Laser Microscope Multiphoton Excitation Multi-Point Observation
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Solution Overview
Problem
The fluorescence observation method based on multiphoton excitation faces challenges in simultaneously irradiating a large number of ultrashort-pulse laser light beams and achieving high signal-to-noise ratio observations with low invasiveness.
Innovation Solution
A laser microscope that applies different modulations to ultrashort-pulse laser light beams, focuses them simultaneously onto various positions of a sample, and uses a fluorescence detecting device with a demodulation unit to separate and extract fluorescence signals, enabling simultaneous observation of multiple points with a high signal-to-noise ratio and low invasiveness.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If fluorescence observation is performed by collecting scattered fluorescence from focal position via objective lens, then deep tissue observation with low invasiveness is achieved, but simultaneous multi-beam observation is not possible
Solution Approach 1:
The fluorescence detection system is segmented into multiple independent detection paths, each corresponding to a specific focal position. The fluorescence collection objective lens is positioned to receive fluorescence from multiple focal points simultaneously, and the detection device includes multiple photodetectors arranged to detect fluorescence from different spatial locations. This segmentation allows independent detection channels for each focal position, enabling simultaneous multi-beam observation while maintaining low invasiveness through the use of scattered fluorescence collection.
2Productivity
If multiple ultrashort-pulse laser beams are focused onto different positions simultaneously, then multi-point observation capability is improved, but signal separation and identification becomes difficult
Solution Approach 1:
Each ultrashort-pulse laser beam is modulated with a distinct periodic modulation signal before being focused onto its target focal position. The modulation frequencies are chosen to be different for each beam, allowing the fluorescence signals generated at different focal positions to be distinguished through frequency demultiplexing. The detection device includes demodulation circuits that can separate the mixed fluorescence signals based on their unique modulation frequencies, thus enabling simultaneous multi-point observation while maintaining clear signal identification.
3Device complexity
If a single detection device is used to detect fluorescence from multiple focal points, then device complexity is reduced, but measurement precision and signal-to-noise ratio deteriorate
Solution Approach 1:
The detection device is segmented into multiple independent detection channels, with each channel including its own photodetector and demodulation circuitry. Each detection channel is dedicated to detecting fluorescence from a specific focal position, allowing for optimized signal processing and noise filtering for each channel. This segmentation maintains relatively simple overall device structure while significantly improving measurement precision and signal-to-noise ratio through specialized detection paths for each focal point.
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 allows for the simultaneous observation of fluorescence generated by multiphoton excitation using multiple beams and multiple points with a high signal-to-noise ratio, reducing invasiveness and improving observation capabilities.
Implementation Method 1
a modulation unit that applies different modulations to a plurality of ultrashort-pulse laser light beams of the same type emitted from a light source unit
Implementation Method 2
a fluorescence generating unit that generates fluorescence by multiphoton excitation at a focal position of each ultrashort-pulse laser light beam
Implementation Method 3
an illumination optical system that simultaneously focuses the plurality of ultrashort-pulse laser light beams, to which the different modulations are applied by the modulation unit, onto different positions of a sample
Implementation Method 4
a fluorescence detecting device that detects fluorescence generated at a focal position of each ultrashort-pulse laser light beam and performs photoelectric conversion of the fluorescence
Implementation Method 5
a demodulation unit that demodulates an output from the fluorescence detecting device based on modulation information from the modulation unit
Data Source
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AI summary
Fluorescence generated by multiphoton excitation can be observed simultaneously using multiple beams, and multiple points can be observed simultaneously with a high signal-to-noise ratio with low invasiveness. Provided is a laser microscope 1 including: modulation units 5 and 6 that apply different modulations to a plurality of ultrashort-pulse laser light beams of the same type emitted from a light source unit 3; an illumination optical system 7 that simultaneously focuses the plurality of ultrashort-pulse laser light beams, to which the different modulations are applied by the modulation units 5 and 6, onto different positions of a sample O; a fluorescence detecting device 8 that detects fluorescence generated at a focal position of each ultrashort-pulse laser light beam and performs photoelectric conversion of the fluorescence; and a demodulation unit 9 that demodulates an output from the fluorescence detecting device 8 based on modulation information from the modulation units 5 and 6.