Primary cells non6cancerous non6transformed can be isolated and can also be cultured to study cell6speci c func ons neutrophils lymphocytes neurons etc Transformed i e cancerous cells can be grown con nuously in culture HeLa cells cervical cancer cells for example have been growing con nuously since the 1950s Many transformed cell lines duplicate key features of normal cells and can be used to study important processes Some model basic cell func ons others retain specialized func ons What are the cells that cell biologists typically study is one of the core tools of cell biology Light microscopes are limited in contrast magni ca on and resolving power limi ng what can be detected with them Light microscopy Light microscopes magnify a specimen using one or more lenses In a transmiOed light microscope white light passes through the specimen before being collected Cells are mostly transparent They neither re ect nor absorb much light unless colored e g chloroplasts or stained so contrast is poor and liOle detail can be made out Contrast is the di erence in intensity between an object and its background To bring out detail enhance contrast we need to exploit changes in the phase of light or stain the object to make it darker Transmi ed light microscopy Di erenFal Interference Contrast DIC Phase contrast BrighKield transmiOed light only For more see hOp micro magnet fsu edu primer techniques index html Chemical stain Modulate phase of light Modulate contrast Techniques for enhancing contrast Resolu on of conven onal light microscopes assuming a perfect lens is the wavelength of light being used The wavelength of light shorter wavelength beOer resolu on Proper es of the lenses used speci cally the numerical aperture N A the width of the light6cone the objec ve gathers Resolu on how far apart two objects have to be to be seen as two separate objects It is not directly related to magni ca on although magni ca on has to be high enough It is determined by Magni ca on is the amount you blow up the ini al image It s dependent on the lenses you use Resolu on is dependent upon the proper es of light and how it interacts with the specimen Magni caFon and ResoluFon What s really there What you can see depends on spacing It is not the same as resolu on which is how much detail you can make out Magni ca on is how much you blow up an image 200 300 400 500 Actual object size nm 100 An object 200 nm or smaller will appear to be 200 nm regardless of its actual size If you see a 200 nm spot in a microscope it could be 1 200 nm or smaller object Mul ple smaller objects close together What all this means pracFcally Fluorescence when a molecule absorbs light of one wavelength and then re6emits it at a longer wavelength Fluorophores uors can be linked to various chemicals or biological molecules so as to label speci c structures Because speci c molecules glow brightly against a dark background the contrast of epi uorescence microscopes is excellent hOp www shsu edu chemistry chemiluminescence JABLONSKI html Epi uorescence Microscopy dramaFcally improves contrast and allows speci c structures to be labeled An bodies are immune proteins that bind to speci c proteins To make an an body you obtain pure protein of interest and inject it into an animal The animal recognizes it as foreign and mounts an immune response Individual B6cells will each produce a unique an body that recognizes a speci c 8612 amino acid sequence the epitope Monoclonal anFbodies are made by cloning a single an body6 producing cell and thus recognize a single epitope All the an body molecules produced are iden cal Polyclonal anFbodies are a mixture of di erent an bodies produced by the host animals immune response against various epitopes of the target protein and isolated from blood serum A digression from microscopy anFbodies are all around useful molecules Add the an body to the xed permeabilized cell and allow it to bind its target Use a uorescent secondary an body an an body against an bodies to bind to the primary an body if the primary is not directly labeled direct vs indirect Immuno uorescence Fix add chemicals xa ves that cross6link everything in the cell to nearby molecules Permeabilize add a detergent to perforate the membrane so an bodies can enter To immunolabel intracellular structures you must x and permeabilize the cell Immunocytochemistry uses anFbodies to visualize proteins in cells CMV GFP CMV GFP PTS GFP Tubulin C CMV GFP Tubulin CMV promoter is a strong ubiquitously expressed promoter N GFP Back to microscopy Fluorescent proteins are gene cally encoded uorescence markers that can be fused to proteins of interest at the DNA sequence level Allows live imaging Transfec on can be transient expression from plasmid or stable DNA integrates into genome heritable Transgenic lines of animals can be generated by stable transfec on of germ cells Protein expression can be general in all ssues or under the control of a ssue6speci c promoter brain heart etc Proteins can also be knocked out Molecules that aren t normally present in a cell Mutant molecules that are consFtuFvely acFve6 i e ac ve all the me Mutant molecules that are dominant negaFve6 i e don t func on right and block the func on of the cell s own version of the molecule TransfecFon to infect a cell with foreign DNA to cause a foreign protein to be expressed in a cell In addi on to GFP6tagged versions of molecules present in a cell you can express either tagged or not Another digression TransfecFon of cells is a useful cell biology tool Small interfering RNA siRNA new technologies exploit exisFng mechanisms to allow us to knock out speci c proteins of interest in cells http www scbt com gene silencers html Laser6scanning Confocal microscopy uses pinholes to deblur Digital deconvoluFon uses computa onal methods to deblur Light above and below the focal plane you are interested in blurs images Methods to deblur images remove out of focus light STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis Katrin I Willig Silvio O Rizzoli Volker Westphal Reinhard Jahn and Stefan W Hell Nature 440 935 939 13 April 2006 doi 10 1038 nature04592 A last word on light microscopy The resoluFon limit of light microscopy can been broken with some really fancy and opFcs Although not yet suitable for live cell work this may be a huge breakthrough Scale bar 500 nm Scanning Electron microscope SEM 6 images electrons scaOered by an intact object Depth