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14 Pages

parke_ch7_10_11

Course: CHAPTER 7, Fall 2009
School: Utah State
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to 1 Introduction Biophotonics Section 7.10 - 7.11 TIR fluorescence microscopy & Near-field microscopy Module: Bioimaging Lesson: TIRF 2 Introduction to Biophotonics TIR fluorescence microscopy viate TI RF m d icroscopy Abbre rnal fle sce Total Inte Re ction Fluore nce st aging ce at thesurfaceand up to 100nm lls Be for im s Advantage al xposureof ce to light e ct at surface lls xpe Minim e ry...

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Coursehero >> Utah >> Utah State >> CHAPTER 7

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to 1 Introduction Biophotonics Section 7.10 - 7.11 TIR fluorescence microscopy & Near-field microscopy Module: Bioimaging Lesson: TIRF 2 Introduction to Biophotonics TIR fluorescence microscopy viate TI RF m d icroscopy Abbre rnal fle sce Total Inte Re ction Fluore nce st aging ce at thesurfaceand up to 100nm lls Be for im s Advantage al xposureof ce to light e ct at surface lls xpe Minim e ry sce Ve low background fluore nce ittanceof ce you want adm lls itting only haveadm sce No out of focus fluore nce hange in distanceas sm as 1 nmcan bede cte s all te d. C ll-substratecontact and adsorption of m acrom cule to ole s study ce surface s Module: Bioimaging Lesson: TIRF 3 Introduction to Biophotonics How it works d rnal fle Base on Total Inte Re ction d Oncepasse thecritical anglethewaveis totally re cte fle d Module: Bioimaging Lesson: TIRF 4 Introduction to Biophotonics How it works sce Evane nt Wave e n s fle d Form d whe sinusoidal wave aretotally re cte at an inte rface . nsity de e cays xpone ntially frominte rface inte Module: Bioimaging Lesson: TIRF 5 Introduction to Biophotonics How it works Module: Bioimaging Lesson: TIRF 6 Introduction to Biophotonics How it works ous Thecritical angleof glass slideand Aque solution approx. 62 de e gre s ne vane nt Distanceof 100 nmof pe tration of e sce wavedueto ce m r and glass slide ll atte . Module: Bioimaging Lesson: TIRF 7 Introduction to Biophotonics Examples singlem cule of them ole s utant prote GFP-Rac trafficking along thin filopodia of ce growing on a substrate in lls Module: Bioimaging Lesson: TIRF 8 Introduction to Biophotonics Examples GFP-actin filam nts we obse d surrounding fluore ntly labe d pinocytic ve s and pulling the into thece in a stre of e re rve sce le sicle m ll am actin during e ndocytosis Module: Bioimaging Lesson: TIRF 9 Introduction to Biophotonics Near-field microscopy ar-fie optical te ld chnique can beapplie to re s d solutions Ne be ath 100 nanom te ne e rs. om s xtre e solution C with bine thee m ly high topographic re good characte ristics of optical m icroscopy such as: significant te poral re m solution polarization characte ristics, spe ctroscopic capabilitie s, S nsitivity e fle xibility Module: Bioimaging Lesson: TIRF 10 Introduction to Biophotonics Near-field microscopy Module: Bioimaging Lesson: TIRF 11 Introduction to Biophotonics Operation portant C pone om nt Most im ub le rture S wave ngth ape d re r Produce using a tape d optical fibe solution dueto two factors Re rture Probeape m r tte solution S alle be r re ple Probesam distance e nsurechangein signal due Ke p optical probeat constant distanceto e to variation in topographic fe ature not probesam distance , ple d Maintaine using al e Norm forcefe dback he e e S ar forcefe dback, or tuning fork fe dback. Module: Bioimaging Lesson: TIRF 12 Introduction to Biophotonics Operation Module: Bioimaging Lesson: TIRF 13 Introduction to Biophotonics Modes Transmission mode imaging. Thesam is illum ple inate through theprobe and thelight passing through thesam is d , ple colle d and de cte cte te d. Reflection mode imaging. Thesam is illum ple inate through theprobe and thelight re cte fromthesam surfaceis d , fle d ple colle d and de cte cte te d. Collection mode imaging. Thesam is illum ple inate with a m d acroscopic light sourcefromthetop or bottom and theprobeis , use to colle thelight fromthesam surface d ct ple . Illumination/collection mode imaging. Theprobeis use for both theillum d ination of thesam andfor thecolle ple ction of the re cte signal. fle d Module: Bioimaging Lesson: TIRF 14 Introduction to Biophotonics References http://www.cstl.nist.gov/div838/crds_we b/atr_fig1.gif http://www.olym pusam rica.com g_se e /se ction/te chnone wsfile s/Am LabTI RFM.pdf http://www.nanonics.co.il/m ain/twole ls_ite 1.php?ln=e ite _id=34& m ve m n& m ain_id=14 http://www.olym pusm icro.com /prim r/te e chnique arfie s/ne ld/ne ldintro.htm arfie l Module: Bioimaging Lesson: TIRF
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