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.

The invention discloses a programmable annular LED illumination-based high efficiency quantitative phase microscopy imaging method, the proposed method comprising the following steps: the derivation of system optical transfer function in a partially coherent illumination imaging system; the derivation of phase transfer function with the weak object approximation under the illumination of tilted axially symmetric coherent point illumination source; the extension of illumination from an axially symmetric coherence point source to a discrete annular point source, and the optical transfer function can be treated as an incoherent superposition of each pair of tilted axially symmetric coherent point sources. The acquisition of raw intensity dataset; the implementation of deconvolution for quantitative phase reconstruction. The invention derives the system phase transfer function under the tilted axially symmetric point light source in the case of partially coherent illumination, and promotes the optical phase transfer function of the discrete annular point light source. The programmability characteristic of LED array enables the annular illumination aperture to be flexibly adjustable, being applicable to different microscopic objects with different numerical apertures, and improving the compatibility and flexibility of the system.

The invention discloses a programmable annular LED illumination-based high efficiency quantitative phase microscopy imaging method, the proposed method comprising the following steps: the derivation of system optical transfer function in a partially coherent illumination imaging system; the derivation of phase transfer function with the weak object approximation under the illumination of tilted axially symmetric coherent point illumination source; the extension of illumination from an axially symmetric coherence point source to a discrete annular point source, and the optical transfer function can be treated as an incoherent superposition of each pair of tilted axially symmetric coherent point sources. The acquisition of raw intensity dataset; the implementation of deconvolution for quantitative phase reconstruction. The invention derives the system phase transfer function under the tilted axially symmetric point light source in the case of partially coherent illumination, and promotes the optical phase transfer function of the discrete annular point light source. The programmability characteristic of LED array enables the annular illumination aperture to be flexibly adjustable, being applicable to different microscopic objects with different numerical apertures, and improving the compatibility and flexibility of the system.

A computational microscope and a method for its operation are disclosed. In some embodiments, the microscope maps points on a sample to point in an intensity pattern on a one-to-many basis. The microscope utilizes illumination angle coding, polarization coding, amplitude coding, and phase coding to capture more information than prior art computational microscopes. Although the resulting intensity patterns are not human-interpretable images of the sample, they contain more information about the sample, by virtue of the aforementioned coding techniques, than is captured by prior-art microscopes. Machine-learning algorithms, such as neural networks, are used to analyze the intensity patterns and extract useful information, such as cellular events or cell behavior.

A computational microscope and a method for its operation are disclosed. In some embodiments, the microscope maps points on a sample to point in an intensity pattern on a one-to-many basis. The microscope utilizes illumination angle coding, polarization coding, amplitude coding, and phase coding to capture more information than prior art computational microscopes. Although the resulting intensity patterns are not human-interpretable images of the sample, they contain more information about the sample, by virtue of the aforementioned coding techniques, than is captured by prior-art microscopes. Machine-learning algorithms, such as neural networks, are used to analyze the intensity patterns and extract useful information, such as cellular events or cell behavior.

An illumination system includes a surface configured to have an imaging target placed thereon, a light source, a beam splitter and at least a first mirror. The beam splitter is configured to split the beam of light from the light source and the first mirror is configured to reflect a first beam from the beam splitter onto the surface with the imaging target. An imaging system includes an imaging surface configured to have an imaging target placed thereon, a mirror, and a capturing device. The capturing device is configured to capture an image of the imaging target through a path of emitted light that extends from the imaging target, reflects off of the mirror, and to the capturing device. The mirror, the capturing device, or both are configured to move in a diagonal direction with respect to the imaging surface to reduce a length of the path of emitted light. Systems and methods to calibrate an imaging system to remove or reduce non-uniformities within images of samples due to imaging system properties.

An illumination system includes a surface configured to have an imaging target placed thereon, a light source, a beam splitter and at least a first mirror. The beam splitter is configured to split the beam of light from the light source and the first mirror is configured to reflect a first beam from the beam splitter onto the surface with the imaging target. An imaging system includes an imaging surface configured to have an imaging target placed thereon, a mirror, and a capturing device. The capturing device is configured to capture an image of the imaging target through a path of emitted light that extends from the imaging target, reflects off of the mirror, and to the capturing device. The mirror, the capturing device, or both are configured to move in a diagonal direction with respect to the imaging surface to reduce a length of the path of emitted light. Systems and methods to calibrate an imaging system to remove or reduce non-uniformities within images of samples due to imaging system properties.

Apparatus and methods for 3D-Scanless Holographic Optogenetics with Temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of the microscope. Soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression are also provided. The methods use point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a designated neuron's cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D.

Apparatus and methods for 3D-Scanless Holographic Optogenetics with Temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of the microscope. Soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression are also provided. The methods use point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a designated neuron's cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D.

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.