Multi-dimensional scanner for nano-second time scale signal detection
Inventors
Assignees
GENECAPTURE Inc • Gene Capture Inc
Publication Number
US-8981318-B1
Publication Date
2015-03-17
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. The substance applied to a sample holder can be scanned with an optical point source excitation and a read-back optical stage. The sample can be excited at each of a plurality of points with a fast, nanosecond-scale pulse of light, and the resulting fluorescence is 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.
The patent addresses the problem of detecting very short decay time fluorescence signals from high performance fluorescent probes with lifetimes on the order of nanoseconds. These lifetimes are so short that previous gating methods, which delayed the data collection until after excitation ended, would cause significant signal loss. Additionally, it is challenging to reduce excitation light leakage into the emission detection channel, which lowers the signal-to-noise ratio. Traditional methods either require turning the detector on and off, which is slow and complicated, or have fixed and lengthy delays that reduce sensitivity.
Claims Coverage
The patent contains several independent claims that delineate inventive features related to a device and method for mapping fluorescence signals with precise timing control and spatial resolution.
Device for mapping fluorescence with timing-controlled detection and spatial mapping
A device comprising a light source directed at a sample containing a fluorophore that generates a fluorescence signal; a pulse generator producing light source pulses; an active detector with data acquisition rates between approximately 10^4 and 10^7 pulses per second and image acquisition pixel resolutions between 10^-6 m × 10^-6 m and 10^-4 m × 10^-4 m; a timing circuit generating a delay between about 10^-12 s and 10^-10 s; a control circuit generating a duration; a high speed switch controlled by the delay and/or duration to selectively direct the fluorescence signal to the active detector; and a stepping motor moving the sample to map the fluorescence signal at different positions.
Use of a Field Effect Transistor switch for high speed gating
The device uses a Field Effect Transistor (FET) as a high speed switch controlled by timing and control circuits to gate and selectively direct the fluorescence signal to the detector after a specified delay and during a defined duration.
Control circuitry selection based on BoxCar Integrator
The control circuit selects the high speed switching circuit based on a BoxCar Integrator that defines the integration window for signal collection.
Fluorophore half-life specification
The fluorophore used has a half-life between approximately 10^-9 seconds and 10^-8 seconds, suitable for nanosecond time scale fluorescence detection.
Pulse generator and timing specifications
The pulse generator operates between approximately 10^4 and 10^7 pulses per second, with light source pulse durations between about 0.5×10^-9 s and 10^-8 s, and excitation separations between about 10^-6 m and 10^-4 m.
Detector response and transit time
The active detector exhibits response times and transit times between approximately 0.5×10^-9 s and 2×10^-9 s, supporting rapid accurate detection of short-lived fluorescence signals.
Method for mapping fluorophore labeled samples
Steps include receiving a sample with fluorophore half-life of approximately 10^-9 to 4×10^-9 seconds; applying voltages to an active photomultiplier tube; spatially orienting the sample; irradiating the sample with pulsed light; generating precise delay and duration control signals; directing fluorescence using a switch according to the delay and duration; detecting fluorescence at each spatial location; scanning through multiple locations and samples; and summing fluorescence data to map the sample.
System for mapping fluorescence with precise timing and stepping
A system includes a light source for excitation, a pulse generator for light pulses, an active photomultiplier tube detector, a timing circuit to generate delays between 10^-12 s and 10^-10 s, a control circuit for duration, a FET switch to gate detection based on delay and duration, and a step generator to raster scan the light source over the sample location.
The claims cover a comprehensive system and method for high-speed fluorescence detection and mapping using timing-controlled gating with FET switches, enabling detection of short lifetime fluorophores with nanosecond-scale pulse excitation and spatial scanning.
Stated Advantages
Improved signal-to-noise ratio by off gating the fluorescence detection until after excitation light has ceased, reducing excitation light leakage.
Capability to detect very short fluorescence lifetimes on the order of nanoseconds without signal loss due to gating delay.
Continuous operation of the photomultiplier tube without the need to switch on and off its high voltage, simplifying circuitry and reducing complexity.
Increased sensitivity enabling detection of very low analyte concentrations, reportedly as low as femtomolar concentrations.
Flexibility to adjust timing parameters to start detection at any desired point relative to excitation pulse for optimal signal collection.
Documented Applications
Measuring multidimensional distribution of samples tagged with short life fluorescent labels, including protein or other substances.
Scanning samples with optical point source excitation for two-dimensional or three-dimensional fluorescence mapping.
Protein microarrays for identifying protein interactions, receptor ligands, substrates of protein kinases, or transcription factor activators.
DNA sequencing using fluorophores attached to oligonucleotide primers representing different bases.
Live cell imaging utilizing fluorescent proteins as biosensors for monitoring intracellular phenomena such as pH, metal-ion concentration, apoptosis, membrane voltage, and neuronal pathway tracing.
Flow cytometry detection by labeling cellular features with different fluorophores.
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