Lecture 11. Microscopy

Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope. The most recent development is the digital microscope which uses a CCD camera to focus on the exhibit of interest. The image is shown on a computer screen since the camera is attached to it via a USB port, so eye- pieces are unnecessary.

TEM The conventional electron microscopy is nowadays called TEM (transmission electron microscopy). We will therefore start with its construction. The ray of electrons is produced by a pin-shaped cathode heated up by current. The electrons are vacuumed up by a high voltage at the anode. The acceleration voltage is between 50 and 150 kV. The higher it is, the shorter are the electron waves and the higher is the power of resolution. But this factor is hardly ever limiting. The power of resolution of electron microscopy is usually restrained by the quality of the lens-systems and especially by the technique with which the preparation has been achieved. Modern gadgets have powers of resolution that range from 0,2 - 0,3 nm. The useful resolution is therefore around 300,000 x.

SEM The path of the electron beam within the scanning electron microscope differs from that of the TEM. The technology used is based on television techniques. The method is suitable for the depiction of preparations with conductive surfaces. Biological objects have thus to be made conductive by coating with a thin layer of heavy metal (usually gold is taken). The power of resolution is normally smaller than in transmission electron microscopes, but the depth of focus is several orders of magnitude greater. Scanning electron microscopy is therefore also well- suited for very low magnifications.

Fluorescence microscopy acquiring microscopic images of samples (often biological materials) using fluorescence within the sample, which is usually excited with a sharply focused diffraction-limited laser beam. focus of the laser beam is raster scanned through the sample (e.g. using galvo mirrors), and the fluorescent light excited in the sample is collected with some optics and monitored with a photodetector. Residual laser light can be eliminated with an optical filter before the detector. The fluorescence microscope is usually operated with a computer (a PC or a laptop), which controls the scan, records the fluorescence intensities, and finally generates, processes and stores the obtained images.