Lecture 4 Notes - Microscopy & Methods
Lecture 4 Notes - Microscopy & Methods BIL 255
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This 3 page Class Notes was uploaded by Elizabeth Mompoint on Tuesday September 22, 2015. The Class Notes belongs to BIL 255 at University of Miami taught by Dr. Mallery in Fall 2015. Since its upload, it has received 15 views. For similar materials see Cellular & Molecular Biology in Biology at University of Miami.
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Date Created: 09/22/15
Cell and Molecular Biology Lecture 4 Microscopy Mir 39thth39alf39ld39 39 c oscopy 1s e cc me 16 us1ng mlcroscopes THE MGHTMICROSCOPE mm to view small cellular objects 2 major classes of microscopy Light optical Microscopy amp Electron Microscopy Light Microscopes involves the diffraction eye magni cation r objective 2 15M a molar lens 1m some 1500 Eyepiece refraction or re ection of light interacting with a prepared sample amp subsequent collection of the scattered radiation light to build up magnified images of small objects using a compound lens 77 resolution near limits of light 02 pm or 200nm Types of Light Microscopy O Brightfield light passes thru a specimen with little contrast A light source the light path in a light microscope Pnn l 11ainmi 1 ml P39l lltuiiiiiial ntLI lb39nllnmi 4th Hl ltk m nmi lrlmm im I39Ll Panel l ia rim 1 ml Ill Fiil ii li l ill Slalom dlh f l39lnlm failmud Momre milti Types of Light Microscopy amp is colorless amp translucent incident light is out of phase with amp transmitted cell is 70 water J quotiiP 4quot i I 0 Phase contrast light When iL interference lens 0 Differential microscopy uses shadow casting 0 Darkfield only scattered i and tL phases are synchronized by an Bright eld good contrast results interferencecontrast ie Nomarski 7 interference between polarized light producing images giving pseudorelief illuminating light is directed from the side so ight enters the lens thus cell appears lite against 39F39n a ntl Iblie rence Contrast background Light microscopy methods 0 Autoradiography 1924 Lacassagne produced an autoradiograph which is a light microscope image on photographic Dark e39d 39 50m film or emulsion produced by the pattern of radioactive decay emissions eg beta particles or gamma rays from a distribution of a radioactive substance 0 Fluorescence microscopy a form of light 0 Conventional light microscopy microscopy where a component of interest in a Fluorescence mlcmsmpquot specimen has been specifically labeled with uorescent molecules 0 Immuno uorescence microscopy uses Proteins l l iftrigzajt it1lii ii39tgini alto antibodies uorescently tagged to bind specifically to a corresponding antigen as a probe for identifying a particular molecule in cells tissues or tissues or biological uids 0 Confocal uorescence microscopy 1957 Minsky uses pinpoint illumination of uorophore in l fti fl lihi tg 1 or one focal plane to eliminate outoffocus uorescence fCDiNiFCiC L MliiC DSEDPY A confocal microscope is a fluorescence microscope with a laser as its source of illumination This is ecu see onto a sin pie point at e sp eeific depth in the specin39ienw and a pinhole aperture in the detector allows on lir fluorescence emitter from the exact point or focus to be incliuded in the image scanning th e laser beam across the specimen generates a sheep twodimensional image of the plane offlocus A 39 series of optical sections at different riepthe allows a thr eeseimensionai line he to be EleCtron constructed An intact insect e mibriro is shown here stained with a uorescent probe for seen is Wpe of p roteiini nu Convention all fine reecence rriirroscopnr generates a blurw image tine to the preset es of fluorescent structures e bore and Mlcroscopy 39 bellow the plane of liocus Bi Gonfocal hticrosooInr provides a crisp optical section USCS ELof the cells in the emibrro it counes39r of Richard Warm Emptiness of Peter Show 39 quot a bCam Of electrons to create an image 0 TEM transmission electron microscopy passes e39s through a specimen onto a viewing screen magnify up to million fold resolution theoretical 0005nm but effective resolution is 01 to 02 nm or larger I Ruska 1931 1st transmission electron microscope I PaladePorter 1952 1st TEM pics amp EM stains imaged via scattering of e39s by molecules Within specimen specimens must be very thin 50 nm or less sections are cut via microtome I Robertson 1957 all membranes look alike I Tagging 1981 antibody tagging With gold particle in electron microscopy I Tomography 2001 computer image averaging allows 3D modeling 0 fF EM cryoelectron microscopy an aqueous specimen is frozen in liquid Freon 196 C I SteereMuhlethaler 1964 develop freeze fracture EM I Cryoelectron tomography 2004 specimens are rotated in electron beam amp Ebgo individual images computationally make i ii itfi i i fgti39iii iil no stilts Ji iir xiii quot f2ii iiquot littti itt placed in xative tang 050 7 plastic em bedid ing plastic media in a tomogram ieggilut areldeihydel Dehydration giggli id contarans rrthin 0 SEM scanning EM uses metal shadowing amp coating of specimens to reveal surface tomography his I Charles Oatley 1965 1st scanning 395 EMs Block containing tissue is Plastic in trial polymerizes tri rrirrietl1 to prepare for sectioning into a solid block with tissoe at the bottom edgeof the block ssure is sliced into sections approximately ilOlJ nm thick as blocik moves down across the sharp edge of a glass or diamond knife Sections float in n trough TRA NSMiSSlGN ELECFRDIM 39 Grid Dme h avr gt 7 metal Stain MICROSCOPY I V electron 7 5 all 7 a v39 7 EM grid containing sections Sections are stainedi Closeup View of ready to be stained stairreel with it cum at Philips E litman mics Wl mi 3 sections in a ribbon heavy rnetalnpl aced in a grid holder and condenser E oating in trough examined in the electron microscope lens 5 Figure 39l il 1 Cell and Molecular Eioloqv le it Rim John Wiley Er Sons 0 specimen 7 g V v objective SDBCV Kn n t i G 39 torecled 5 The electron micrograph below a Signalman PM 375mg i999 9i 339 ill m The transmission electron microscope TEM is in principle similar 39 Drojzector 39 quot 3 PIECE Of TEStIS The 55119 has to a light microscope out It uses a beam of electrons Instead of a 39 especimen lens I C liem39jca l ll39 fli iedi beam of light and magnetic coils to focus the beam instead or Em bedd dhm Plastic 3quot all piglets lenses The Specimen which is placed In a vacuum must be H 5 mm VET 5 In 59am 31 WE fiery thin Contrast is usual introduced by staining the specimen 39 r T V 1 C 111 m crolome Specimen able r then been Stained with 53113 of with electron ems navy meta st at ocali y absorb or scatter Em will 39mg photag phic uranium arid lead lCOiII39TES 0f electrons removing them from the beam as it pastes through lm v Danlel 5 Frieu d the specimen The TEM in as a useilui magni cation of up to a milliontold and with biological specimens can resolve details as specimen is fractured with misretems knife th mush small as about 2 nm interior ofi lllFllld bilayer plane of least resistance fractured specimen is shadow coaterl with platinum Moral replica structural detail exposed by the fracture plane is than visualized hy39vacuum depusition ofplatinum carbon to make a replica nr examination in TEM SCANNING ELECTRON mmquot m mace MHCRQSCQ PY In the scannlng ene ron m Icrosclope SIEM the specimen whlch has 39 Evaporation been coated With a very thin f m of a heavy metal Is scanned by a 7 at metal 1mm Madmen v r Hz quot 1 1 Metal wice I gm beam of elle r rus hmught m a focus an the speclmen by the 7 platmum me electromagnetlc coils that In eMeclron microscopes act as lenses Muriel 31m e The quantity of electrons scattered er emitted as the beam mamam 0m Heated amen Evacuated E bombard each successive point cm the surface 131 the specimen electrode an we chamber g a I measured by the detector and 39Is used to control the intensity 39 39 39 e e f SUCCESSWE palms In an Wage bull up an a vmdleo scureen The Mem39cp g microswpe creates striki ngwrimages 0f three dimensional i g objects with great depth of focus and cam resolvedetal ls down Carbon Wm Wigwam E to samewhale bet rem 3 nm and 20 nm dependmgi on the Item ovennew eleclroaq 3 x d E cmdiensec quot m Instrument V if Metal mph L J 5 13 Specimen leaching sc am Eng mm mmkzroguph away 7 a ma aremini mugm V hem a heirmu In the may gay v Malai replica Hem Fe companion m r 7 me SfLI39ruCl ul39a ms srmm by him mamaw 3 W imi t 9139 m leaowl ltmm of MN Camper gm mom and mm Humth electrons from smimen specimen lny linemanyam n elven mun
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