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

Methods and apparatus for inferring protein binding based on rotational diffusion of a collection of fluorophores. One example of a method includes applying a first light pulse to excite a plurality of fluorophores in the collection of fluorophores to produce a plurality of excited fluorophores, the first light pulse having a first polarization and the plurality of excited fluorophores having a component of their orientation aligned with the first polarization, applying a second light pulse to stimulate emission by the plurality of excited fluorophores, the second light pulse having a second polarization orthogonal to the first polarization, after a time delay following application of the second light pulse, applying a third light pulse of the second polarization to further stimulate emission by the plurality of excited fluorophores, detecting polarized emissions from the plurality of excited fluorophores, and inferring the rate of rotational diffusion of the collection of fluorophores based on the detected polarized emissions.

The present invention provides for metallic structures comprising a sulfhydryl or amino-terminated hydrophilic coating to provide a layer of hydrophilic character on the surface of the metallic structures thereby allowing the use of low volumes of aqueous solvents of fluorophores that have the ability to “spread out” across the surfaces of the metallic structures and to provide for a more uniform surface coating of fluorophores attached to or near the metallic structures.

A method and optode for determining a concentration of an analyte in a sample liquid is provided. The method comprises a radiation source, where excitation radiation is directed onto a carrier unit which is in contact with the sample liquid and has immobilized molecules of a sensor dye that is sensitive to the analyte. The excitation radiation induces luminescence radiation of the sensor dye. This radiation is detected by a radiation detector, which generates an output signal. The analyte concentration is ascertained from the detector output signal using an evaluation routine. This uses a property of the luminescence radiation on the interaction of the concentration of the analyte in the sample liquid used. The dependence of the examined property of the luminescence radiation on an indirect exchange interaction between the individual molecules of the sensor dye, which interact with each other over particles of the analyte.

The present disclosure provides a method for detection of an analyte in a sample, where the sample is introduced into an analytic chamber along with droplets of an emulsion or gel beads. In another aspect, the present disclosure provides designs for formulations of emulsion drops or gel beads such that they are useful for detection of analytes in a massively parallel manner. Formulations that contain specific combinations of fluorescent particles allow optical determination of the identity of each fluorescent particle. The combinations are based on particle fluorescence emission wavelength, fluorescence excitation wavelength, and particle count.

A fluorescent image analyzer stores a reference fluorescent-sample image and a subject fluorescent-sample image. The reference fluorescent-sample image is an image obtained by illuminating a reference fluorescent sample about which relation of in-plane fluorescence intensities is known with linearly polarized light and capturing a first specific polarization component of fluorescence from the reference fluorescent sample. The subject fluorescent-sample image is an image obtained by illuminating a subject fluorescent sample with linearly polarized light and capturing a second specific polarization component of fluorescence from the subject fluorescent sample. The fluorescent image analyzer is configured to determine correction coefficients to correct non-uniformity in measurement of light intensities among pixels of a captured image based on the reference fluorescent-sample image, and correct light intensities of the pixels of the subject fluorescent-sample image based on the correction coefficients.

The disclosure may relate to a method for the characterization of the 3D orientation of an emitting dipole within a specimen. The method comprises splitting a light beam emitted by the emitting dipole and exiting an objective lens into a first and a second beams; spatially filtering said first beam by using a spatial frequency filter; splitting each of said filtered first beam and said second beam into two beams linearly polarized using polarizing beam splitters; detecting with an optical detection unit four beams linearly polarized in a detection plane optically conjugated with the front focal plane of said microscope objective lens; determining, from four intensity images, in a predefined frame of the specimen, the mean orientation and the angular aperture of the distribution of the 3D orientation of the emitting dipole, during an acquisition time of said four intensity images.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

Swept polarization optical beams are directed to a sample to produce fluorescence. typical as a plurality of single photon detection events. Based on frequencies associated with the polarization sweeps, orientation of a sample can be determined. Using polarization sweeps at first and second frequencies and wavelength, donor fluorophore orientation can be established based on the first frequency and wavelength and acceptor orientation can be established based on a sum or difference of the first and second frequencies and the second wavelength.

Systems, devices and methods for determining an orientation and a rotational mobility of the single point emitter using a duo-spot point spread function (PSF) phase mask are disclosed. The duo-spot PSF phase mask includes at least three partitions, in which each partition includes a phase delay ramp aligned along one of two phase delay axes. Each phase delay ramp includes a gradient of phase delays. Each partition includes a subset of a total area of the phase mask and the two phase delay axes are oriented in different directions. The duo-spot PSF phase mask is configured to produce a duo-spot PSF that includes two light spots. The relative brightness of the two spots encodes an orientation and a rotational mobility of the single point emitter.