Lecture 4 - Microscopy

Contents
1. Compound (optical) microscope 2. The magnification of a microscope
M = mob mey = L 25 cm fo fe

O b F
O

E y

F F
O E

F
E

L the distance between the lenses fo the focal length of the objective fe the focal length of the eyepiece 3. Resolving power of a microscope 1 n sin D= =

- the smallest distance between two object details still distinguishable in the microscope - the wavelength, n refractive index of the medium between the specimen and the objective - half of the aperture angle
The magnification of a compound microscope is limited by its resolving power. 4. Transmission Electron Microscopy The electrons passing through the object behave as the so-called matter wave with the wavelength where h is Plancks constant and mv is an electron momentum. The electrons h = leaving the source are accelerated. The accelerating voltage is usually several ten mv thousands volts, so is in range even 1/100 nm. 5. Scanning electron microscope The surface of a thicker object can be examined by the reflection of the electron beam from the surface. A beam spot is scanned across the specimen like in a television tube.
Secondary Electron Imaging shows the topography of surface features a few nm across. Films and stains as thin as 20 nm produce adequate-contrast images. More secondary electrons can escape from a tip than from a cavity, therefore convex areas are brighter then concave ones. Low energy electrons (ca. 5eV) can be emitted from very thin film, therefore the resolution is high. Materials are viewed at useful magnifications up to 100,000x without the need for extensive sample preparation and without damaging the sample. Backscattered Electron Imaging shows the spatial distribution of elements or compounds within the top micron of the sample. Areas of bigger atomic number scatter more electrons and are brighter then areas composed of atoms of lower atomic numbers. Backscattered electron images have the resolution smaller then secondary electron ones. Features as small as 10 nm are resolved and composition variations of as little as 0.2% determined.

6. Fluorescence Microscopy The fluorescence microscopy is used to study specimens, which can be made to fluoresce. The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. The sample can either be fluorescing in its natural form like chlorophyll and some minerals, or treated with fluorescing chemicals. The emitted light is of lower energy and has a longer wavelength. Hence, to become visible, the emitted light is separated from the much brighter excitation light in a color filter. Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity. For example, certain antibodies and disease conditions or impurities in inorganic material can be studied with the fluorescence microscopy. 7. Confocal microscopy The confocal microscope is designed to accept light only from a thin slice within the tissue and to reject light reflected and scattered from other regions. Light reflected from the focal point emerges from the objective as a parallel beam. The beam is focused by the collecting lens at the exit aperture and creates the final image. 8. Scanning Tunneling Microscopy The STM is based on the concept of quantum tunneling. When a conducting tip is brought very near to a metallic or semiconducting surface, a voltage between the two can allow electrons to tunnel through the vacuum between them. For low voltages, this tunneling current is a function of the local density of states at the Fermi level, Ef, of the sample. Variations in current as the probe passes over the surface are translated into an image. STM requires extremely clean surfaces and sharp tips. 9. Atomic Force Microscopy A feedback mechanism is employed to adjust the tip-to-sample distance to maintain a constant force between the tip and the sample. The sample is mounted on a piezoelectric tube, that can move the sample in the z direction for maintaining a constant force, and the x and y directions for scanning the sample. Advantages relative to SEM Unlike the electron microscope which provides a two-dimensional projection or a twodimensional image of a sample, the AFM provides a true three-dimensional surface profile. Samples viewed by AFM do not require any special treatments (such as metal/carbon coatings) that would irreversibly change or damage the sample. While an electron microscope needs an expensive vacuum environment for proper operation, most AFM modes can work perfectly well in ambient air or even a liquid environment. This makes it possible to study biological macromolecules and even living organisms. Ultra-high vacuum (UHV). UHV AFM is comparable in resolution to STM and TEM. Disadvantages The SEM can image an area on the order of millimetres by millimetres with a depth of field on the order of millimetres. The AFM can only image a maximum height on the order of micrometres and a maximum scanning area of around 150 by 150 micrometres. The quality of an image is limited by the radius of curvature of the probe tip, and an incorrect choice of tip for the required resolution can lead to image artifacts. Traditionally the AFM could not scan images as fast as an SEM, requiring several minutes for a typical scan, while an SEM is capable of scanning at near real-time (although at relatively

low quality) after the chamber is evacuated. The relatively slow rate of scanning during AFM imaging often leads to thermal drift in the image.