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Opening a new perspective in high resolution imaging. Boosting research in the biomedical world by combining two advanced imaging techniques: electron microscopy and fluorescence microscopy

Application | Thermofisher Scientific/Delmic correlative imaging. The microscopy solution is used by analysts in a research and development environments in the Biological, Medical and Life Science sectors.
Market segment | Analytical & Lifesciences: Correlative imaging; augmenting electron microscopy imaging with fluorescence details.
Business activities | Concept development, optical and opto-mechanical design, prototyping and series production.

 

Electron microscopy is known for its very high resolution. It, however, lacks the ability to clearly show the composition of the imaged sample. Fluorescence microscopy is known for its ability to clearly show and discern fluorescent labeled materials, albeit with an optically limited spatial resolution. Combining these techniques in one apparatus yields images with high resolution and detailed molecular information. This in turn opens up new perspectives for analysts in an R&D environment.

 

Application context

Electron microscopy yields high resolution monochromatic images showing the detailed structure of the sample. However, with biological samples it lacks essential information about the composition of these structures. This information can be obtained by using fluorescence microscopy and fluorescent labeling of proteins or RNA sequences. Integrating a fluorescence microscope within an electron microscope, both observing the same sample at the same location and time enables Correlative Light and Electron Microscopy (CLEM).

CLEM requires simultaneous access to the sample by the electron beam and by the high numerical aperture microscope objective of the fluorescence microscope. This can be realized by locating each on either side of the ultra-thin sample or by having an accurate way of flipping the sample between two positions.

Sub-micron image overlay is important, as well as having the microscope objective and its actuators for focusing and alignment to operate in the high vacuum environment of the electron microscope. Furthermore, charge build-up on the optical microscope objective has to be avoided, since it could affect the electron beam and resulting image. This requires charge draining measures and coatings.

 

Business benefits

Correlative imaging provides structural and composition information of samples in perfect overlay and in a single step. Obtaining comparable results with a classical electron microscope and a fluorescence microscope is time consuming and laborious. As such Correlative Imaging saves valuable R&D time and speeds up research in biomedical analysis. It ultimately provides more insight and more accurate results.

Used techniques

It is the combination of optical design and mechanical construction that are essential: Vacuum compatible, high numerical aperture objective, compact manipulator for alignment and focusing with micrometer resolution.

Key characteristics:

- a diffraction limited performance with high NA and low image distortion
- high power density UV-LED illumination (>150mW/mm2)
- vacuum compatible objective (no outgassing)
- ITO-coated (Indium Tin Oxide) optical objective lens to avoid charge build-up
- vacuum compatible XY and focusing actuators
- X-ray shielding using lead components

 

 

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