Transcript Document
Concept: Biologists use microscopes and the tools of biochemistry to study cells • first compound microscope – Zacharias Jansen in 1590 • three important parameters of microscopy – Magnification: the ratio of an object’s image size to its real size – Resolution: the measure of the clarity of the image, or the minimum distance of two distinguishable points • inversely related to the wavelength of the radiation a microscope uses – Contrast: visible differences in parts of the sample Human height 1m • – • • Chicken egg 1 cm Frog egg 1 mm 100 m in compound microscopes – more than one lens – – • e.g. magnifying lens 0.1 m ocular and objective lenses improves resolution and allows for more than one magnification LMs can magnify effectively to about 1,000 times the size of the actual specimen this allows for individual cells within a tissue to be visualized Human egg Most plant and animal cells 10 m 1 m 100 nm Nucleus Most bacteria Mitochondrion Smallest bacteria Viruses Ribosomes 10 nm Proteins Lipids 1 nm 0.1 nm Small molecules Atoms Superresolution microscopy Electron microscopy • in a light microscope (LM) - visible light is passed through a specimen and then through glass lenses the lenses refract (bend) the light - so that the image is magnified in a simple microscope - there is one lens for magnification Light microscopy • Length of some nerve and muscle cells Unaided eye Studying Cells: Microscopy 10 m Light Microscopy (LM) • various techniques enhance contrast of a LM and enable cell components to be stained or labeled • BUT - most subcellular structures, including organelles, are too small to be resolved by an LM • LMs cannot resolve detail finer than 0.2um - regardless of magnification 50 m Brightfield (unstained specimen) Brightfield (stained specimen) Phase-contrast Differential-interferencecontrast (Nomarski) Fluorescence 10 m • to improve resolution and magnification – allowing for imaging of subcellular structure - development of two other microscopes in the 1950s • called electron microscopes (EMs)- use a focused beam of electrons rather than light • resolution increase – due to the shorter wavelength of the electron beam • two types: – 1. Transmission Electron Microscope (TEM) – 2. Scanning Electron Microscope (SEM) • 1. Scanning electron microscopes (SEMs) – electron beam is focused onto the surface of a subject Blood cells – providing images that look 3D – SEM electron beam excites the electrons of the gold on the subject’s surface – several kinds of electrons are produced – these electrons are detected by the scope and projected onto a video screen as a magnified image that appears 3D – can be colorized Pollen grains • 2. Transmission electron microscopes (TEMs) focus a beam of electrons through a specimen – – – – – – subject is sliced into a very thin layer so TEMs are used mainly to study the internal structure of cells subject is stained with heavy metals that adhere to the internal structures of the cell so some parts of the cell become more electron dense than others the electron beam passes through those less dense and scattered/reflected by the more dense regions the electrons that pass through hit a piece of film negative or hit a detector for displaying the image