Systems and methods for detecting lifetime of luminescent molecules using photodetectors configured to perform photon counting are described. The systems and methods may involve an array of photodetectors for detecting photons emitted from a sample, which may include the luminescent molecules, and detection circuitry associated with the array of photodetectors. The detection circuitry may be configured to count, during at least a first time period and a second time period, a quantity of incident photons at a photodetector in the array of photodetectors.

An example system for inspecting a surface includes a laser, an optical system, a gated camera, and a control system. The laser is configured to emit pulses of light, with respective wavelengths of the pulses of light varying over time. The optical system includes at least one optical element, and is configured to direct light emitted by the laser to points along a scan line one point at a time. The gated camera is configured to record a fluorescent response of the surface from light having each wavelength of a plurality of wavelengths at each point along the scan line. The control system is configured to control the gated camera such that an aperture of the gated camera is open during fluorescence of the surface but closed during exposure of the surface to light emitted by the laser.

An example system for inspecting a surface includes a laser, an optical system, a gated camera, and a control system. The laser is configured to emit pulses of light, with respective wavelengths of the pulses of light varying over time. The optical system includes at least one optical element, and is configured to direct light emitted by the laser to points along a scan line one point at a time. The gated camera is configured to record a fluorescent response of the surface from light having each wavelength of a plurality of wavelengths at each point along the scan line. The control system is configured to control the gated camera such that an aperture of the gated camera is open during fluorescence of the surface but closed during exposure of the surface to light emitted by the laser.

An emission lifetime measuring method, in particular for measuring a mean lifetime of electronically excited states of a sample, comprises the steps of illuminating the sample with at least one excitation light pulse, time-resolved detecting an emission response from the sample and creating a temporal detector response function, and calculating the mean lifetime of the electronically excited states on the basis of the detector response function, wherein the at least one excitation light pulse is shaped such that the sample achieves an equilibrium excited steady-state including a linearly increasing or constant number of the electronically excited states, the detector response function has a linear response function section with a constant slope, and the mean lifetime () of the electronically excited states is calculated on the basis of at least one of a time position of the linear response function section relative to a reference time of the at least one excitation light pulse and the slope of the linear response function section. Furthermore, an emission lifetime measuring apparatus (100) is described.

A method for analyzing a biological sample (1002) comprises: Providing a plurality of markers (1612), each marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) comprising a fluorescent dye (1320) unique to the marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) and an affinity reagent (1310 to 1319) unique to the marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526), the affinity reagent (1310 to 1319) being configured to attach to a predetermined structure (1706 to 1714) within the sample (1002). Staining the sample (1002) by introducing the plurality (1612) of markers into the sample (1002). Directing first excitation light having a first wavelength spectrum onto the sample (1002) in order to excite the fluorescent dyes (1320) of a first set of markers (1614). Generating at least one first image from fluorescence light emitted by the excited dyes of the first set (1614), the first image comprising at least two channels, each channel corresponding to one marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) of the first set of markers (1614). Directing at least one second excitation light having a second wavelength spectrum onto the sample (1002) in order to excite the fluorescent dyes (1320) of a second set of markers (1616), the second set (1616) being distinct from the first set (1614). Generating at least one second image from fluorescence light emitted by the excited dyes of the second set (1616), the second image comprising at least two channels, each channel corresponding to one marker (1300 to 1309, 1400 to 1422, 1500 to 1508, 1518, 1526) of the second set of markers (1616).

An emission lifetime measuring method, in particular for measuring a mean lifetime of electronically excited states of a sample, comprises the steps of illuminating the sample with at least one excitation light pulse, time-resolved detecting an emission response from the sample and creating a temporal detector response function, and calculating the mean lifetime of the electronically excited states on the basis of the detector response function, wherein the at least one excitation light pulse is shaped such that the sample achieves an equilibrium excited steady-state including a linearly increasing or constant number of the electronically excited states, the detector response function has a linear response function section with a constant slope, and the mean lifetime () of the electronically Diode current and pulse width control excited states is calculated on the basis of at least one of a time position of the linear response function section relative to a reference time of the at least one excitation light pulse and the slope of the linear response function section. Furthermore, an emission lifetime measuring apparatus (100) is described.

The disclosure features methods of analyzing a fluid extracted from a reservoir, the methods including introducing a first composition featuring a first complexing agent into a reservoir at a first location, extracting a fluid from the reservoir at a second location different from the first location, combining the fluid with a second composition featuring a concentration of a lanthanide ion to form a third composition featuring a concentration of a complex formed by the first complexing agent and the lanthanide ion, exposing a quantity of the complex to electromagnetic radiation for a first time period ending at a time to, detecting fluorescence emission from the quantity of the complex for a second time period starting at a time t.sub.1>t.sub.0, where t.sub.1?t.sub.0 is greater than 2 microseconds, and determining information about a fluid flow path between the first location and the second location.

The disclosure features methods of analyzing a fluid extracted from a reservoir, the methods including introducing a first composition featuring a first complexing agent into a reservoir at a first location, extracting a fluid from the reservoir at a second location different from the first location, combining the fluid with a second composition featuring a concentration of a lanthanide ion to form a third composition featuring a concentration of a complex formed by the first complexing agent and the lanthanide ion, exposing a quantity of the complex to electromagnetic radiation for a first time period ending at a time t.sub.0, detecting fluorescence emission from the quantity of the complex for a second time period starting at a time t.sub.1>t.sub.0, where t.sub.1?t.sub.0 is greater than 2 microseconds, and determining information about a fluid flow path between the first location and the second location.

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

The present disclosure provides methods, apparatus and systems for time-gated fluorescent-based detection. Time-based fluorescence analysis can be used in certain biochemical assays by measuring the emitted photon flux from fluorophores after an individual excitation pulse.