Microscopy is the science of investigating small objects and structures using a microscope. Microscopy allows amplification of samples that are too small to be seen by the naked eye. It works by the reflection, refraction and diffraction of electromagnetic radiation and electron beams that interact with samples, followed by the collection of scattered signals that create an image.
The different types of microscopy include:
- Optical Microscopy
- Electron Microscopy
- Scanning Probe Microscopy
Optical microscopy is also known as light microscopy and was the first type of microscopy discovered. The microscope contains refractive glass and multiple lenses, which produce enlarged images of samples when they are in the focal plane.
Optical microscopes are split into categories depending on their design, including compound microscopes, stereo microscopes and confocal microscopes. Optical microscopes can also have ultraviolet, IR and fluorescence filters attached for a more in-depth analysis of samples.
Fluorescence microscopy is a type of optical microscopy developed later due to the increase in need for the analysis of fluorescent probes. Fluorescently labeled proteins and chemical stains are used to research proteins and cells.
Samples are illuminated with light that ranges from ultraviolet to visible wavelengths, and these are absorbed by the fluorophores in the sample. The absorption causes the samples to emit light of longer wavelengths that can then make up an image.
Atomic Force Microscopy
Atomic Force Microscopy (AFM) is a type of scanning probe microscopy. It was first discovered in 1986 by Binning, Gerber and Quate to overcome the disadvantages of scanning tunneling microscopy (STM).
Scanning probe microscopy scans the surface of samples using a probe to measure fine surface shapes and properties and then it generates an image. AFM has a fine silicon or silicon nitride probe attached to a cantilever. As the probe travels across the sample surface, it measures the morphology on an atomic scale. During scanning, the force between the tip and the sample is measured by monitoring the deflection of the cantilever. To investigate different surface properties, the tip can be modified.
AFM applications include imaging of molecules, tissues and cells, materials science, chemistry, nanotechnology applications for imaging polymers and nanostructures, physical and biophysical applications such as measuring force between the AFM tip and the sample surface.
Combining AFM and Optical Microscopy
The combination of AFM and optical microscopy has many different advantages. The AFM probe can be optically navigated to regions of interest and the additional 3D high resolution gives structural information of cells and molecules.
The combined mechanics of both allows for mechanical probing of elasticity, affinities and intramolecular forces and manipulation with optical observation. The tools are ultraprecise for nanolithography high-resolution AFM topographs, as well as providing overlays of optical and fluorescence images.
Optical microscopy’s chemical specificity and ability to image live processes within the depth of a sample complements the higher resolution capability of AFM. A popular use for combining the techniques includes identifying internal components of cells to utilize multiple fluorescent markers so specific binding.
When you overlay AFM data directly onto the optical data it allows for correlation, and the higher-resolution of AFM resolves structures that are not composed of the target molecules for fluorescence, or those that are weakly labeled or too small.
Combining AFM with optical systems has given the option to allow researchers to interact with samples and physically manipulate them. Researchers at UC Berkeley used a carbon nanotube modified tip to penetrate the cell membrane and physically inject molecules of interest without damaging or killing the cell.
Transmitted light optical microscopy was used to guide the nano-surgical tool to the area of interest within the cell and fluorescence microscopy was used to confirm the localization, internalization and lifetime of the molecules for several hours after injection into the cell.
While combining correlative AFM and optical microscopy has many advantages, there are still many challenges to overcome. For true meaningful correlation between AFM and optical microscopy, the resolution of the two techniques must be matched.
Having unmatched resolutions between AFM and fluorescence microscopy has produced difficulty when correlating the structural information with the distribution of specific components at a single-molecule level. To be able to overcome this, super-resolution fluorescence microscopy that achieves nanometer resolution has to be combined with AFM.
References and further reading
- Geisse, N. (2009). AFM and combined optical techniques. Materials Today, 12(7-8), pp.40-45.
- Chen, X., Kis, A., Zettl, A. and Bertozzi, C. (2007). A cell nanoinjector based on carbon nanotubes. Proceedings of the National Academy of Sciences, 104(20), pp.8218-8222