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1. Light Microscopy.pdf

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McGill University
Anatomy & Cell Biology
ANAT 262
John Presley

Light Microscopy: We know so much about cells thanks to three main biological techniques: Microscopy (light and electron) o Light microscopy was the earliest technique used to view cells as a whole Biochemical techniques Genetic techniques (particularly in yeast) o Both techniques above are used to determine what specific proteins do Combinations of all three of the above Light microscopy traces back as early as the 1950, when the technology involved little more than a lens and a mirror Light microscopy was the most important technique until the 1950s o The technology was limited to looking at pictures of cells as a whole Electron microscopy was introduced in the 1940s and became the dominant technique from the 1950s to the 1970s o This allowed scientists to look at components which make up the cell on a molecular basis A combination of biochemistry and yeast genetics dominated the 1980s to present o Genetics has even moved to more complex organisms, including us Light microscopy, however, has made a recent comeback as a dominant technique thanks to a new ability to follow dynamics of proteins in living cells o This is due to very recent techniques in the field of light microscopy, which we will cover later The first evidence of a light microscope came from Antony von Leeuwenhoek, a Dutch scientist His device was a crude, simple microscope, consisting of only one lens. These are NOT the common type of microscopes we use today o The simple microscope used by von Leeuwenhoek was essentially a very powerful magnifying glass By putting samples into his simple microscope, he was able to draw designs of very small, almost microscopic organisms o These structures included yeast and rotifers, which were a type of flagella These drawings were so incredibly detailed that most people thought that von Leeuwenhoek was making his results him o We know now, however, that von Leeuwenhoek, drew his findings with incredibly true details o This method would even be difficult today, and we have found more practical methods to avoid it Meanwhile, in England, Robert Hooke was coming up with the design of a compound microscope, which contains more than one lens The compound microscope is the type of microscope used today, though modern designs are obviously more detailed and slightly more complex o The compound microscope are able to resolve more detailed images than a simple microscopes, making them more practical By looking at thin slices of cork and wood, Hooke came up with the names cells to name compartments of the wood fiber o The name cell came from the fact that they resembled cells in monasteries or prisons o The cork cells in this image are dead and they have no nucleus, but the compartments can clearly be seen The high magnification powers of the light microscope created the need for a microscopic unit of measure; the micron The micron (technically called the micrometer), denoted by the unit m, which is one thousandth of a millimeter (mm) o Therefore, there are 1,000,000 microns in a meter A human hair, a visible structure, is around 100 microns in diameter for the average person o In comparison, a large cell is around 30 microns while a small cell, like a red blood cell, is around 7 microns in diameter Taking this immunofluorescent image of a large cell, we can see two stained proteins, tubulin, a cytoskeletal protein, and a protein of the Golgi o The resolving power of a light microscope is 2/10 of a micron, meaning that two objects will be seen distinctly as separate objects only if they are 2/10 microns apart or more o Also, any object smaller than 2/10 microns in diameter will look like a 2/10 micron disc, no matter how small it is Due to this resolving power, the tubulin strands that are close together look like a smear o Tubulin is made of two helical subunits, but the light microscope can only see it as one line because the strands are below the resolving power of the lenses Electron microscopy has brought in an even smaller unit of measure, the nanometer; there are 1000nm in one micron At this much higher resolution power, individual components of the cell can be resolved o For example, the microtubule is around 24nm in diameter, and can each be resolved in EM, unlike in LM where they are smeared o Microtubules are also viewed at their true diameter in EM, unlike in LM where they are viewed at 2/10 micron discs o Electron microscope samples, however, are much more difficult to prepare For example in this axon, which is about 1 micron in diameter, is completely resolved and its individual components, such as microtubules, can be seen Individual components of the cell such as organelles can also be resolved o In LM, only the nucleus and other really large organelles can be seen because of its size and staining properties Taking a look at the EM picture of a lipid bilayer of the plasma membrane, using osmium tetroxide as a stain o The hydrophilic heads and hydrophobic tails can be seen separately, and with special tools it is even possible to see the individual membrane proteins The first type of light microscopy we will discuss is called transmission LM, which is the typical kind of microscope found in high school labs In biology labs, compound microscopes are used, which are just more sophisticated versions of the ones found in high school labs o Transmission LM means that the light is shone from underneath the sample, while compound microscopes have multiple lenses Fluorescent and confocal microscopes are now more commonly used, and we will discuss them later o Confocal microscopes are just a specialized type of fluorescent microscopes Conventional transmission microscopes contain many components, but the most important are the light source, condenser, stage, slide, objective lens, ocular lens and eyepiece o Light comes from the light source and is condensed into a dense bean by the condense o The light then passes through the sample, placed on a slide which rests on the stage, and is magnified and flipped 180 degrees by the objective o The image is then magnified once more, and flipped back to the original orientation by the objective lens and is then viewed by the eyepiece The other details arent really important, but the focus knob is used to move the stage up and down to focus the image in the eyepiece Cells are very thing and transparent, meaning that with normal transmission LM, called brightfield illumination, cells are very difficult, almost impossible, to see To make them more visible, we can either stain them or increase the contrast of the image o Common stains include hematoxylin and eosin (H&E) which can stain different cellular components different colors o Special optical tricks allow us to increase the contrast of the image which makes the cell visible. There are 3 main methods Phase contrast Differential-interference-contrast (DIC) or Nomarski Dark field Cell samples are usually pieces of tissue which have been fixed and embedded in paraffin or plastic, and then sectioned o LM does not allow you to see 3D structure because of a limited plane of section, which means thin sections of around 5 microns must be used Tissue cultures are cells which are grown outside of the organism, and usually have the shape of a fried egg o They are much easier to work with, but they tend to lose specific tissue and cell properties Depending on the technique, tissue cultures can either be fixed or alive. Fixed tissues are the most common and most practical, but live tissue can also be used when necessary o It is difficult, but possible, to be kept alive while being worked with under the microscope o Another problem with tissues is their 3D structure. Due to the narrow depth of focus, everything above and below the plane of focus of the microscope will look blurry A single cell layer is usually fine, but multi- layered tissues are a nightmare There are advantages to viewing unstained cells under an LM is you are willing to give up detailed visibility They allow prolonged observation of live cells, which allows, for example, the study of movements of cell division and intracellular structures o These movements can also be filmed in a process called micro-cinemato
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