Integrated fluorescence and transmission electron microscopy
Alexandra V Agronskaia1, Jack A Valentijn, Linda F van Driel
1Molecular Biophysics, Department of Physics, Faculty of Science, Utrecht University, Princetonplein 1, 3584 CC Utrecht, The Netherlands.
Journal of Structural Biology
|July 31, 2008
Summary
This study presents an integrated correlative microscopy system combining fluorescence and electron microscopy. This novel approach simplifies specimen searching and significantly reduces experimental time for biological imaging.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biophysics
Background:
- Correlative microscopy merges fluorescence microscopy (FM) and electron microscopy (EM) for enhanced biological imaging.
- Current correlative workflows are hindered by complex, time-consuming procedures involving separate microscopes.
- Bridging the gap between FM's broad searching capability and EM's high resolution is crucial.
Purpose of the Study:
- To develop and validate an integrated correlative microscopy system.
- To streamline the correlative workflow by combining FM and EM into a single platform.
- To reduce the time and complexity associated with correlative sample analysis.
Main Methods:
- Integration of a laser scanning fluorescence microscope within a transmission electron microscope.
- Utilized AlexaFluor488-conjugated wheat germ agglutinin for labeling rat intestine cells.
- Employed immunolabeling with anti-catalase antibodies and protein A-gold for rat liver peroxisomes.
Main Results:
- Demonstrated a simplified search for specimen features within the integrated system.
- Significantly reduced the overall experimental time compared to conventional methods.
- Successfully imaged labeled rat intestine cells and liver peroxisomes at room temperature.
Conclusions:
- The integrated correlative microscopy approach offers a more efficient and user-friendly method for biological research.
- This system overcomes limitations of separate FM and EM setups, enhancing workflow efficiency.
- The presented technology holds significant potential for advancing high-resolution cellular and subcellular imaging.
More Related Videos
08:29Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells
Published on: October 17, 2025
09:17Scanning Transmission Electron Microscopy Tomography in Virology: 3D Imaging of High-pressure Frozen, Freeze-substituted Samples
Published on: August 6, 2025
Related Concept Videos
01:15Transmission Electron Microscopy
In 1931, physicist Ernst Ruska—building on the idea that magnetic fields can direct an electron beam just as lenses can direct a beam of light in an optical microscope—developed the first prototype of the electron microscope. This development led to the development of the field of electron microscopy. In the transmission electron microscope (TEM), electrons are produced by a hot tungsten element and accelerated by a potential difference in an electron gun, which gives them up to 400 keV in...
01:25Overview of Electron Microscopy
The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
01:37Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
01:05Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
01:07Electron Microscope Tomography and Single-particle Reconstruction
Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
01:22Overview of Microscopy Techniques
The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...