and an image sensor, includes: illuminating an area of the sample; imaging and magnifying the sample onto the image sensor and capturing the image using a predetermined number of pixels; providing a plurality of different comparison sample areas; for each comparison sample area, performing a reference measurement, wherein the comparison sample areas are illuminated, imaged and magnified onto the image sensor and captured with the predetermined number of image pixels as a reference image; determining a brightness-correction image with the predetermined number of image pixels by determining the value for each image pixel of the brightness-correction image from the values of allocated image pixels of the reference images, and correcting the image of the area of the sample captured based on the brightness-correction image and outputting it as a corrected image.

A method for automatically ascertaining illumination brightnesses to be adjusted of at least two light sources for exciting at least one respective fluorophore in a sample to be imaged in a fluorescence microscope includes separately controlling, in terms of illumination brightness, each of the at least two light sources, detecting an image intensity of a microscopically imaged sample with at least two detectors, and automatically ascertaining the illumination brightnesses to be adjusted of the at least two light sources in such a way that a predefined setpoint of a signal-to-noise ratio is reached for each fluorophore. In order to ascertain the illumination brightnesses of the at least two light sources, cross-talk of a detector for different emission spectra of the fluorophores and/or cross-excitation of a fluorophore for different illumination spectra of the light sources are/is taken into account.

Methods and apparatus for transverse sheet illuminated multiple plane imaging that can achieve simultaneous imaging of multiple z-planes in a laser scanning confocal fluorescence microscope.

A re-scan optical system scans a light spot over a plane to form an image and includes an illumination system for directing, and optionally focusing, light providing an illumination light spot. A directing element scans the spot over and/or through the sample, de-scans sample light from the sample and scans the sample light. A detection system directs the de-scanned light along a path running from the directing element back to the directing element so that the directing element scans the sample light. A prism inverts and/or reverts the light, and/or the re-scan system comprises one or more elements to cause at least two foci of the light in said path, and/or the path is provided with a deflecting prism to deflect the light without inverting the light and/or to deflect the light without reverting the light.

A multiplexing module provided herein is configured to perform operations of receiving a plurality of laser pulses from a pulsed laser source; splitting each laser pulse into a plurality of beamlets; introducing a delay between each adjacent beamlet of the plurality of beamlets, such that the plurality of beamlets associated with a respective laser pulse of the plurality of laser pulses is distributed equally across a pulse repetition period associated with the pulsed laser source; changing a divergence of each subsequent beamlet of the plurality of beamlets associated with each respective laser pulse to introduce a distinguishing feature between each beamlet of the plurality of beamlet to cause each beamlet to focus on a different axial plane or lateral position of the sample; and outputting the plurality of beamlets associated with each respective laser pulse.

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 microscope including an illumination assembly, an image capture device and a processor can be configured to selectively identify regions of a sample including artifacts or empty space. By selectively identifying regions of the sample that have artifacts or empty space, the amount of time to generate an image of the sample and resources used to generate the image can be decreased substantially while providing high resolution for appropriate regions of the computational image. The processor can be configured to change the imaging process in response to regions of the sample that includes artifacts or empty space. The imaging process may include a higher resolution process to output higher resolution portions of the computational image for sample regions including valid sample material, and a lower resolution process to output lower resolution portions of the computational image for sample regions including valid sample material.

A confocal microscope is provided that includes one or more lasers focused by an optical system into a line on the surface of a sample mounted to a stage. The microscope further includes, at least one linear array detector that is optically conjugated to the focused line. The stage permits movement of the sample with respect to all other components of the microscope, which remain stationary.

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.