1933 Ernst Ruska and Max Knoll first TEM

1938 von Ardenne first STEM

1942 V. Zworykin, J. Hillier and G. Snyder first SEM

1985 H Rohrer and G Binnig first scanning tunneling microscope
 

Transmission Electron Microscope (TEM) (Fig. 1.1, 4.6) uses a wide beam of electrons passing through a thin sliced specimen to form an image. This microscope is analogous to a standard upright or inverted light microscope. Stained areas of the sample absorb or scatter the beam, producing dark spots; unstained areas appear light (Fig 1.3).
 
 

Scanning Transmission Electron Microscope (STEM) uses a focused beam of electrons scanning through a thin sliced specimen to form an image. The STEM looks like a TEM but produces images as does an SEM (one spot at a time). The images are produced a spot at a time, as in the scanning electron microscope, rather than all at one time as in the TEM. It is most commonly used for elemental analysis of samples.
 
 

Scanning Electron Microscope (SEM) (Fig. 1.4, 5.3) uses focused beam of electrons scanning over the surface of thick or thin specimens. This microscope is analogous to the stereo or dissecting light microscope. Images are produced one spot at a time in a grid-like raster pattern. Areas reflecting lots of electrons appear bright; areas not relecting many electrons appear dark (Fig 1.4).
 
  Boston Museum of Science SEM animation: http://www.mos.org/sln/sem/sem.mov

Scanning Probe Microscopes (SPMs) are a family of instruments used for studying surface properties of materials from the atomic to the micron level. All these microscopes (atomic force, scanning tunneling microscope, etc.) use a probe tip that measures changes in surface characteristics as the tip scans over the surface of an object. Different kinds of images can be created depending on the nature of the probe tip. For example, in a scanning tunneling microscope, a fixed voltage is maintained between the tip of a probe and the conductive sample surface. If the surface dips, the voltage drops exponentially as the electron shells between probe tip and sample get further apart (tunneling current). The probe moves up or down to keep the voltage constant and thereby records the surface topography. The atomic force microscope uses a laser reflecting off the back side of the probe tip to determine the position of the probe tip. Ridges and valleys on the non-conductive sample cause the probe tip to rise and fall as the negative electron shells of the sample repel the negative electron shells of the probe tip (similar charges repel). These microscopes, although they may use interactions between electron shells, are not electron microscopes as used above.(Fig 5.29 -5.31)

Scanning probe microscopy of membrane bound protein complexes (from http://www.ornl.gov/sci/GenomestoLife/areas/imaging.shtml)
atomic force microscope

Diagram of atomic force microscope

Adapted from Hansma 2001. Ann. Rev. Phys. Chem 52:71-92

. . The scanning probe microscope (SPM) is ideally suited for characterizations at or near the cell surface). The nanometer scale resolution of atomic force microscopy (AFM) can be used to image membrane bound proteins in a liquid environment and in many cases sub-molecular structure of individual proteins can be resolved. In addition to imaging, sensitivity of the AFM cantilever can be exploited to measure forces required to rupture bonds between biomolecules, within single protein molecules, and by tethering specific probe molecules to the cantilever to identify single molecule interactions with specific target molecules on surfaces.

Laser scanning confocal microscope (LSM)

http://www.confocal-microscopy.com/website/sc_llt.nsfl

uses an aperture to limit the images observed to a single focussed slice of a 3D fluorescent sample.  Using a motor attached to the focus knob, a computer captures a series of these slices and reconstructs them into a 3D image. The aperture blocks a lot of light, so a high intensity source (a Laser) is necessary to provide sufficient illumination. The laser is focussed to a spot, and mirrors are used for Scanning the laser spot through the sample in a grid-like raster pattern. A pinhole aperture restricts the image collected by the detector to be the same focussed image observed in an optical slice of the sample (Confocal (same focus) images). All this is an accessory to a standard fluorescent Microscope.

Type of machines and illumination
 

• flood-beam:     TEM

• raster:              SEM, STEM

Resolution is the ability to see two closely placed objects as discrete structures. It is the smallest distance one can see between two closely placed objects

under the best of conditions:   human eye     => 0.2mm

                                                light scope     => 0.2um
 
                                                SEM               => 3nm

                                                TEM             => 0.2nm

 

wavelength: the peak to peak or trough to trough distance
  radios => miles to mm

infrared => mm to 800nm

light => 400-800nm

electrons => 20kV 0.008 nm

wavelength _electron=1.23/acc.volt ^1/2                                                                          80kV == 0.004 nm

A useful rule of thumb from the light microscope is that maximum theoretical resolution
l/2 wavelength

light scope 550 nm/2 = 275 nm

UV scope 0.35nm/2 =175 nm

wavelength of an electron will vary depending on accelerating voltage

electron =1.23/acc.volt^1/2 1.23==>de Broglie's constant

electron =1.23/80,000^1/2 ==>1.23/282 ==>0.004nm

max resolution of microscope operating at 80kV is electron /2 ==>0.004/2=0.002

We can compare it to looking down from a cliff onto waves on the shore:

when there are big waves, only big objects can be seen in the water
 

--the smaller objects do not deflect the wave enough
 

with smaller waves, smaller objects as well as big objects can be seen

Electron beam illumination/ interaction with the TEM specimen

Beam of electrons passing through a thin slice interacts with sample in passing. The ratio of transmitted and scattered electrons forms the image and sets contrast in the TEM absorbed few electrons are completely absorbed--produce heat buildup   unscattered don't hit anything   inelastic interact with electrons of atoms and suffer small deflection, but large loss in energy   elastic interact with nuclei of atoms and undergo large deflection,but little or no loss of energy

Resolution in the scanning electron microscope
Since the sample is illuminated with a spot that scans across the sample, the resolution of this microscope is determined by a number of different factors. Of prime importance is how small the spot is that exits the sample surface, and the magnification of the image for a given spot size. The wavelength formula is not applicable to this instrument. Resolution is 3 nm tungsten, 1.5 nm field emission
Each spot in the SEM will show only an intensity of black/grey/white. If the spot if larger than the detail of interest, the detail will be lost. To see the stubs on this pollen grain, the spot size must be much much smaller than the small spont on the left.

Electron beam illumination/ interactionwith a SEM specimen
Beam penetrates the surface layer of a bulk sample and interacts with atoms of sample This interaction region is pear shaped, referred to as the Monte Carlo configuration (interaction volume Fig 5.6-5.8)   It will vary with accelerating voltage and atomic number
A number of different interactions may take place within the sample to produce signals that can be captured by the appropriate detector:

Auger e-	low energy, at surface, results from inner shell displacement
Secondary e-	low energy, mostly close to surface
Backscattered e-	high energy, deeper in sample
X-ray photons	deepest in sample
Absorbed e-	measure of conduction of electrons through sample
Cathodeluminescence	interactions causes sample to glow