Cells are s- m - a- l - l, and cells are c o m p l e x Because of their small size, complexity, and the fact that we are essentially working at the molecular level, Cell Biology is Technology-intensive “ There is a paradox in the growth of scientific knowledge. As information accumulates in ever more intimidating quantities, disconnected facts and impenetrable mysteries give way to rational explanations, and simplicity emerges from chaos.” Alberts et al. Molecular Biology of the Cell, 1st ed., 1984. This complexity, and the constant need for the development of new experimental tools and techniques notwithstanding, tremendous strides have and are being made in cell and molecular biology today.
Antonie van Leeuwenhoek “... my work, which I've done for a long time, was not pursued in order to gain the praise I now enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in most other men. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof.” Antony van Leeuwenhoek. Letter of June 12, 1716 Brass single lens microscope, Univ. of Utrecht Brass replica showing size
The Cell Theory Schwann and Schleiden collaborated on the first statement of the cell theory in 1839. Their work, along with the realization by Virchow: “Omnis cellula e cellula, ” published in in 1858 constitute the “Cell Theory:” the cell is the fundamental functional unit of life; all organisms consist of cells; and cells arise only from preexistent cells. Theodore Schwann Matthius Schleiden Rudolf Virchow
Spontaneous Generation -The theory with nine lives Francesco Redi 1668 John Needham 1745 Lazzaro Spallanzani 1768 Louis Pasteur 1859 The controversy over the “Spontaneous” Generation of life from dead material has an ancient origin. First disproved by Redi for macroscopic life, it became an issue again after the discovery of microbes. Over 100 years later it was finally laid to rest through the work of Spallanzani and the more comprehensive and elegant experiments of Pasteur. From Redi’s work in 1668 through that of Pasteur in 1859, the question concerned scientists for almost 200 years.
There were two versions of the spontaneous generation idea: Abiogenesis: Some life forms arose spontaneously from non-living matter in the past or can arise in the present time (a version of this is still held today by most biologists). Heterogenesis: From decaying or decomposing organic material you can get well-organized forms of life.
Francesco Redi and the disappearing maggots In 1666 Grand Duke Ferdinando II appointed Francesco Redi as his First Physician and director of the grand-ducal "Spezieria" (Pharmacy): positions in which he was confirmed by Cosimo III in 1670, when he became Grand Duke. So Redi, a doctor, poet, and scientist, spent much of his life at the Medici's Court and was, after Galileo, a unique example of scientist and courtier. Redi placed some pieces of snake, fish, some “eels of Amo” and a slice of milk fed veal into jarss. Flies were seen to lay eggs on the meat and on the netting. Only the meat in the open jar developed maggots. The meat covered with parchment and securely sealed with wax, and that in the netting-covered jar did not.
John Tuberville Needham John Needham studied theology at the English College in Douay France, and was ordained a priest in 1738. He headed a Catholic school in Winchester in 1764 and was elected to the Royal Society in 1767. He eventually moved on to positions in Paris and Brussels. About 1748 Needham, at the urging of the French naturalist Georges Louis Leclerc, comte de Buffon (known as Buffon) attempted to repeat the work of the Frenchman Louis Joblot (1645-1723) who had done experiments showing that boiling broth and sealing the container precluded microbial growth in the medium until the container was reopened. Needham made a mutton broth, some of which he boiled. The broth grew bacteria, whether it was boiled or not. The problem was the length of time of boiling, but Needham concluded that every bit of mater contained a vegetative force.
Lazzaro Spallanzai Spallanzani was educated in the Jesuit College of Reggio and, after an attempt at studying law, turned to the study of science at the University of Bologa. He became professor of logic, metaphysics and Greek at the University of Reggio, then at Modena and Pavia. All his free time was spent in pursuing biological questions. He did work on digestion, limb and organ regeneration in invertebrates and amphibians, fertilization, and echo-location in bats.
Spallanzani discovers the importance of boiling time Spallanzani made infusions from several types of seeds; he first boiled these for 30 minutes and placed them into loosely corked flasks. All grew microbes of different sorts. He then explored boiling his infusions in sealed flasks for different periods of time and found that those kept sealed while boiling for at least 30-45 minutes remained sterile. When his critics, including Needham, suggested that long boiling periods destroyed the ability of his infusions to support life, Spallanzani showed that microbes would grow again in his infusions once they were reopened to the air.
Into the 19th century Many famous naturalists of the time favored some form of spontaneous generation. The Frenchman Lamark, and Charles Darwin’s grandfather Erasmus among them. The Director of the Natural History Museum in Rouen, Felix Archimede Pouchet (1800-1872), began presenting a series of papers in 1855 to the Academy of Sciences in Paris, purporting to prove spontaneous generation, and to show not only that it happened, but under what circumstances. He named his subject heterogenesis, which was the title of a massive volume he published on the subject in 1859.
Pasteur to the rescue Pasteur, who was trained as a chemist, was a giant in the field of microbiology and infectious disease. He developed vaccines for anthrax and rabies, linked fermentation to yeast, developed pasteurization, and discovered the role of enantiomers in biochemistry. The French Academy of Science offered the Alhumbert Prize of 2500 francs to whoever could shed "new light on the question of so-called spontaneous generation". Pasteur won it in 1862 for his famous essay of 1861, "Memoire sur les corpuscules organises qui existent dans l'atmosphere", published in their Annales the next year.
Pasteur’s experiment (conclusion) Interestingly enough, Pasteur’s notebooks, made available for the first time in 1970, show that he achieved these results only about 10% of the time. He rejected results to the contrary on the basis of error.
Meanwhile, back at the optics lab... Ernst Abbe is considered the father of modern microscopy. Abbe was a German physicst. A professor at Jena, he was hired by Carl Zeiss to improve the process of optical lens manufacturing and in the process worked out (in the late 1800s) much of the mathematical foundations of modern optics. Describing his work in his own words he said: Most optical work of the time (and since) has centered on optimizing microscopes to provide the greatest detail possible. For the most part this involves minimizing the limitations imposed by the fact that microscopes must work with light passing through glass lenses.
The problem with light and lenses I The villains in this story are the diffraction seen when light waves interact with opaque edges, and the refraction that occurs when light passes from one medium to another. The diffraction that gives us picturesque sunsets as light peeks through slits in the clouds also contributes to the blurring of a microscope image. Diffraction
The problem with light and lenses II The villains in this story are the diffraction seen when light waves interact with opaque edges, and the refraction that occurs when light passes from one medium to another. Refraction The refraction that produces odd optical effects as we look through a glass of water (due to light bending at the interface of two different media) also contributes to the blurring of a microscope image.
An airy disc As a result of these factors, the detail points of a specimen appear as blurred airy discs and those that are closely spaced tend to merge in a microscope image, with a resultant loss of image detail. The evil “Airy Disc”
The limitation imposed by the diffraction and refraction limit our ability to preserve or “Resolve” detail in a magnified image. Ideally, we would want the minimum detectable (“see-able”) distance (D or R) between any two image points to be as small as possible.
As a result of all of this, the “light collecting” ability of a lens system and the wavelength of light used greatly affect resolution Don’t panic, this all reduces to: the better the lens light-collecting ability, the higher the value for NA, and the better the resolution (smaller the “D” or “R” number becomes) as detailed on the next few slides.
Our minimum distance (D) can also be expressed in terms of Resolving power “R”(sometimes referred to as the Abbe limit) Don’t panic! See the next summation slide
What this all comes down to: So to minimize R (D) - remember smaller is better, must be as __________ as possible, and NA must be as__________ as possible. R _ _ NA ( where the symbol means: “is proportional to”)
R _ _ NA So to minimize R (D) - remember smaller is better, must be as small as possible, and NA must be as large as possible. The problem is that the useful range of is limited to 700-400nm, and lenses, being made of glass, can only have an NA within fixed limits as well. So the upshot is that R has an upper limit of about 0.200 m (200nm) at best. So detail that is smaller than that (200nm) is simply lost!
Over the years, modifications of the standard “bright field” microscope have been developed that allow us to “fudge” a bit: Phase contrast microscopy Differential Interference Contrast (DIC) or Nomarski optics Fluorescence microscopy Confocal fluorescence microscopy
Phase contrast overview Translation: parts of a living cell image that affect the phase orientation of light passing through it - edges and cell walls for example - appear brighter.
DIC Overview Translation: differences in cell content densities are used to create a pseudo 3D effect
Phase contrast and DIC Human Epithelial cells Cyanobacterium (Tolypothrix)
Phase contrast and DIC HeLa cells Whipworm eggs (Trichuris)
An Epi-Fluorescence Microscope A filter cube with a beam-splitter mirror allows uv light to be applied from above, and allows only selected visible light to reach the viewer’s eye from the fluorescing specimen. Different filter cubes are used with different stains based on the maximum excitation and fluorescence emission wavelengths of each stain. eye specimen UV light
Fluorescence example 3 Transformed Green Monkey Kidney Fibroblast Cells (COS-1 Line)
Fluorescence example 4 Normal African Green Monkey Kidney Fibroblast Cells (CV-1 Line)
Fluorescence “Tagging” Specimen fluorescence can be... - Natural; as in plant cells wall autofluorescence due to their natural content of phenolic materials.
Fluorescence “Tagging” Specimen fluorescence can be... - produced by differential fluorescent stains such as DAPI that selectively binds DNA. Candida albicans (yeast) with DAPI stained nuclei
Fluorescence “Tagging” Specimen fluorescence can be... - produced using specific antibodies linked to fluorescent stains. fluorescein One or two antibodies can be used to selectively tag a cellular component. At least one of the antibodies must be conjugated to a fluorescent compound such as fluorescein.
Ernst Ruska and the electron microscope Ernst Ruska was a German physics student pursuing his Ph.D. involving electron beam lens development when he developed the first primitive electron microscope in 1931. De Broglie and others had already developed quantum theory and demonstrated that an electron beam, like a photon beam (light), could have both particle and wave properties. Ruska’s early work was with cathode ray tubes (like those in oscilloscopes and TVs) that use an electron beam to trace a line on a phosphor screen. The first EM with resolution better than a light microscope was produced by Ruska in 1933. Further development by many others was necessary to produce the useful instruments of today. Ruska won the Nobel prize in physics for this work in 1986.
Light vs Electrons Olympus confocal microscope Modern TEM
From the top: Euglena as imaged by differential interference contrast light microscopy (uses polarized light to create a pseudo 3D image). Surface detail as revealed by S.E.M. and internal detail as revealed by T.E.M.
TEM In TEM a specimen embedded in plastic is sliced super-thin, stained with heavy metal salts, and placed into an electron beam on a fine copper grid. A high voltage and a vacuum must exist within the electron “gun” to produce the electron beam which is focused with electromagnets. An image of the stained specimen is produced on a phosphor screen.
SEM SEM specimen chamber Dried specimens are surface coated with a fine metal film before being subjected to SEM. An electron beam is scanned across the specimen surface which creates secondary electrons that are collected and used to form an image.
SEM Bamboo vessel cross section; Light M (left) and SEM (right)
Cell fractionation via Centrifugation depends on angular velocity to generate force F = ma or F = m 2 r where The larger the radius of rotation r the more force is applied for a given rpm r A fixed angle centrifuge rotor (head)
A nomograph such as this can be used to determine the force - expressed as Relative Centrifugal Force (RCF) being exerted by spinning a rotor of radius r at a given rpm. The rpm alone is of limited value as the rotor radius r has a large effect on the force generated, so two rotors of different radius will produce a different force, even though they are used at the same rpm.
Alternatively, RCF can be calculated using a simple formula RCF has “g” units where 1 g = the force due to gravity
Differential centrifugation involves successive re-centrifuging of the supernatant from a previous run at a higher speed (“g” force). The pelleted material of each run is not “pure,” as some random material always starts out already at the bottom of the tube, so each pellet is said to be “enriched” for a given cellular component.
A finer separation of cellular components can be achieved using a gradient of some solute that is denser than water. This usually requires the use of an ultracentrifuge that can generate rpm of 100,000 or better thereby generating hefty “g” forces. Sucrose is often used in zonal separations that must be timed to avoid pelleting. Cesium Chloride gradients can be spun indefinitely as it is dense enough for the migrating material to reach a zone of density equilibrium where it will move no further.