Systems and methods for distinguishing stimulated emissions as a means of increasing the signal of fluorescence microscopy
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9791371-B2
Publication Date
2017-10-17
Expiration Date
2035-10-29
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Abstract
Embodiments of a fluorescence microscopy system that employs a technique for distinguishing stimulated emission as a means for enhancing signal strength of fluorescent markers are disclosed.
Core Innovation
The invention describes fluorescence microscopy systems and methods that utilize a technique for distinguishing stimulated emission as a means to increase the signal strength of fluorescent markers. The system employs separate excitation and stimulation light beams positioned along axes that are perpendicular to each other, involving different wavelengths to stimulate fluorescent molecules in a sample. By selectively blocking the stimulating light beam and allowing the stimulated emission to pass to a detector, the fluorescence microscopy system enhances detection sensitivity and brightness of the fluorescent markers.
The problem addressed is the fundamental limitation in fluorescence microscopy due to the nanosecond-scale lag between absorption and spontaneous emission, which restricts how much light a fluorescent marker can emit per second. This limitation hinders high-speed and high-precision measurements such as observing individual molecular steps that require fluorescent markers emitting thousands of photons per millisecond. While stimulated emission can produce much brighter signals faster than spontaneous emission, it is difficult to distinguish from the stimulation beam itself due to identical color, phase, polarization, and direction, causing noise and background that negate its advantages. No existing method adequately separates the stimulated emission from the stimulation beam for detection.
The disclosed embodiments solve this by arranging excitation and stimulation beams at perpendicular axes with different wavelengths and incorporating features such as a stimulation light beam block component at the focal point of an objective lens to block the stimulating beam while capturing the stimulated emission. Alternatively, a system embodiment combines excitation and stimulation beams at a right-angle intersection illuminating the sample and captures the stimulated emission along a third, distinct axis, allowing clean separation from the stimulating light. These configurations allow earlier and clearer detection of the stimulated emission signal from fluorescent markers, thus overcoming the prior limitation of distinguishing stimulated emission for enhanced fluorescence microscopy.
Claims Coverage
The claims include two independent fluorescence microscopy system claims and one independent method claim, each comprising inventive features related to the orientation, wavelength differentiation, and separation of excitation, stimulation, and stimulated emission light beams for enhanced fluorescence detection.
Perpendicular excitation and stimulation light beams with different wavelengths
The system employs an excitation source generating an excitation light beam along a first axis and a stimulation source generating a stimulation light beam along a second axis positioned perpendicularly to the first axis, the beams having different wavelengths.
Use of stimulation light beam block to separate stimulated emission
A stimulation light beam block component is positioned at the focal point of an objective lens oriented on the stimulation light beam axis to block the stimulation beam while allowing the stimulated emission to pass to a detector.
Combined excitation/stimulation light beam generation by intersection and perpendicular detection
The system generates a combined excitation/stimulation light beam from the intersection of the excitation and stimulation beams, captures it via an excitation objective lens, then captures the stimulated emission emitted by the sample with a detection objective lens oriented perpendicularly to the excitation objective, allowing spatial separation of stimulated emission from excitation/stimulation beams.
Method for fluorescence microscopy distinguishing stimulated emission
A method involving illuminating fluorescent markers with excitation and stimulation beams along perpendicular axes of different wavelengths, focusing the stimulation light and stimulated emission through an objective lens, blocking the stimulation beam at the focal point of the lens, and detecting the stimulated emission by a detector.
These inventive features collectively enable clean and efficient distinction of stimulated emission from the stimulation light beam by spatially separating axis orientations, using different wavelengths, and incorporating blocking or objective lens arrangements, thereby enhancing fluorescence microscopy signal detection.
Stated Advantages
Greatly increasing the brightness and photostability of fluorescent markers, enabling high speed, high precision measurements which are currently impossible.
Stimulated emission can be several orders of magnitude brighter than spontaneous emission, providing a stronger fluorescence signal.
The arrangement allows the stimulated emission to be detected earlier and with less noise by effectively separating it from the stimulating beam.
Documented Applications
High-resolution, high-speed, protein-specific imaging in living cells, tissues, and animals using fluorescence microscopy.
Observing individual steps of RNA polymerase on DNA strands which require fluorescent markers emitting thousands of photons per millisecond.
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