Multi-dimensional scanner for nano-second time scale signal detection
Inventors
Assignees
Gene Capture Inc • GENECAPTURE Inc
Publication Number
US-9835557-B1
Publication Date
2017-12-05
Expiration Date
2032-12-30
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Abstract
A device and system for measuring the multidimensional distribution of a sample tagged with a short life fluorescent label. The substance applied to a sample holder can be scanned with an optical point source excitation and read back optical stage. The sample can be excited at each of a plurality of points with a fast, e.g., nanosecond pulse of light. The resulting fluorescence can be detected after the excitation is extinguished. A detection gate window can be optimized to maximize the fluorescence signal detected for a predetermined amount of time.
Core Innovation
The invention relates to a device and system for measuring the multidimensional distribution of a sample tagged with a short life fluorescent label. A sample applied to a sample holder can be scanned using an optical point source excitation with read-back optical stage. The system excites the sample at multiple points with a fast, e.g., nanosecond pulse of light, and detects the resulting fluorescence after the excitation is extinguished. A detection gate window can be optimized to maximize the fluorescence signal detected for a predetermined time.
The invention addresses the challenge of detecting very short decay time fluorescence, especially from fluorophores with lifetimes on the order of a few nanoseconds. The system incorporates a high speed switching circuit, such as a Field Effect Transistor (FET) switch, controlled by a timing circuit to selectively direct the desired component of a repetitive fluorescence signal to the detection mechanism. This gating approach allows the photomultiplier tube (PMT) detector to remain continuously on while selectively collecting fluorescence signals after the excitation pulse, improving the signal-to-noise ratio (S/N) by effectively excluding excitation light leakage and ambient background.
The problem being solved stems from the difficulty in detecting fluorescence signals from high performance probes with lifetimes of 3-4 nanoseconds, where traditional off-gating methods with delays longer than the fluorophore's lifetime cause loss of signal. Furthermore, background noise from excitation light leakage through imperfect optics reduces sensitivity. The invention overcomes these problems by implementing a high speed, precise gating mechanism with nanosecond switching speed that enables detection starting shortly after or even before the end of the excitation pulse, thereby achieving improved sensitivity and allowing detection of extremely low concentrations of fluorescent molecules.
Claims Coverage
The patent includes multiple independent claims describing devices and methods for identifying fluorescence signals using precise timing and high speed switching. The main inventive features focus on the use of high speed switching circuits, timing control for gating detection, and specific configurations of the components.
High speed switching circuit controlled by timing for selective fluorescence detection
A device comprising a light source, pulse generator, active detector, timing circuit generating a delay between approximately 10⁻¹² s to 10⁻¹⁰ s, control circuit generating a duration for decay time, and a high speed switching circuit controlled by the delay and duration to selectively direct the fluorescence signal to the detector after the delay and during the duration.
Use of Field Effect Transistor as the high speed switching circuit
The high speed switching circuit is specifically a Field Effect Transistor (FET) switching circuit that can switch in less than 5 nanoseconds, enabling efficient gating of fluorescence signals with very short lifetimes.
Control circuit incorporating BoxCar integrator for gating
A control circuit selects the high speed switching circuit operation based on a BoxCar integrator, allowing integration of signal segments over precise time windows.
Fluorophore half-life range specification
The devices and methods are designed to detect fluorophores with half-lives between approximately 10⁻⁹ s and 10⁻⁸ s, enabling detection of very short lifetime fluorescent signals.
Pulse generator rate range and light source specifications
The pulse generator operates at rates between approximately 10⁴ to 10⁷ pulses per second, with the light source being a light emitting diode and pulse durations between approximately 0.5×10⁻⁹ s and 10⁻⁸ s.
Detector response and transit time specifications
The active detector (e.g., PMT) has response times, transit times, and response to background light all in the range of approximately 0.5×10⁻⁹ s to 2×10⁻⁹ s, enabling rapid and sensitive detection.
Detection capability per excitation cycle and high resolution acquisition
The control circuit can detect fluorescence signals within a single excitation cycle comprising a single light pulse, gate delay, and detection period, with image acquisition pixel resolutions ranging from approximately 10⁻⁶ m × 10⁻⁶ m to 10⁻⁴ m × 10⁻⁴ m.
Method for detecting fluorophore labeled samples using timed gating and FET switching
A method comprising receiving a fluorophore labeled sample, applying voltages to an active PMT, irradiating the sample with a pulse, providing controlled delay and duration timings generated by a timing circuit and control circuit respectively, directing the fluorescence through a switch after the delay and for the duration, and detecting the fluorescence with suitable data acquisition and spatial resolution.
The claims define inventive features centered on the use of a FET high speed switching circuit controlled by precise timing circuits to gate fluorescence signals with nanosecond scale delays and durations, enabling sensitive detection of short lifetime fluorescent probes with high acquisition rates and spatial resolution.
Stated Advantages
Improved signal-to-noise ratio by gating detection to exclude excitation light and ambient background, especially important for short lifetime fluorophores.
Capability to detect fluorescence signals with nanosecond lifetimes as short as 3-4 nanoseconds without significant loss of signal due to gating delays.
Continuous operation of the photomultiplier tube avoids complications and cost of switching high voltage power on and off, resulting in a simple, direct, and low-cost implementation.
Flexibility to adjust timing parameters to bring the detection window to any desired point before, during, or after the excitation pulse enhances adaptability to different fluorophores and sample conditions.
High spatial resolution scanning with ability to scan samples at very high data acquisition rates (up to millions of pulses per second) enabling detailed multidimensional mapping of samples.
Sensitivity sufficient to detect extremely low concentrations of fluorescent molecules, down to femtomolar levels, without amplification.
Documented Applications
Scanning and measuring fluorescence intensity of multi-dimensional samples tagged with short life fluorescent labels, including protein or other biomolecules.
Two-dimensional fluorescence scanning for detecting very low concentrations of analytes such as fluorescent dyes in buffer, exemplified by femtomolar concentration detection.
Fluorescent microarray scanning and protein or gene distribution mapping with nanosecond-scale fluorescence detection.
Application with biological samples such as DNA sequencing using fluorescently labeled oligonucleotide primers and protein microarrays.
Potential three-dimensional scanning using two-photon excitation and focal point collection by moving optics to scan planes above or below a data set.
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