Fluorescence Microscopy Illumination Localization for Vitrified Sample Preservation
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Solution Overview
Problem
Intense illumination in fluorescence microscopy can cause heating and damage to vitrified samples due to absorption of excitation light by the carbon support film, leading to crystallization or sublimation.
Innovation Solution
The method involves using a carbon film with holes or thickness variations to create position-dependent absorbance, ensuring that areas of the sample are illuminated only over low-absorption regions, while avoiding illumination of high-absorption areas to minimize heat dissipation, utilizing a spatial light modulator to localize intense illumination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If intense illumination is used in fluorescence microscopy, then the fluorescent signal can be collected in short time periods and signal-to-noise is improved, but heating occurs that damages or destroys the specimen
Solution Approach 1:
The carbon support film is designed with spatially varying properties - holes or thickness variations create position-dependent absorbance. The sample areas to be inspected are positioned over low-absorption regions, while high-absorption regions are avoided during illumination. This local differentiation allows intense illumination of specific areas without heating damage.
Solution Approach 2:
The carbon support film is segmented into regions with different optical absorbance characteristics through holes or thickness variations. This segmentation allows selective illumination of low-absorption regions while excluding high-absorption regions from the illumination path, resolving the contradiction between intense illumination and heating damage.
2Illumination intensity
If the sample is illuminated with intense light, then the fluorescent signal is enhanced, but the carbon support film absorbs the light and converts it to heat, causing crystallization or sublimation of the ice
Solution Approach 1:
The carbon support film exhibits local quality variations through holes or thickness variations, creating position-dependent absorbance. The sample areas are positioned over low-absorption regions, ensuring that intense illumination does not convert to heat in the carbon film, thus preventing crystallization or sublimation while maintaining high illumination intensity for fluorescence detection.
3Strength
If a continuous carbon support film is used, then the sample is well-supported, but the entire film absorbs excitation light and generates heat uniformly, damaging the sample
Solution Approach 1:
The continuous carbon support film is segmented into regions with holes or thickness variations. This segmentation maintains sample support in low-absorption regions while creating high-absorption regions that are avoided during illumination. The segmentation allows the carbon film to provide mechanical support without causing uniform heating across the entire sample area.
Solution Approach 2:
The carbon support film is designed with local quality variations - holes or thickness variations - to create position-dependent absorbance. The sample areas are positioned over low-absorption regions that maintain support, while high-absorption regions are strategically avoided during illumination to prevent uniform heating and sample damage.
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 intense illumination without damaging the sample, maintaining the sample's vitrified state by controlling heat generation and dissipation, enabling high-quality imaging while preventing crystallization or sublimation.
Implementation Method 1
the supporting carbon film shows holes or thickness variations resulting in position dependent absorbance of the excitation light
Implementation Method 2
The heat that is generated in the carbon reaches the sample area by thermal conduction and in this way causes a temperature rise of the sample
Implementation Method 3
the illumination is localized such that the parts of the supporting carbon film with a high absorption of the excitation light and bordering the parts of the sample to be inspected are not illuminated
Implementation Method 4
the fluorescence microscope illuminating the sample with excitation light to generate fluorescence or phosphorescence
Implementation Method 5
the fluorescence microscope illuminating the sample with excitation light to generate fluorescence or phosphorescence
Implementation Method 6
The heat that is generated in the carbon reaches the sample area by thermal conduction
Implementation Method 7
a cryogenic vitrified sample is irradiated with light
Implementation Method 8
freezing the sample to a temperature below the glass transition temperature of water of approximately 130 K
Data Source
Figure 1A
Figure 1B~1C
AI summary
The invention relates to a method of inspecting parts of a sample on a TEM grid (12) with a fluorescence microscope, as arises when performing correlative microscopy, more specifically for samples on a holey carbon grid or . A problem occurs when imaging vitrified ice (20) with sample material (22) therein as the ice is heated by the light used. The invention is based on the insight that especially the absorption in the carbon (16) is responsible for the heating, as ice hardly absorbs light. By localizing the illumination of the fluorescent microscope to the parts of the sample that are above a hole (18) in the carbon, heating of the ice is lowered. The localization can be achieved by e.g. passing the light though a LCD type Spatial Light Modulator.