SLAM Microscopy Single-Wavelength Dual-Fluorophore Excitation
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Solution Overview
Problem
Current multiphoton microscopy techniques face challenges in simultaneous and efficient excitation of autofluorescence and multiharmonic processes due to the need for sequential excitation bands, leading to increased acquisition time and photodamage risk, as well as weak intrinsic contrast from endogenous fluorophores at longer excitation wavelengths.
Innovation Solution
The development of a simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy platform that uses a single excitation wavelength across 1080-1140 nm, employing near-transform-limited excitation pulses with broad bandwidth and low pulse repetition rate to enhance peak power, allowing for simultaneous visualization of various molecular contrasts via spectrally resolved detection channels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Power
If sequential excitation bands are used for autofluorescence and multiharmonic processes, then signal generation efficiency is improved, but acquisition time increases and photodamage risk increases
Solution Approach 1:
The patent combines multiple excitation processes (autofluorescence and multiharmonic generation) into a single excitation band at 1080-1140 nm, allowing simultaneous excitation of both red and blue fluorophores through different photon orders (n and n+1), thereby eliminating sequential scanning and reducing acquisition time while maintaining signal generation efficiency
Solution Approach 2:
The single excitation wavelength band (1080-1140 nm) serves multiple functions by enabling both n-photon excitation for red fluorophores and (n+1)-photon excitation for blue fluorophores, making the excitation source universal for detecting multiple molecular contrasts simultaneously
2Power
If sequential excitation bands are used for autofluorescence and multiharmonic processes, then signal generation efficiency is improved, but photodamage risk increases
Solution Approach 1:
By merging multiple excitation processes into a single simultaneous excitation band, the total exposure time is reduced, which directly lowers the cumulative photodamage risk to biological samples while maintaining efficient signal generation through optimized peak power of the pulsed laser
Solution Approach 2:
The patent employs pulsed laser excitation with optimized duty cycle and peak power, using periodic action to deliver high instantaneous power for efficient signal generation while allowing intervals between pulses to minimize cumulative photodamage to the sample
3Length of stationary object
If longer excitation wavelengths are used, then penetration depth is improved, but intrinsic contrast from endogenous fluorophores decreases
Solution Approach 1:
The patent changes the excitation parameters by using a specific wavelength band (1080-1140 nm) that balances penetration depth and signal generation, and employs mixed photon-order excitation (n and n+1) to enhance the excitation efficiency of endogenous fluorophores at these longer wavelengths, thereby maintaining intrinsic contrast while achieving deep tissue penetration
Solution Approach 2:
The detection system uses composite spectral analysis by simultaneously detecting multiple photon orders and separating signals through spectral resolution, effectively extracting weak intrinsic contrast signals from deeper tissue by combining information from different emission channels
4Loss of time
If single wavelength band excitation is used, then acquisition time is reduced and photodamage is reduced, but simultaneous excitation of multiple fluorophores becomes difficult
Solution Approach 1:
The patent applies local quality by exploiting the different photon order requirements of different fluorophores: red fluorophores are excited via n-photon absorption while blue fluorophores are excited via (n+1)-photon absorption using the same excitation band, allowing selective excitation of different molecular targets through their distinct nonlinear optical properties
Solution Approach 2:
The patent utilizes color changes in the emitted fluorescence signals to differentiate between fluorophores excited by the same wavelength band, detecting red fluorophores at longer emission wavelengths and blue fluorophores at shorter emission wavelengths, thereby achieving multiplexed detection through spectral separation
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly reduces photodamage and acquisition time while improving signal generation efficiency, enabling real-time label-free imaging with enhanced contrast and reduced phototoxicity.
Implementation Method 1
exciting the red fluorophore via absorption of a photon order of n by a single wavelength band of light... exciting the blue fluorophore substantially via absorption of a photon order of n+1 by the single wavelength band of light
Implementation Method 2
employing near-transform-limited excitation pulses with broad bandwidth and low pulse repetition rate to enhance peak power
Implementation Method 3
simultaneous detection of light emitted by the red fluorophore and the blue fluorophore... spectrally resolved detection channels
Data Source
AI summary
A method is provided for characterizing a biological sample having a plurality of fluorophores, including a red fluorophore and a blue fluorophore, comprises exciting the red fluorophore via absorption of a photon order of n by a single wavelength band of light that has longer wavelengths than a typical wavelength band of light known to excite the red fluorophore would have. The method further comprises exciting the blue fluorophore substantially via absorption of a photon order of n+1 by the single wavelength band of light. The method also comprises simultaneously detecting light emitted by the red fluorophore and the blue fluorophore. The method further comprises creating an image or a temporal series for sensing from the light detected in the plurality of orthogonal colors.