Variable Diaphragm and Filter for Simultaneous Darkfield Fluorescence Imaging
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Solution Overview
Problem
Current optical microscopes, such as electron and ultraviolet microscopes, are inadequate for observing living, unaltered biological specimens in real time due to sample preparation damage, high costs, and the inability to view processes like cellular transport and interactions, limiting understanding of cellular biology and potential treatments for diseases.
Innovation Solution
A method and apparatus for simultaneous observation of high-resolution darkfield images and fluorescence using a variable diaphragm and filter, allowing adjustment of light intensity and frequency to optimize imaging, enabling the observation of both darkfield and fluorescent images simultaneously without damaging living specimens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If electron microscopes are used to achieve high resolution, then imaging precision is improved, but the specimen is damaged by vacuum dehydration, heat, and electron beam
Solution Approach 1:
The patent uses inexpensive glass optics instead of expensive quartz optics required for UV microscopes, and employs standard optical components that can be replaced or adjusted easily. The system uses a broadband light source with filters rather than expensive UV lasers, making the system more accessible and less damaging to specimens.
Solution Approach 2:
The patent changes the wavelength parameter from ultraviolet to visible light range, allowing the use of glass optics instead of quartz optics. This parameter change enables observation of living specimens without UV damage while maintaining sufficient resolution through the use of darkfield and fluorescence imaging techniques.
2Measurement precision
If ultraviolet light is used to achieve finer resolution and better magnification, then imaging precision is improved, but living biological specimens are damaged or killed
Solution Approach 1:
The patent shifts the operating wavelength from ultraviolet to visible light range (400-700 nm), which eliminates UV damage to living specimens. The system compensates for the reduced resolution by using darkfield illumination and fluorescence techniques that enhance contrast and imaging capability at visible wavelengths.
Solution Approach 2:
The patent introduces fluorescent dyes as intermediaries that absorb visible light and emit fluorescence, enabling high-contrast imaging of cellular structures without direct UV exposure. The fluorescent markers allow visualization of specific cellular components using visible light excitation, avoiding the harmful effects of ultraviolet radiation on living specimens.
3Illumination intensity
If darkfield illumination is used to observe scattered light, then contrast is improved, but fluorescence observation is compromised due to overwhelming scattered light
Solution Approach 1:
The patent segments the optical path into distinct channels: a darkfield condenser for scattered light observation and a fluorescence excitation path with separate filters. The beam splitter divides the illumination, allowing darkfield illumination for one channel and fluorescence excitation for another, enabling simultaneous observation without interference between the two techniques.
Solution Approach 2:
The patent uses a beam splitter as an intermediary device to separate the darkfield and fluorescence optical paths. This allows independent optimization of each imaging mode while enabling simultaneous observation, with the beam splitter directing scattered light to one detector and fluorescence to another, resolving the conflict between high contrast darkfield imaging and sensitive fluorescence detection.
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
Enables the observation of living cells and their processes at high resolution, facilitating research in cellular biology and potential disease treatments by allowing for real-time, unaltered imaging without the need for skilled technicians or expensive equipment.
Implementation Method 1
fluorescence when illuminated with ultraviolet light
Implementation Method 2
A first filter transmits only exciting radiation, and the second filter transmits only emitted fluorescent light
Implementation Method 3
A light source provides broadband light that is collimated and filtered in frequency
Implementation Method 4
separate a weak emitted fluorescence from the excitation light, so that the emitted fluorescence can be observed
Data Source
AI summary
An annular diaphragm and filter used for the simultaneous observation of darkfield images and fluorescence. The diaphragm has a variable diameter controlled by a lever and a removable filter. The diaphragm is used to adjust the amount of unfiltered incident light which produces darkfield images when directed on a sample. The removable filter is used to filter light to a particular frequency for producing fluorescence images. An Acousto-Optical Tunable Filter, or other such tunable filter may be used with the diaphragm. A method of using the diaphragm and filter is also disclosed.