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

Studying Cells: Microscopy 1 cm 1 mm 100 m 10 m 1 m 100 nm 10 nm 1 nm 0.1 nm Atoms Small molecules Lipids Proteins Ribosomes Viruses Smallest bacteria Mitochondrion Most bacteria Nucleus Most plant and animal cells Human egg Frog egg Chicken egg Length of some nerve and muscle cells Human height Unaided eye Light microscopy Electron microscopy Super- resolution 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 e.g. magnifying lens in compound microscopes – more than one lens 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

Brightfield (unstained specimen) (stained specimen) 50 m Differential-interference- contrast (Nomarski) Fluorescence 10 m Light Microscopy (LM) Phase-contrast 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 Figure 6.3 Exploring: Microscopy

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)

Blood cells 1. Scanning electron microscopes (SEMs) – electron beam is focused onto the surface of a subject 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