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Bioinstru & Signal Proc

by: Annie Collins DVM

Bioinstru & Signal Proc BIM 289A

Annie Collins DVM
GPA 3.79


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Class Notes
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This 63 page Class Notes was uploaded by Annie Collins DVM on Tuesday September 8, 2015. The Class Notes belongs to BIM 289A at University of California - Davis taught by Staff in Fall. Since its upload, it has received 34 views. For similar materials see /class/191771/bim-289a-university-of-california-davis in Engineering Biomedical at University of California - Davis.

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Date Created: 09/08/15
Membrane BiophoTonics Andr39eia Michelle Smi rh Biophysics Gradua re Gr39oup CBST Par39ikh Labs par39ikhucdavisedu Importance of the cell Membrane Membranes constitute 1500 of the eukaryotic cell 3000 of the human genome codes for membrane proteins 8000 prescription drugs target membrane components 999 Primary regulator of signaling transport adhesion and communications functions of a cell Darnell et al Mm w H Freeman Company New York Surface of a Biological Cell quotM 5 3939 39a 1 gm f 63 v vi 39 J Kag n 5 u 53 vi 0255 l quot 3quot339 wmm Figure 1 The F uidMosaic Water Hydrophobic tails u cHrMcsz Hvdrophmc I head E UP U phosphate 39 Onltrogen Water g 3 2 9127917012 l g yceml I Gpmspnoms o o 39 i0 i0 ooxygen Figure 3 Basic structure of H2 H2 1 l the lipid bilayer EH 1 r Ocarbon Phospholipids the lipid type that 2 2 7 constitutes the majority of the g 1 u 55 Ohydroqen cell mem rane are made u 5 C H doub e 0 from a phosphate head circles 3 CH bond that like water and li id tail 3 cuZ lines that hate it These 50 LH2 lled 39pathic molecules line up so as to limit t e exposure of the h drophoblc port ons to he C CH both sides of the membrane summit MULTILAMELLAR gallsli llillgLLAR VESICLE AMPHIPHILE BILAYER 39 39 t 39 5 LIQUIDCRYSTALLINE a LAYERED STACKS mum s i 3wmv 1 10 1 00 1 000 LOG LENGTH SCALE NM Figure 4 Primary Structure and the selfassembling property A Synthetic Model of the Cell Membrane gig Supported Bilayer Lipid Membranes The cell membrane being a selfassembled fluid can be reconstituted from its components such as shown lt By selecting appropriate lipids and proteins a synthetic control of membrane structure and function can be achieved Supported Membrane Systems Modeling platform for cell membrane dynamics 2 D diffusive glass supported lipid bilayer Composite and fluidity properties can be manipulated Fluorescence Lipid probes from molecular probes Location and orientation of representative uorescent membrane probes in a phospholipid bilayer A DPH 2202 B NEDCG HPC N3786 C bis pyrenePC B3782 D DiI D282 E cisparinaric acid E36005 F BODIPY 500510 C4 C9 B3824 G N RhPE L1392 H DiA D3883 and 1 C12 uorescein 2109 Experiment Setup Sample Fluorescently labeled artificial fluid bilayer Imaging Inverted Fluorescent Microscope Equipped with CCD camera with fluorescence filter set Microscope Objective 39 Shortpass Dichroic Mercury Lamp Filter Set CCD Fluorescent Recovery After Photobleaching FRAP What do FRAP curves tell us Kota Miura EMBL Heidelberg Germany Based on the EAMNET Practical Course 20 April 2004 To proceed press Page Down or click the mouse button To go back Press page Up Clicking hyperlink blue fonts leads you to a website in the Internet 1 What is FRAP Fluorescence Recovery After Photobleaching FRAP 00000 00000quot0000 0050 0quot 00060030 COOquot O OOOO 000000 Then the strong irradiation BLEACHES the fluorescence at that spot WeiSdbinkaa tituogemommuleoldmdtqpedsddttadadiett White circles represent the molecules 1 What is FRAP Fluorescence Recovery After Photobleaching FRAP CO 00 0 Q0000 000529 Oquot 000Q39OO OOOOO39 39 000 0000 Since molecules are moving driven by diffusion or active transport bleached molecules exchange their place with unbleached molecules Then the average intensity at the bleached spot recovers 1 What is FRAP Fluorescence Recovery After Photobleaching FRAP Bleach a spot Gradual Fluorescence Recovery In practice the FRAP process looks like the following Above is a microscope field filled with fluorophores 2 Quantitative Analysis of FRAP Fluorescence Recovery After Photobleaching FRAP To gain information on molecular dynamics time course of the fluorescence recovery must be Measure the temporal Changes of measured the fluorescence Intensity 2 Quantitative Analysis of FRAP Frap Curve lmmamle traction Figure 1 Fluorescence recovery me phatohleaching FRAP When a region here lxmic lulu ere tlm 39 indica r e erran a i we actinn can be calculated me m in 1 mm the mini llume y The tlunre um alter mu retau ery m e e a e in W and wit alter 4 mm m dilmsmn mm The t This is an example of FRAP curve a result of measuring the fluorescence intensity at the bleached spot Xaxis is time Yaxis is the fluorescence intensity 2 Quantitative Analysis of FRAP u 00 Terms Used in FRAP analysis easori of every W at couid be tner tnis incornpiete rec Firsti ne nai ife tne takesfor intensity to reacn nait irnurn ottne ateauievei Tne recovery i5 not cornpiete piateau being iower tnan tne prebieacn intensity iiii 30 erai inrorrnation out Wing iines o OO it Half Life rm ID 20 Time sec We can extract sev ottnis curve by dra Tne actuai measurement ot uorescence intensity tends to resuit in a grapn We tnis 2 Quantitative Analysis of FRAP Mobile Fraction and Immobile Fraction Immobile molecule The incomplete recovery is due to some fraction of the molecules that are immobilized at the BLEACHED spot We call these molecules the immobile fraction Rest of the molecules are contributing to the fluorescence recovery We call them the mobile fraction The time constant and mobile limmobile fractions lmmobile Fraction Mobile Fraction in me FRAP curve the lmmoblle amp moblle fractlon can easured by determlnlngtne plateau leve O 00 r o l l l l o 30 ID Tlme sec Half Life rm 7 a 3 Curve Fi Subsmuuon of m by AZ Ha f We can be Ca cu ated from 1 re ated to heoFERfP kmeucs Mm m 05 lL Tmswsme ned curve 3 A more automauc Way of obtammg Ha f w Curve A V K g meg 0 2 VZA A 5 the meme fracuon O o o O and A 5 me mmob e fracuon o 10 m curve mggg parameters Wm equauon are Half Life 5 me Ume Tm Igg wrmzed by computer Here We use a 5mm en the recoyery 5 exponenua equauon for mng theha fofA by de nmon Half 9 Tm Patterning Current Patterning Technique UltraViolet Patterning Light directed patterning 1020 min process CK Yee et al JACS 12643 1396213972 2004 Mechanistic basis of hole formation within membranes 34257 um LW lighl u OrEBS u I in mm m f quot iiiiguii l il quot ii lt29 Ozonegenerating lt1 Molecular decomposition at lt Hydrophobic chains UV light mainly UV exposed regions to seal upquot to minimize 184nm 254nm small molecules C02 H20 interfacial tension N2 etc C Physical Mask quartz amp Chrome 184 nm O2 03 102d 254nm I Rx 01 R X39 ions radicals excited species 0 R x 3gt co2 H20 N2 102d Shapes Sizes Density and Distributions conveniently controlled using appropriate masks C Epifluorescence images of membranes red and Lithographically derived holes gray Preserved fluidity of the unexposed bilayer Fluorescence Recovery after Photobleaching FRAP T0 min T 10 min 60X 10 min Membrane Patterning with Ultrafast Lasers Andreia Michelle Smith Biophysics Graduate Group Thomas Huser PhD Dept of Internal Medicine Atul Parikh PhD Dept of Applied Science University of California Davis Supported Membrane Systems JM Johnson T Ha S Chu SG Boxer Biophys J 83 33713379 2002 Modeling platform for cell membrane dynamics r2 D diffusive glass supported lipid bilayer Composite and fluidity properties can be manipulated Patterning Membrane Systems 39 39 o I o 39 39 g I o 0 na Newly introduced f 550 Small unilbniella esi dssfSUV 39 039 ng39g gt m W tw W ww 39339 a5 3 OK Yee ML Amweg JAcs 12643 13962 13972 2004 Backfi39ingz Patterning Techniques Current Patterning Technique UltraViolet Patterning J i Light directed patterning 4020 min process Disadvantages 39Mask dependent vMultiple steps 39Size restriction 5pm Using Alternative Light Source Near Infrared Red Ultrafast Laser NIR transparent to biological samples 1 03915 3 pulse width femtosecond Spectrally broad 15nm FWHM quotquotquotquotquot Generate high intensity Tightly focused beammin 1013WIcm2 269nm a 3 and 4 photon absorption EBOOHm Maskless patterning with sub micron resolution Experiment Setup Sampb Fluorescently labeled artificial fluid bilayer quotnagmg Inverted Fluorescent Microscope Equipped with CCD camera with fluorescence filter set Microscope ObjectiveNA 145 Oil Cavity Titanlur nSapphIre Dumper ND Filters Mercury Lamp Filter Set Pump Laser 800nm 540kHz27kHz rep rate 350fs pulse width Wmasm d TAPEE VHFJE l gl EM FJQJJQ i r 10 Unmodified bilaver Laser exnosure Recoverv after exnosure Writing Barriers Writing Translating stage Maintain focus at bilayer 055 for 100um line 25ms per 05um Bar ers s T 5min 7 T 9mm 39T 9min Diffusional obstacles that coral lipids Modification of Barriers Functionalizing Barriers 39 quotf lncubated with FitC labeled BSA I gt Red Filter set for redZTas Red labeled lipidsBlue Filterlsejt for greenFitC labeled BSA Repetition Rate Dependence Low reprate 27 kHz Creation of barriers rep rate 200x more pulse per second Creation of barriers and Modi cation of Barriers Sample 97 POPC 3 NEDPE 54QkHz Rep rate 3rd Erect and Erasing Diffusional Barriers in Real time Modi cation of Barriers Sample 97 POPC 3 NBD PE 540kHz Rep rate 3n Writing and Erasing Barriers Added functionally to odei mebrane system Dynamically modi compartmentalization Peripheral 3 v freely dii iusivg and undeng n ian 7 miitigon iSJ Sihger and GJL Nickolsldh Sick766175 72014731 4972 Writing and Erasing Barriers Added functionally to model membrane system Dynamically modify compartmentalization Dynamically structured mosaic model i I 39 39 GV ereb etaiPNAs 100 z80 53810582003 Most membrane roteins do not en39o the continuous unrestricted lateral difosi on Instead prot ein l s diffuse in a more complicated way that indicates icnsi derable lateral heterogeneity in membrane structure at least on a nanoscale K Jacobson E Sheets and R Simsonr Spiencet2468 14414 4421 1995 Acknowledgements Parikh Research Group UC Davis httpparikhucdavisedu James Chan PhD and Nan Shen PhDLawrence Uvermore National Labs This work was supported by Center for BiophotonicsScience and Technology CBST under Cooperative Agreement No PHY 0120999 Work at LLNL was performed under the auspices of the US Department of Energy by the University of California Lawrence Livermore National Laboratory under contract No w 7405 Engs48 Study of Bilayer Edge Conformation with Model Membrane Systems Andreia Michelle Smith Biophysics Graduate Group Atul N Parikh PhD Department of Applied Science University of California Davis UV patterned1 Texas Red 99 DMPC Start temp 10 C ending temp 50 C Heating of UV Patterned Bilayer A patterned bilayer is heated above the transition temperature of the lipid The shape holds even though the bilayer itself is mobile and uid It only loses its shape almost 20 C above the transition temperature of DMPC Does the edge stabilize the structure Di 18 to c Figure l DiIC18 is a lipid probe known to have a preference for more ordered states when exposed to a mixture of liquid and gel phase lipids The picture on the right shows the uorescent image of a patterned stamped with PDMS lipid bilayer that comprises of a single lipid component and DiIC18 at room temperature It demonstrates an apparent DiIC18 preference for the edge of the patterned bilayer mac H3 H3 CH3 1H CH CH N N I I W CHM CH3 CH3 0 04 CH Spink MD Yeager GW Feigenson Biochimica et Biophyisca Acta 1020 2533 1990 7 quot 18 39ge quot1graton n ayers 1 DiC18 99 POPC on patterned OTS 1 DiC18 99 DPPC on patterned OTS DiICIS doped POPC and DPPC patterned monolayerbilayers shows non uniform uorescence There is a preference for the edge regardless of lipid type Is the edge is more ordered than bulk Llnescan from 10d 69 to ZSdeg Intensity an 0 D 20 0 200 Patterned H 995 DMP Mlcmns Figure 2 Linescans of timelapse uorescent images showing the progression of dye movement in a supported bilayer over a temperature range starting from 10 C to 25 C Increased intensity was seen over a 10 micron area around the edge of the bilayer Linescan from 3Sdeg to 45deg Intensity auJ b O 12 ml39ns 20 a a mlns Micron Figure 3 The temperature was increased from 35 C to 45 C The linescans show the gradual broadening of highlighted edge to a point of uniform uorescence across the bilayer region at 37 C Fi ure 4 The sample was then allowed to cool to room temperature The uorescent images show DilC18 returning to edge These set of experiments point to the edge being more gel like in nature and undergoes a melting transition at temperatures much higher than the center of the bilayer i 3va C 439 Ii mw Mc 23m 325 30 35 23113 396 I3940C 39 at em Frap Recovery Vs Temperature In order to rule out DiIC18 nucleation a frap recovery experiment was conducted using Texas Red DHPE and DMPC At gel state a laser was used to photobleach spots at various locations ie edge and center of a patterned m on OI ay er bilayermonolayer The temperature was then increased and the uorescent recovery of e spots was monitored Monolayer Bulk Monolayer Edge Bilayer Edge Bilayer Bulk At 174 C the frap spot at the monolayer edge starts to recover Monola er Bulk Monolayer Edge Bilayer Edge Bilayer Bulk Frap Recovery vs Temperature At 179 C the spots at the monolayer bulk and bilayer bulk both recover simultaneously However the spot at the edge remains ci39cnnlgr V quot Monolayer Bulk Mohol yer Bilayer Edge 7 Bilayer Bulk quot atern Frap Recovery vs Temperature The temperature was increased to the point where spot at the edge of the bilayer started to lose its mor39OIayequot de nition and start to recover This was achieved at 343 C Monolayer Bulk Monolayer Edge Bilayer Edge Bilayer Bulk The edge of the bilayer quotmeltsquot at a significantly higher temperature than the center of the bilayer 5n 39Ll 2 III Ell It39ll Eli Bl l1 iii a E M in 5 The photobleached spot at the edge of the bilayer started to recover17deg C higher than the photobleached spot at the center of the bilayer con rming an inherent difference in phase properties Note the monolayer edge and monolayer bulk behave differently than the bilayer bulk and edge Schematic Illustration of a ATRFTIR crystal with a lipid bilayer on the surface The multiply bounce of the Infrared light inside the crystal gives multiple sampling of the surface giving information of the overall the membrane system The two major absorptions peaks can be straightforwardly assigned to the methylene symmetric and asymmetric stretching modes The position of these peaks can give direct information of the conformational order of the lipid carbon chains The symmetric methylene stretching d of the alkyl chains absorbs between 2848 cm1 and 2850 cm1 when in a gel state and shifts to higher ranges when in a liquid disordered state 10 2922 2921 wavenu mber d 2919 2915 2920 2917 2Wn 4 Plain DMPC 0 Patterned DMPC 30 Celsius 2353 IH 39 26525 e 2352 23515 1 2551 E I E E t a I MW 4 t Pattemed DMPC I l 20 25 30 35 40 Celsius d cm l 25505i I39I39II Plot of dv peak position ofa plain DMPC bilayer versus patterned DMPC bilayer obtained by ATR FTIRThere is a slight shift in the melting curve of a patterned bilayer campared to an unpatterned bilayer Bilayer edge is more ordered thus more densely populated in nature regardless of bulk lipid This ordering at the edge forces the phase transition to be shifted to a higher temperature The tensed hernisphere edge can than serve as barrier for uid bilayer Special Thanks Sarah Sherlock Funding NSFCenter for BiophotonicsScience and Technology Molecular Organization in Micelles and Vesicles Ken A Dill and Paul J Flory PNAS 78 2 676680 1981 a a Flat 1 a Conventinnal representation of model representation Because the diagrams a micelle b Lam are two dimensinnal Other biophotonic things with membranes Total Internal Reflective Fluorescence TIRF Coherent Anti Stokes Raman Scattering CARS Second Harmonic Generation SHG Fluorescent Lifetime Imaging Microscopy FLIM Third Harmonic Generation Issues with fluorescent probes PROS 1 Can see stuff 2 Easy to use and anything can be tagged 3 Can report environment FLIM CONS 1 MAY NOT JUST BE A REPORTER BUT A PARTICIPANT Spectroscopy From Wikipedia the free encyclopedia Spectroscopy was originally the study of the interaction between radiation and matter as a function of wavelength A A plot of the response as a function of wavelength or more commonly frequency is referred to as a spectrum see also spectral linewidth Spectrometry is the measurement of these responses and an instrument which performs such measurements is a spectrometer or spectrograph although these terms are more limited in use to the original field of optics from which the concept sprang Spectroscopy is often used in physical and analytical chemistm for the identification of substances through the spectrum emitted from or absorbed by them


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