Cell Biology Microscopy Microscopy founding method of cell biology o 1655 Robert Hooke cells o 1674 Anthony van Leeuwok o 1838 Schleiden and Schwann cell theory Why did nothing happen until now in the past 200 years New stages for textile industry Disagreement over one cell type brain tissue Reticular Theory brain tissue more like vascular system not cells Neuronal Theory brains were made of cells correct o 1873 Abbe w Carl Zeiss important figure in optics Good lenses Resolving power can t use same type of microscope to see all kinds of distances o 100 micrometers plant cells see w eye o 10 microns animal cells o 1 micron mitochondria bacteria limit of Abbe s equation o 100 nanometers viruses ribosomes o 10 nanometers proteins Approach o 1 Select microscope Question dictates choice o 2 How to prepare the specimen Look at living cells Dead fixed How to stain them 50 60 different stains to choose from Resolution o Ability to see 2 points as 2 points Increase in resolution could be one dot now appearing as 2 d defines distance btwn 2 dots o Theoretical limit of resolution best Dictated by the optics Abbe s equation Image processing o Practical limit of resolution really see Cells tissues worst candidates to see through microscope Cannot approach theoretical limit easily using cells as specimens due to problems Problems Low contrast cells are 85 90 water Organic compounds ring compounds absorb illumination generate heat Leads to thermal instability Look at 10 15 micron section of tissue thick o Abbe resolution more or less diffraction d 61 lambda nsinthetea d distance lambda wavelength of illuminating source not necessarily white light may be electrons n refracting index of the medium sin theta light gathering ability of lens example d 61 400nm 1 5 87 200nm the distance between two objects is half the wavelength of the illuminated source P 1 most microscopes are diffraction limited governed by Abbe s equation Abbe s Rule Overcome in 2014 3 men received Nobel Prize can make microscopes that are not diffraction limited using fluorescence of molecules monitor interplay btwn individual molecules inside cells track cell division nano level Cell Biology Microscopy Contrast stain o No dyes manipulate the light to generate artificial contrast o Dyes Colorimetric dyes absorb select wavelengths instead of others Late 1800s preferred by pathologists b c they are diagnostic fixed tissue Fluorescence o Computers 1 Bright field microscope compound light o Oldest but most common o Used to process the tissue from a biopsy 1st step fix the tissue Kill the tissue so it looks like it was alive Uses formaldehyde crosslinks proteins 2nd step dehydrate the tissue Swap the water with something else ethanol 3rd step swap ethanol w different solvent xylene 4th step embed in paraffin candle wax Swap xylene w liquid paraffin Use microtome to slice into thin sections 10 15 microns 5th step Go back to steps 4 2 section on the slide w no wax 6th step Stain the tissue o Problem takes a long time to accomplish Solution alternative way to make sections cryosections Cryostat freezes tissues equivalent to paraffin embedding 10 15 micrometers o Fast but not as a good histology Pathologist o Story about atypical blue nevi pathology is an Interpretive science not an absolute science 2 Phase microscopy o No fixation no dyes o Takes differences in refractive indices in cells translates it into contrast Wavelength goes through nucleus becomes delayed lambda Microscopes take delay generates images not colorful o Uses living cells in real time o Routine following of growing cells 3 Nomarski or differential interference microscope P 2 Cell Biology Microscopy o Looks at living cells o Not routine difficult to set up o 3 D effect o One application single cell electrophysiology Record electo changes in liquid media w pipette hook up to 20k system Look millivolts measures resting potential of neurons look for drug that drives membrane potential in opposite direction down from 70 to 90 see cell surface must have cell survive technique delicately 4 Dark Field Microscope o Uses dark field black background o Has unusual condenser lens o Illuminate spots see diagram moves light into very fine cone Images specimen obliquely light goes through hits small particule e g lysosome Images present as bright spots o Microbiologists use this for bacteria same size as mitochondria o Eukaryotic cell biologist 5 Polarizing Light Microscope o Designed to look structures that are highly ordered and parallel e g actin rayosin microtubules that run through axon o see diagram o Light emits from lightbulb in all different planes goes through polarizing filter plane polarized light Plane polarized light goes through specimen anything Plane polarized light goes through analyzer to you o Fibrosis liver lungs tissue Tissue death fibroblasts infiltrate fibroblasts secrete extracellular collagen Use polarizing light microscope image to come up w fibrosis index 1980 s predicted very few to no advances in microscopy microscopy is dead o b c revolution in microscopy due to confocal microscope Confocal microscope microscopy now alive o History first patent 1957 first attempt in making microscope 1970 s first commercial microscope 1988 o Why did it take so long to develop Data computers Sums spots o Key operating parts Lasers Reagan funded monocaromatic e g 488 nm used derived beam Confocal pinholes Computers image is summed spot by spot instead of all together o See figure in textbook Confocal Microscopy o Benefits 1 Decreases stray fluorescence by 50 2 Can optically section cell create a Z stack by building those images together P 3 3 Because of this you can make 3D stereoimages assign each cross section different colors 4 Colorimetric or fluoro dyes 5 Double triple labels to see things at the same time o Two Different Types of Confocals 1 Laser Scanning Confocal Microscope Cell Biology Microscopy Slow compared to alternative Goes point by point Excite a fluorescent dye emits different colors Bright image Problems photobleaching Heat is necessary difficult to use with living cell 2 Spinning Disk Confocal Microscope Collets multiple points at same time Faster Involves less heat Image not as bright less photobleaching Dynamic phenomenon 1millisec o Lucid Corporation Caliber Imaging Developed clinical hand held version of confocal microscope Allowed for non invasive imaging of skin detect skin cancer o Other applications Art forgeries Vital Fluorescence Microscopy