Structural Biochemistry/Microscopy

Background
Microscopy is a technique that is oftentimes used to study the cell (inside and outside). The two most common types of microscopy are light microscopy and electron microscopy.

The three types of light microscopy are fluorescence, phase-contrast, and confocal.

1) Fluorescence shows the location of molecules in the cell by tagging them with fluorescent dyes or antibodies.

2) Phase-contrast enhances contrast in cells by amplifying variations in density.

3) Confocal uses lasers and special optics to optically section fluorescently-stained cells.

The two types of electron microscopy are scanning and transmission.

1) Scanning shows a 3D image of the surface of a cell or specimen.

2) Transmission is used to section through a cell or specimen.

The difference between light microscopy vs. electron microscopy is that election microscopy uses a beam of electrons instead of light.

The key to excellent microscopy is magnification and resolution (clarity) to be able to see what you're trying to find in a cell.

Scanning Electron Microscopy
Scanning Electron Microscopy works by having an electron beam scan over the entire object. This process can be related to a 3D scanner that takes the positioning data of each atom on the object to create a computer generated model that reflects the data. This positioning data is retrieved by the detecting the collision behavior of the particles when an incoming electron causes the ejection, scattering, or excitation of other electrons. Other data such as the velocity and amount of x-ray packets that are produced by electron scattering can also be used to provide more details for the computer model.

In order to provide maximum magnifying capabilities for the microscope, an electromagnetic field is maintained by fluctuating voltage contained in copper coils surrounding critical portions of the beam. This magnetic is used to maintain a very precise and linear beam of electrons to scan the object with. These “scanning” coils are also what allows the beam to direct and adjust itself accordingly to properly scan the specimen.

Electron microscopy is particularly popular amongst scientists visualizing minute specimens, some of which are smaller than the smallest frequency of light physically smaller. It is this inherent problem of light microscopy that prevents normal microscopes from viewing nanoscopic structures, since the “light” used to too big to view anything that small. Given that the de Broglie wavelengths of electrons are capable of being created thousands of times shorter than normal light, electron beams are the preferred wave particles for observing molecular structures.

Atomic Force Microscopy
Atomic force microscopy (AFM) is a type of high-resolution imaging with resolution on the nanometer scale. Interestingly, this is a resolution that is 1000 times more than the expected optical diffraction limit. The original AFM is a derivative of the scanning tunneling microscope that was engineered by Gerd Binnig and Heinrich Rohrer in Zurich, Sweden in the 1980s. Today, the AFM is widely used to investigate on the subcellular, nanometer scale.

The AFM has a cantilever with a sharp tip at the end. This tip is used to scan the surface of interest, which is mounted on a glass surface. The cantilever is brought close to the surface of the sample. Tiny forces act on the principle of springs (Hooke's law) caused by interaction between the tip and the sample through chemical interactions such as van der waals, capillary forces, electrostatic forces, and magnetic forces to name a few. These various forces causes a measurable deflection of the cantilever. As the cantilever tip taps along the surface of the sample, the components of the cantilever is continuously measured in the x, y and z direction to create an image that is computationally constructed based on the amplitude, phase, and other features of the cantilever tip movement. From the tracking of the cantilever movement, the surface of the sample can be analyzed and 3D images can be produced such as amplitude and phase traces.

Atomic Force Microscopy is widely used in the academic lab setting. A research group at the University of California, Davis headed by Dr. Tina Jeoh focuses on using atomic force microscopy to explore the surface chemistry and structure of cellulose, a polysaccharide component of the cell wall in plants. With the investigations of the cellulose structure, the aim is to access the mechanisms by which enzymes such as cellulases act on the cell wall to release soluble, fermentable sugars in order to optimize efficiency of converting the sugars into second generation cellulose-based biofuels. The group uses chemical vapor deposition with methyltrimethoxysilane (MTMS) and centrifugation to adhere purified macro-algae based cellulose to silanized glass. The wet cellulose is imaged under AFM along with cellulase. The time-lapse AFM gives an insight to the mechanism by which cellulase breaks down cellulose into beta glucan rings through imaging.

References 1. http://www.nanoscience.com/education/afm.html 2. Santa-Maria, M. and T. Jeoh. 2010. "Molecular-scale investigations of cellulose microstructure during enzymatic hydrolysis." Biomacromolecules 11(8):2000 - 2007. 3.http://bae.engineering.ucdavis.edu/pages/faculty/jeoh.html

Transmission Electron Microscopy:
Transmission Electron Microscopy operates similarly to scanning electron microscopy, but rather than letting an electron beam individually scan the surface of a specimen, the microscope takes electrons and transmits them through an ultra-thin specimen. The resulting electron collisions are then detected by electron detectors to create a model of the imaged specimen. Fundamentally, a Transmission Electron Microscope is similar to the Scanning variant, they only differ in how the image is created. A disadvantage for this system is that it requires the creation of very thin specimens for the electron beam to pass through, which is a very meticulous and laborious process. There is also the risk of damaging thin biological specimens with the beam.

Electron Gun
The electron source used to image the specimen with. They come in two types. Thermoionic: This type of electron source utilizes thermal energy in a filament to eject electrons from. Field emission: Uses an electrical field to eject electrons

Lenses
The lenses of a scanning electron microscope are the components that concentrate the electrons coming from the gun into a fine linear beam. These lenses use magnetism rather than optical prisms to fine tune the electron beam.

Sample Chamber
Specimens are stored within the sample chamber and provide a focal point for the electron gun as well as electron detectors.

Detectors
These line the sample chamber to observe any scattered electrons and any resulting rays from electron collisions.

Vacuum Chamber
The case for practically the entire electron microscope. A vacuum is required to operate the electron microscope because anything less would cause unintended electron collisions and interference for the electron beam.