Autophagy genes are discovered by Yoshinori Ohsumi for the greatest benefit to mankind

Autophagy has been known for over 50 years but its fundamental importance in physiology and medicine was only recognized after Yoshinori Ohsumi's paradigm-shifting research in the 1990's.

The experiment by 2016 Medicine Laureate Yoshinori Ohsumi demonstrated that autophagy exists in yeast. Ohsumi studied thousands of yeast mutants and identified 15 genes that are essential for autophagy. But even more importantly, he now had a method to identify and characterize key genes involved in this process. This was a major break through and Ohsumi published the results in 1992. First key publication: Takeshige, K., Baba, M., Tsuboi, S., Noda, T, and Ohsumi, Y. (1992). Autophagy in yeast demonstrated with proteinase-defifient mutants and conditoins for its induction. Journal of Cell Biology 119, 301-3011

The term "autophagy" was coined by Christian de Duve in 1963. Our bodies are made up of cells that contain organelles, components with various functions, Albert Claude's research with the newly developed electron microscope and his methods for separating the various parts of pulverized cells using a centrifuge opened up new opportunities for studying cells in detail. In 1995, Christian de Duve discovered previously unknown organelles in the cell, lysosomes. These have important functions in decomposing different types of materials, such as bacteria and parts of cells that have worn out. In 1974 de Duve shared the Nobel Prize in Medicine for discovering the lysosome.

Thanks to Ohsumi and others following in his footsteps, we now know that autophagy controls important physiological functions where cellular components need to be degraded and recycled. Autophagy can rapidly provide fuel for energy and building blocks for renewal of cellular components, and is therefore essential for the cellular response to starvation and other types of stress. After infection, autophagy can eliminate invading intracellular bacteria and viruses. Autophagy contributes to embryo development and cell differentiation. Cells also use autophagy to eliminate damaged proteins and organelles, a quality control mechanism that is critical for counteracting the negative consequences of aging.

Disrupted autophagy has been linked to Parkinson's disease, type 2 diabetes and other disorders that appear in the elderly. Mutations in autophagy genes can cause genetic disease. Disturbances in the autophagic machinery have also been linked to cancer. Intense research is now ongoing to develop drugs that can target autophagy in various diseases.

Microscope

Electron microscope

Accelerated electrons behave in vacuum just like light. They travel in straight lines and have wavelength which is about 100, 000 times smaller than that of light.
Electric and magnetic fields have the same effect on electron as glass lenses and mirrors have on visible light.

Transmission electron microscope (TEM)
Transmission electron microscope can be compared with slide projector. In TEM, light source is replaced by an electron source tungsten filament heated in vacuum. Glass lenses are replaced by electromagnetic lenses and projection screen is replaced by fluorescent screen which emits light when struck by electrons. The whole trajectory from source of electron to screen is under vacuum and the specimen has to be very thin to allow the electrons to penetrate it. Electron are easily stopped or deflected by air molecules. That is why the microscope has to be evacuated and why specimens for TEM have to be very thin in order to imaged with electrons.
Unlike glass lenses, electromagnetic lenses are variable. By varying the current through the lens coil, the focal length, which determines the magnification, can be varied. In light microscope, variation in magnification is obtained by changing lens or by mechanically moving lens.
Electron gun comprises s filament and an anode. The tungsten filament is hairpin shaped and heated to about 2700 OC. By applying a very high potential difference between filament and anode, electrons are extracted and accelerated towards anode. Anode has a hole through which electron beam emerges. The beam is condensed by condenser coarse and fine condenser, then focused on specimen through condenser aperture that knocks out high angle electrons far from optic axis.

When electrons impinge on specimen, a number of things can happen
Some of the electrons are absorbed
Some are scattered over small angles
In crystalline specimens, electrons are scattered in very distinct directions
Some of impinging electrons are reflected (backscattered electrons)
Impinging electrons cause the specimen to emit electrons (secondary electron)
Impinging electrons cause the specimen to emit X rays
Impinging electrons cause the specimen to emit photons or light (cathodoluminescence)
Transmitted

Transmitted portion is focused by objective lens into an image. Objective aperture and selected area aperture enhance contrast by blocking high angle diffracted electrons. Image is passed through intermediate and projector lens being enlarged all the way. The image strikes the phosphor image screen and light is generated allowing user to see the image. Darker areas of image represent the area on sample that transmitted fewer electrons (thicker or dense). Light areas of image represent those areas of sample through which more electrons were transmitted (thinner).

Specimen preparation
In biology, tissues are treated as follows
First, there is a chemical treatment to remove water and preserve the tissue as much as possible in its original state.
It is then embedded in a hardening resin
After the resin has hardened, slices with an average thickness of 0.5 µm are cut with an instrument called ultramicrotome equipped with glass or diamond knife.
Tiny sections thus obtained are placed on specimen carrier coated with a structureless carbon film



Scanning electron microscope (SEM)
The electron gun produces an electron beam which is focused into a fine spot less than 4 nm in diameter on the specimen. The stream of electron is coarsely condensed by first condenser lens. It works in conjunction with condenser aperture to eliminate high angle electrons from the beam. Second condenser lens focus electron into finely thin beam. Objective aperture further eliminates high angle electron from the beam. The beam is scanned over the specimen in a series of lines and frames called raster (like in CRT) by set of coils. The final lens, objective focuses the scanning beam onto the very small area of the specimen. Several things may happen to these electrons. Commonly image formation is by means of low energy secondary electrons. An image is built up simply by scanning the electron beam across the specimen in exact synchrony with the scan of the electron beam in CRT. Magnification results from the ratio of the area scanned on the specimen to the area of television screen. Therefore, magnification is increased in SEM by scanning electron beam over a smaller area of the specimen. Detectors for secondary electron are usually scintillation detector or solid state detector.

Specimen preparation
Specimens must be able to withstand the vacuum of the chamber and the electron bombardment. Many specimens can be brought into the chamber without preparation of any kind. If the specimen contains any volatile components such as water, this should be removed using a drying process or in some circumstances it can be frozen to solid. Specimens must be electrically conductive, at least at the surface. Non conducting specimens will charge up under electron bombardment and need to be coated with a conducting layer. Since heavy element like gold produces good secondary electron and also yields good quality image, this is favorable element for coating. All in all, preparation of specimen to be investigated by SEM is not as complicated as the preparation of specimen for TEM.

Disadvantages of electron microscope
Expensive to build and maintain
Require extremely stable high voltage supplies, extremely stable current to each electromagnetic coils/lens
Continuously pumped high or ultra high vacuum required
As they are sensitive to vibration and external magnetic field, EM should be housed in stable buildings (sometimes underground) with special services such as magnetic field canceling system


TEM has the best resolution