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.

The present invention relates to a fixation and body temperature maintaining apparatus for fixing a small animal in order to generate a high-resolution micro image of a predetermined tissue of the small animal within a biomicroscope with an object lens, which includes: a plate heater with a heat wire installed therein, on which the small animal is enabled to be directly placed; a glass heater holder fixed to a hole of the plate heater; and a glass heater fixed to the glass heater holder, and located above the tissue of the small animal and maintaining flatness of the tissue, and increasing a temperature of the tissue itself, in which a cover glass serving to adjust a refractive index of the object lens and a heat wire heater are integrally formed. In an embodiment of the present invention, a body temperature of a live small animal which is an object to be observed with a biomicroscope is maintained to be constant.

Systems, devices, and methods for producing an optimized phase mask for use in a single-molecule orientation localization microscopy (SMOLM) imaging system are disclosed.

A Raman spectrometric imaging method, including: placing a sample on a two-dimensional translation stage; emitting a first light beam by a first optical comb light source; dividing the first light beam into a pump light beam and a depletion light beam to illuminate the sample; guiding the pump light beam to illuminate a region of the sample to excite molecules of the sample in the region; guiding the depletion light beam to the region of the sample to make excited molecules at a periphery of the region to return into a vibrational ground state; emitting a second light beam as a probe light beam by a second optical comb light source to the remaining excited molecules to generate a CARS signal; recording the CARS signal for imaging; moving the two-dimensional translation stage to scan other regions of the sample to form an image of the sample.

A microscope for imaging a sample is disclosed that may include at least one illumination objective arranged to eject an illumination light beam along an illumination path to illuminate the sample; an imaging objective arranged to receive detection light including at least a portion of the light ejected from the sample, wherein the detection light is propagated along a detection axis and the imaging objective has an imaging focal plane; an adjustment arrangement to linearly displace the illumination light beam and the imaging focal plane relative to each other along the detection axis; a sample holder arranged to receive a sample and having a portion which is transparent to the illumination light beam and to the detection light; and a holder support arranged to receive the sample holder and displace the sample holder relative to the imaging objective such that the imaging focal plane is positioned inside the sample holder.

A method for illuminating samples in microscopic imaging methods, wherein a number m of different wavelengths λ.sub.i, with m>I and i=I, . . . , m, is selected for the illumination. For each of the wavelengths λ.sub.i a target phase function Δφ.sub.i(x, y, λ.sub.i) is predefined, wherein x and y denote spatial coordinates in a plane perpendicular to an optical axis z and each target phase function Δφ.sub.i(x, y, λ.sub.i) is effective only for the corresponding wavelength λ.sub.i. The target phase functions Δφ.sub.i are predefined depending on the structure of the sample and/or the beam shape and/or illumination light structure to be impressed on the light used for illumination. A total phase mask is then produced which realises all target phase functions Δφ.sub.i(x, y, λ.sub.i). This total phase mask is then illuminated simultaneously or successively with coherent light of wavelengths λ.sub.i such that the predefined structure of the illumination light is generated in the region of the sample.

Described herein are methods and systems for the optical imaging of a physical specimen of interest that is in contact with, or in close proximity to, the backplane of a high refractive index solid-immersion lens (SIL), wherein the specimen comprises features of interest that act as a local high-refractive index regions. The SIL lens preferably comprises fiducial markers.

A laser scanning microscope includes a light source configured to emit an illumination light beam. The illumination light beam has a transverse light intensity profile comprising an intensity minimum. The laser scanning microscope further includes a scanning device configured to scan the illumination light beam along a closed trajectory in a target area of a specimen, and a detector configured to detect fluorescence light emitted by a fluorophore within the target area of the specimen. The fluorophore is excited by the illumination light beam. The laser scanning microscope further includes a processor configured to determine an intensity distribution of the fluorescence light as a function of time and to determine a position of the fluorophore within the target area based on the intensity distribution of the fluorescence light.

An optical device, such as a microscope, is disclosed that can be assembled from flat materials. The optical device can be assembled via a series of folds of a flat material. The optical microscope can include a stage for supporting a sample, an optic stage, and a light source. The optic stage can include one or more lenses. The optical microscope can be capable of obtaining simultaneous images from different forms of microscopy. The optical microscope may have bright field and filter field viewing capabilities wherein a user shifts from bright field to filter field by lateral movement of the stage containing a lens and a light source that cooperate to provide either the bright field or the filter field.

A high content imaging system and a method of operating the high content imaging system are disclosed. A microscope has a first objective lens and a second objective lens, and an objective lens database has first and second transformation values associated with the first and the second objective lenses, respectively. A microscope controller operates the microscope with the first objective lens to develop first values of acquisition parameters. A configuration module automatically determines second values of the acquisition parameters using the first values of the acquisition parameters, first transformation values associated with the first objective lens, and second transformation values associated with the second objective lens. The microscope controller operates the microscope using the second objective lens and the second values of the acquisition parameters.