Education Centre

Nature has a fundamental diffraction limit which restricts the level of detail we can acheive when using fluorescence microscopy. This limit is related to the wavelength of light and the optical pathway the light has to pass through. It is important to understand the limitations on conventional microscopy to appreciate the level of detail using SR techniques provides.

 

Fluorescence microscopy is a powerful tool in the study of the physical sciences, enabling researchers to better understand our cellular make-up and providing the fuel to further develop microscopy techniqes. The principles of fluorescence as well as the development of the probes available is covered here.

 

Confocal microscopy is an optical imaging technique that improves on image performance compared to a traditional widefield microscope by acquiring highly detailed information of thin optical sections, a process known as optical sectioning. You can find out more behind the principles of this techniques here.

 

Imaging biological constituents at the plasma membrane can be achived via Total Internal Reflection Fluorescent Microscopy, a method that only excites fluorophores a few 100nm's into the cell. This is advantageous as this minimises out of focus light and gives a high contrast image.

 

Structured Illumination Microscopy, instead of a single beam of light, uses gratings to create a pattern of light which is illuminated onto the sample at different angles. This effect generates an image with greater resolution than would otherwise be acheived using point illumination and can be acheived in multiple colours.

 

Overcoming the diffraction limit can be achieved using a light shaping technique called, Stimulated Emission Depletion Microscopy. This powerful technique uses two lasers to manipulate the point spread function of the sample hence increasing the resolution achieved by the microscope. 

 

Single molecule localisation microscopy relies on the "switching on and off" or the "blinking" of fluorophores and fluorescent dyes to reduce the density of molecules imaged at one time, allowing for single molecule localisation. The improvement in resolution in this technique is 200 fold that of diffraction limited microscopy.

 

This technique uses Time-Correlated Single Photon Counting (TCSPC) to measure the Fluorescence Lifetime of proteins enabling the measure of interaction known as Försters Resonance Energy Transfer (FRET). This imaging tool is highly quantitative and provides information both spatially and temporally.

 

The movement of molecules through a parked laser beam in a sample can provide quantative information about protein or lipid concentration, their diffusion coefficients and other statistically important parameters. This technique can be performed in solution and inside living cells and can be applied to topics outside cell biology.

 

Imaging on the atomic scale is a desirable quality for some projects, and one that ESRIC is proud to offer. Atomic Force Microscopy allows you to generate a 3D topological map as well as perform force mapping to investigate the mechanical properties of your sample.