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.

Provided is a fluorescence reader that uses two excitation channels and can read up to seven different fluorescent dyes in a single run. Each excitation channel has one light source and one single excitation filter and one dichroic mirror. One excitation channel is capable of exciting multiple fluorescent dyes and can be used to distinguish multiple dyes in combination with multiple emission filters. The excitation channels are driven by a motor that can automatically switch the two excitation channels for taking images of up to seven different fluorescent dyes. An algorithm to calibrate the crosstalk between different fluorescent dyes is also provided. Also provided is a method for analyzing digital PCR data using a ratio of two fluorescence emission readings.

A reaction processing apparatus includes: a reaction processing vessel; a first fluorescence detection device that irradiates a sample with first excitation light and detects first fluorescence produced from the sample; and a second fluorescence detection device that irradiates a sample with second excitation light and detects second fluorescence produced from the sample. The wavelength range of the first fluorescence and the wavelength range of the second excitation light overlap at least partially. The first excitation light and the second excitation light flash at a predetermined duty ratio d. The phase difference between the flashing of the first excitation light and the flashing of the second excitation light is set within a range of 2π(pm−Δpm) (rad) to 2π(pm+Δpm) (rad) or within a range of 2π[(1−pm)−Δpm] (rad) to 2π[(1−pm)+Δpm] (rad), where pm=d−d2 and Δpm =0.01*pm.

A multicolor fluorescence analysis device 11 is for detecting fluorescence emitted, as a result of excitation light irradiation, from a plurality of types of fluorophores included in a sample s, and is provided with an irradiation optical unit 520 for irradiating light emitted from a light source 510 onto a sample s as excitation light, a fluorescence condensation unit 530 having a fluorescence filter 531 that transmits light emitted from the sample s and transmits light of transmission wavelength bands different from the excitation wavelength bands, and a two-dimensional detector 554 that has a plurality of types of transmission filters 556 for transmitting prescribed wavelengths of light and detects the intensity of the light of the prescribed wavelength for each transmission filter 556, and the light emitted from at least two fluorophores from among the plurality of types of fluorophores is detected simultaneously and the fluorophore types are identified accordingly.

A structured substrate includes a substrate body having an active side. The substrate body includes reaction cavities that open along the active side and interstitial regions that separate the reaction cavities. The structured substrate includes an ensemble amplifier positioned within each of the reaction cavities. The ensemble amplifier includes a plurality of nanostructures configured to at least one of amplify electromagnetic energy that propagates into the corresponding reaction cavity or amplify electromagnetic energy that is generated within the corresponding reaction cavity.

A method for identifying a microorganism to be identified, which includes the following steps: obtaining an Excitation-Emission EEM of the microorganism to be identified, analysing the main components of the EEM matrix using at least one reference EEMr matrix, projecting the result of the analysis onto a plane defined by two main components, and identifying the microorganism to be identified.

A microfluidic reaction chamber with a reaction chamber circuit includes a microfluidic reaction chamber to contain a reaction fluid for amplification of nucleic acids, and a reaction chamber circuit disposed within the microfluidic reaction chamber. The microfluidic reaction chamber includes a base wall, a top wall parallel to the base wall and defined in part by a transparent lid, a first side wall, and a second side wall. The reaction chamber circuit is disposed within the microfluidic reaction chamber, and includes a top surface, a bottom surface, a first side wall, and a second side wall. The reaction chamber circuit is in fluidic contact with the reaction fluid and includes a photodetector to detect a fluorescence signal from a labeled fluorescent tag in the reaction fluid.

A method for detecting a reversibly photoswitchable chemical species in a sample, includes the steps of: a) illuminating the sample with light suitable to be absorbed by the chemical species triggering a reaction affecting an optical property of the chemical species, the first light being periodically-modulated at a fundamental modulation frequency; b) measuring the evolution of the optical property; c) extracting at least one of an in-phase component at a frequency which is an even multiple of the fundamental modulation frequency; and a quadrature component at a frequency which is an odd multiple of the fundamental modulation frequency of a signal representing the evolution; and d) using the extracted component or components for detecting the chemical species. An apparatus for carrying out the method is also provided.

Methods of deep ultraviolet fluorescence (DUVF) and multi spectral imaging (MSI) detection are disclosed herein for the detection and identification of pathogens on plants. DUV light and visible or near-infrared light are used to illuminate plants or plant leaves such that the light intensity reflected or emitted by the plant or plant leaves can be used to identify the type of pathogen and measure the amount of pathogen on the plant or plant leaves and, additionally, be used to measure the plant's stress response to such pathogen. Also provided herein is a biophotonic analyzer device that uses both DUVF and MSI detection for the monitoring and surveillance of plant health and for the identification and enumeration of pathogens on plants or plant leaves.

A fluorescence observation apparatus (1) includes an irradiation unit (10) that applies a plurality of kinds of excitation light (L11, L21) of mutually different wavelengths to a plurality of spatially or temporally different positions in a biological sample (5) that is labeled with a composite phosphor containing two or more kinds of fluorescent molecules (A, B) at a predetermined composition ratio, a detection unit (20) that detects fluorescence (L12, L22) generated at each of the plurality of positions by application of the irradiation unit (10), and a calculation unit (33) that determines a distribution of pieces of the composite phosphor on the basis of a fluorescence signal (S1, S2) that is obtained from a detection result of the detection unit (20) and that shows a fluorescence intensity corresponding to a position in the biological sample (5) of each piece of the fluorescence (L12, L22).