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Biomedical Imaging

by: Annie Collins DVM

Biomedical Imaging BIM 289B

Annie Collins DVM
GPA 3.79


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This 54 page Class Notes was uploaded by Annie Collins DVM on Tuesday September 8, 2015. The Class Notes belongs to BIM 289B at University of California - Davis taught by Staff in Fall. Since its upload, it has received 33 views. For similar materials see /class/191776/bim-289b-university-of-california-davis in Engineering Biomedical at University of California - Davis.

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Date Created: 09/08/15
Raman Measurements of Organic Molecules and Biological Processes Atomic Force Microscopy AFM cBLs39r AmOld Burger Department of Physics Fisk University Nashville TN CENTER for BIOPHO I ONICS SCIENCE and TECHNOLOGY UCDAVIS January 2 4 2008 Winter 2008 EADBIM 289 6 Outline Introduction to Raman Spectroscopy RS History Principles Advantages Applications Application in Biological Systems Surface Enhanced Raman Spectroscopy Atomic Force Microscopy Introduction and developments Research at Fisk University AFGP Structure Interaction with highly oriented pyrolytic graphite HOPG Interaction with MICA SERS of glucose Quantitative analysis Ag colloid as substrate V nter 2008 EADBIM 289 chT Introduction to Raman Spectroscopy C V Raman discovered the effect in cos wt cos Qt CV Raman s equipment sunlight a colored filter and his eye Classical Simple H0 1928 and was honored with Nobel Prize in 1930 Q I O k in millidynesA E AE Ei ES m in amu s and Vin cmquot Assume k v R a k 5 1o 15 for OH 5 3000 potential energy for Single d0Ubegt CC 15 2100 a diatomic molecule trlple bonds CC 10 1700 V nter 2008 EADBIM 289 C C 5 1200 3 Calculation of the ratio between the vibration frequency of OH and OD bond if vvibOH 2 MOH F vvibOD LJL MOH 275 MOD mOmH 16 mOmD MOH OD m0mH 17 m0mD 18 vvibOH vvibOD 1816 V nter 2008 EADBIM 289 Question If a CH stretch is observed at 3000 cm391 where will the CD stretch in an analogous deuterated molecule be observed V nter 2008 EADBIM 289 ca 7 Q Calculated vs Experimental An example CO stretch fundamental ST W r gm1 1800 1700 1600 l l m XEI Cl ltoc0Hgt2 39 39 D IIoc 0 L0 OC0Ph CFCOR amidel OCNHR v CO 16000mquot E x r calculated I 0CNH2 solution I OCNRR lOCNHR 3939solrcl CCNH2 quot see 69 Gr Socrates Infrared and Raman Characteristic Group Frequencies Wiley 2001 V nter 2008 EADBIM 289 6 O W quotII 1 Functional Groups f I Bond Energy cm391 C H 2850 2960 C H 3020 3100 CC 1650 1670 EC H 3300 CEC 2100 2260 C CI 600 800 C Br 500 600 C I 500 O H 3400 3640 C OH 1050 1150 CH 3030 I 16001500 N H 3310 3500 C N 10301230 CO 1670 1780 COOH 2500 3100 CEN 2210 2260 N02 1540 V nter 2008 EADBIM 289 IR amp Raman are complementary Raman frequency shift VAE and IR absorption peak frequency are identical 4 gt O C 0 IR inactive Raman active gt gt OCO IRactive Raman inactive Raman emphasizes aromatic amp carbon backbone CC CH2 etc O W 4510 Energy Level Diagram for Raman Scattering a Stokes b antiStokes on Virtual E A quotquotKquot quotquotquot quot State Physical reason behind RS For a molecule to be Raman active 7lt Incident Scattered there qut be a maiden photon photon change In the polarzabIIty photon Scattered momentary distortion of the v photo e39distribution around a bond Vibrational V Levels Escat Elaser i Evib b a EhVEhCV Stokes l AntiStokes A AS band is weak at low temperature molecules have to be vibrationally exc ed for a vibrational mode to be IR active there must be a change in the dipole moment Winter 2008 EADBIM 289 8 Units For historical reasons the energies are measured in wavenumbers or cm1 quoti V V no of waves cm wavenumber hc hc hc Em E39ase39 iE m lgt Ascat Alaser i Avib E hv E hcv 7 1 1 1 Ascat laser i Avib vscat Vlaser i vvib Spectra range 25 100 Mm Wntermosuggaggsgesponds to 4000 100 cm1 9 Pi is the induced dipole moment or is the polarizability E is the electric field of the incident wave Linear Nonlinear Raman P Ej 3EJEk rzjklEjEkE 39m l v A l yk CARS Coherent AntiStokes Rayleigh hyper Rayleigh Raman Spectroscopy hyper Raman Resonance Raman effect occurs when the virtual state coincides with one of the molecular eigenstates usually from n0 9 n1 Nonlinear Raman normally requires a large intensity of incident light The elastic scattering is called Rayleigh scattering Most photons are elastically scattered 1 in 107 incident photons undergo the Raman effect V nter 2008 EADBIM 289 10 O W quotM H Normal Raman INCIDENE LIGHT Aro cos 8on cos out paEa0 a 0 2005 2100st cosa Q cosa Q gt VIbratlon STOKES ANTISTOKES V nter 2008 EADBIM 289 H Factors affecting the Raman Intensity Raman line intensities are proportional to EL quHC is the frequency of the incident radiation is the Raman cross section typically 1029 cm2 is the radiation intensity is the energy related to the vibrational energy Qglto lt is the analyte concentration V nter 2008 EADBIM 289 12 Inter 2008 EADBIM 289 4 in 15mm quot390 quot39iquot L I U Example I VD va 4 e ami Sror res Forx7 1000 cm39l T300K9 kT25OCm3919 expEikT e4 55 With comparable cross sections lS is two orders of magnitude larger than lAS CEET t Developments of Raman Spectroscopy In the last two decades great advancement of lasers and multichannel detection techniques has taken place Several advanced Raman techniques Surface Enhanced Raman Spectroscopy SERS Resonance Raman Spectroscopy RRS Coherent Antistokes Raman Scattering CARS MicroRaman and TimeResolved Raman spectroscopy etc Fourier Transform FT Raman Optical trapping Raman spectroscopy made possible the detection of weak bands and weak signal V nter 2008 EADBIM 289 GEL 39 t 3 Raman Spectroscopy is an inelastic light scattering experiment that measures the vibrational frequencies of the sample providing 1 Fingerprint spectra of molecular structures and compositions 2 Information about 3D structural changes 3 Information about intermolecular interactions 4 Molecular dynamics BioRaman For biological systems it is a nondestructive almost real time in vitro measurement of a cell chemical fingerprint Confocal Micro Raman Spectroscopy is a tool developed for the study of materials in localized wavelength cubed diffraction limited space V nter 2008 EADBIM 289 cal Raman vs Infrared IR measurement Raman IR Detector CCD or photomultiplier MCT TGS tube Source UV Visible NearIR Farinfrared Spot Size um mm Mechanism of Vibration modes Polarizability Changes Dipole moment Change Intensities Can be increased by Changing the laser power Both probe the structural information of materials V nter 2008 EADBIM 289 Pros and Cons of Raman Spectroscopy of biological samples Pros Small samples needed easy to prepare compared to HR absorption spectroscopy Nondestructive noncontact and near instantaneous results Offers detailed structural information compared to florescence Resonance Raman and Surface Enhanced Raman can be used to get signals enhanced by a factor as large as 1015 broad range without changing the detector 50 4000 cm391 compared to infrared spectroscopy Relatively narrow lines different se ections rules compared to IR 6 Water scatters weakly the region from 200 2000 cm391 is completely transparent compared to HR absorption spectroscopy Cons Low cross section result in low signal intensity high florescence background sometimes Risk of thermal degradation V nter 2008 EADBIM 289 Confocal Raman Microscope s in BE Video monitor 5 we J chame quot Q 20 sec Spectrometer With CCD camera M 302782 quot4 removable mirror holographic notch filter oi I 300 pm PC cm1 quot confocal aperture r ELM beamsplitter l 3100 j I I I 1315 1 high NA objective xy mapping stage kl Optical Layout of the microRaman system Model LabRam Infinity from JYHORIBA HeNe laser at 63281 nm power ranges from 0011 to 11 mW V nter 2008 EADBIM 289 ll CEST Currently RS is broadly used in chemical systems Pharmaceuticals Polymers Biological and Medical Systems Food Products Forensic applications Materials Semiconductors and Environmental Sciences etc Excellent introduction of RS for living cell applications available at Nondestructive Characterization of Biochemical Processes in Living Cells Thomas Huser Winter 06 Seminar httpcbstucdavisedueducationcourseswinter2006 seminarbioohvsicsthpdf V nter 2008 EADBIM 289 chgt I What is a biological system The basic unit is the cell Elements CHONPS It includes Water Protein Enzymes Lipids Carbohydrates Nucleic acids etc as well as minerals Function groups EtherO HydroxyI OH CarbonylCOH KetoneCO CarboxyCOOH EsterCOO MethyICH3 PhenyIC6H5 AminoNH2 20 V nter 2008 EADBIM 289 cat t r 1 7 2 Raman Applications in Biological Systems Goals Resolve the structure and functions of biological objects by Identifies intramolecular and intermolecular interactions and Elucidates biochemical reaction mechanism Problem The molecular weight of a protein is 30000 corresponding to N4000 atoms and to define the structure 3N6 parameters would be needed huge Simplifications Ignore Hatoms examine general aspects of the structure by identification of functional groups 21 V nter 2008 EADBIM 289 l Amide groups in proteins are generally employed to study protein structure 0 There are 5 atoms in an A amide CONHZ a 3N6 Carbonyl CO GROUP 3x56 9 normal vibration R NH2 modes k Amino NH2 GROUP R H Amide band I C N R 80 00 stretch near 1650 cm1 0 R H Amide band ll o i 60 N H bend and 40 C N stretch near 1550 cm1 0 R R Amide band Ill 0 40 C N stretch 30 N H bend near 1300 cm1 x R IO 22 V nter 2008 EADBIM 289 a i CELfT x65 Raman Application in Biological Systems cont Diagnosis and clinical uses Quality control of pharmaceutical and food products Medical diagnosis skin cancer etc Biosensor SERS technique Medical imaging and monitor 2 D Raman scanning Examples Measure the concentrations of total protein and biological analytes in blood and serum Determine metabolic concentrations Measure blood and tissue oxygenation molecularlevel cancer cervical lung etc Cardiovascular disease such as atherosclerosis diagnosis V nter 2008 EADBIM 289 Raman Spectroscopy of Pollens a BEN 10 aromatic ring stretch C CAR carotenoid 1157 and 1526 cm39l CHL chlorophyll CH2 deformation vibration CH 1442 cm39l I PHE phenylalanine 1003 cm39l PHE CAR CHL CH CAR 28000 24000 39CHLJBEN 20000 Intensity arb Units WI 16000 g I I 560 360 1de who 18k Ramanshi cmquot A BoyainGoitia et al APPLIED OPTICS Vol 42 NO 30 20 October 2003 V nter 2008 EADBIM 289 clay SERS Result of Redwood Pollen it l1 7777 7 L 7 5439 39 llil Mil 397 ill 13 IJU 1 Eli ill 1 CH quot HUD antiwari 3h 7 7 mm in Silver colloidal particles with diameters in the range 10 to 20 nm and with a characteristic ultraviolet visible UVVIS absorption band at 400 nm were used to obtain SERS spectra The peaks are enhanced at 1173 s aromatic amino acids in proteins and at 1240 w N H C N amide III A Sengupta Applied Spectroscopy Volume 59 Number 8 2005 V nter 2008 EADBIM 289 CELST Raman of Wheat Grain Kernels EX Starch LiPid Protein a I I I l 500 1000 1500 Wavenumber cm391l Raman analysis of a starch granule within the wheat grain kernel shows distinct spectral features arising from the starch lipid and protein species present wwwjobinyvoncomusadivisionsRamanapplicationsBi004pdf 26 V nter 2008 EADBIM 289 Raman Microscopic Mapping of the Molecular Components in a Human Tooth T CKW shows that the amount of phosphate in bulk enamel and 01 l bulkidentine is greater than at the enameldentine junction g 0 CB l 5 o iCiIZ g o 00 q 1 ED 3901 g r r w w a C O Elooza o g l 2200 loo 02 o 02 2300 0020 2450 2000 1000 y r 1200 002 v A 800 400 0 C 1200 ibU LCOD 240C 2530 i 7t qECO 4000 no 4500 5200 5630 6000 6400 3000 l 0 Map of the integrated band intensity of VsPO of a transverse section of a human tooth A enamel B enameldentinejuction C dentine E WentrupByrne et al J RAMAN SPECTROS VOL 28 151 1997 x x a A V nter 2008 EADBIM 289 Study of the Effect of Bleach on Human Hair 1040 cm391cysteic acid FT Raman spectra of L l i I k A J reewa 1 human hair 1064 nm i fa A f quoti5 a excitation 8 WrapL ftquot w 39 V i t a 100 mW 400 scans at 4 2 If m K a 39 J cm1 resolution a natural git w Ji untreated hair shaft b g I thwf i bleached hair shaft c hair A I r H kr f y mj 391 is J Vj KxA39 xixxx ax jquot keratin Spectra arranged 439 A I 39 quotW C ad from top down r iW 439 uV39 quotIn 1 RA Irv W b E I a af xf V d avenum er 6 f 39x range 34002600 cm391 i v u 1 v v if v v39 39t7h f r 39T quot50 1125 not 5075 1050 025 mm 975 953 925 SI 91 350 53925 EOE 72 SE Maven nquot W Akhtar et aI Spectrochimica Acta Part A 53 1997 10211031 V nter 2008 EADBIM 289 CEET Ongoing Raman Study at Fisk Antifreeze glycoproteins AFGP structure Polymer of tripeptide n4 40 4 Backbono gt A Q e Hydrophobic CH3 s m at P 3 1 P dig Disaccharido 399 W 39 Wilt 1 i 39 l t quotL l i J r Hydrophilic disaccharides 39 r 4 Ic39c Lalw Antifreeze glycoproteins AFGP structure Y Yeh R E Feeney Chemical Reviews 96 6011996 WATER ICE optical image 60 me60 pm of GLYCO CH3 AFGP on HOPG o V nter 2008 EADBIM 289 93 13quot Interaction Between AFGP and HOPG Ef gy 39v QSUbstrate Tomimatsu et al J Biol Chem 1976 251 2290 r C39EHs senlsitive 80 1003 1361 1460 39 1048 55 391 Ma 004 mgml J 39 g a M v 940 1008 1364 1459 Mb 004 mgml 1 2quot1050108 3 33 1154 Mb I22 05 mgml E 0002 I3 01 mgml E 1002 g 7 s The top four d E quotquot 995 curves are from 86 1458 AFGP solutions 104 1086 1324 F Z Wsw on 827 885 915963973 1061150861101 1270 1320 1457 SUbStrate 799 1 1380 Van v 39 B quot 39 39 B Bulk AFGP 800 1000 1200 1400 Raman Shift cm 1 T I 11 mW 2 Mm diameter AFGP mixture of678 P amide PiCHs asymm Y Cui et a Journal of Raman Spectroscopy 36 11132005 C carbohydrate Pzpolypeptide V nter 2008 EADBIM 289 30 O W U Identifying the Function Groups in AFGP 51 l gt PolyDLAlanine close to the backbone of AFGP includes function group amidel CH2CH3 etc gt DGalactosamine close to the sugar side chain ofAFGP includes function group amide sugar ring and CHZOH gt Stachyose Tetrahydrate includes function group sugar ring and CHZOH with H20 Add together the Raman spectra of the three components above The result is roughly similar to the spectrum of AFGP AFGPs dried gt New peaks from AFGP drop dried sample appear close to 1000 cm1 and 1511 cm391 might be due to Stachyose Tetrahydrate sugar rings build together with the water involved V nter 2008 EADBIM 289 9ng Raman Spectroscopy of AFGP and related groups PCC milUL 000 A AJW IJ Lm39mv vNWAMJ Ar CH will Lian UVJNM JM AJLNI MK J LeriVJMAU L wv wkfw Viv n yr V l39quot DA Add lt1quot 39 1 1000 I 1500 carbohydrate CN II substrate amlde V nter 2008 EADBIM 289 l x amide ll does not show i amide l D Glu Amid HST P Ala AFGP AFGP dried D G111 Amid Tl J W K39ng ill TAssollc w carbohydrate II J V x 139 2L 4 IE 1 i uni AFGP dried l 2000 Wavenumber cm 1 oPAla PolyAlanine oDGlu Amide D Galactosamine HST Stachyose Tetrahydrate oAFGP dried AFGPs solution drop dried on mica Research at Fisk to be published M Guo et al 32 9 e Surface Enhanced Raman Scattering SERS 395 53 K Kneipp et al J C mm WWE The Raman process has a low cross Phys Condens 1quot l0 section SERS has enhanced signal Matter 14 2002 molecules with 0515 intensity by up to 1015 First demonstrated R597 R624 by Jeanmarie and Van Duyne in 1977 i39 HVL lr VL2 x10 um 1005 N m 1m War 3 0 fin x 1 a quotl PSERS S aa slA 3000 20130 b 7 Roman shin tom an Duyne J Elscb39aanal Chem 1939 84 1 A vL and A vs are the enhancement factors oefmthmemllaser and the Raman scattered field vL is the intensity of the laser N is the number of molecules involved in the SERS process 0de is the cross section of Raman process of the adsorbed molecules V nter 2008 EADBIM 289 O U SERS Measurements of aDGlucose Van Duyne group has done SERS measurements of glucose using silver film over nanosphere AgFON They were able to measure glucose concentration as low as a 4W 0 H 20mgdL 1mM after partial least VH r on a square analysis of the data 0 u m N A 10ml 9 g g V quot xv vquotK Vquot squot r1410 g E 1123 F545 I AgFON Surface g 3 E El 1123 1 a 9 a 3 m E El 131115 3 K Shafer Peltier et al J AM CHEM SOC 9 VOL 125 NO 2 2003 r T r r r 1600 1200 800 400 V nter 2008 EADBIM 289 Wavenumber Shift cm i 5 Enhancements 1 Electromagnetic field 2 Chemical first layer effect Challenges Developing stable SERS active substrates Experimental setup for single molecule SERS Insert shows an electron micrograph of typical SERS active colloidal clusters Adapted from K Kneipp Bioimaging 19986 104106 Apparent cross section is 103914 cm2 Glass Slide V nter 2008 EADBIM 289 GELST 4 Research at Fisk r quot 3 39v to be published 5 L Baietal Preparation of Ag Collond k The Ag sol was prepared following the method of Lee et al P C Lee J Phys Chem 86 33911982 with materials shown below Silver nitrate 90 mg water 500 ml Mixed Sodium citrate 100 mg with 10 ml water Kept the mixture boiling for 1 hr Our goal is to be able to obtain SERS of glucose on Ag colloid Glucose is difficult to adsorb on the Ag nanoparticles Self assembled alkanethiol monolayer such as 1decanethiol will be used as a buffer between the Ag colloid and the glucose V nter 2008 EADBIM 289 Ag colloids are Inhomogeneous lEi Enlz 392 l O lum The excitation is not distributed uniformly over the entire cluster but tends to be spatially localized in socalled hot spot areas Therefore the surface of a fractal colloidal cluster structure shows a very inhomogeneous field distribution Field distribution of SERS active silver colloidal cluster Podolskiy V A and Shalaev V M 2001 Laser Phys 11 26 V nter 2008 EADBIM 289 SEM of the Ag Colloids prepared at Fisk showing their inhomogeneous distribution The particle sizes range from 40 to 240 nm V nter 2008 EADBIM 289 SERS of Rhodamine 590 10000 8000 6000 4000 2000 200 400 600 800 1000 1200 1400 1600 Wavenumber cm391 V nter 2008 EADBIM 289 Black curve 105 M Rhodamine 590 Chloride on Ag colloid Red curve 105 M Rhodamine 590 Chloride on Glass slide Indicates significant enhancement over 105 39 995T Quantitative Analysis of DGlucose Concentration at Fisk K De Gussem et al Spectrochimica Acta Part A 61 2005 2896 2908 00 SpectRlMT39V39 slide for Raman signal enhance substrate from Tienta Sciences Inc 00 Based on a chemically inert hydrophobic stainless steel surface to sensitively accumulate the chemical in dilute solutions in polar solvents such as water with its dehydration 00 The different Dglucose concentrations will be in various spot sizes and peak intensities on Raman spectroscopes C6H1206 ocDglucose g 35 um 10007 COH 2 000 on l l K 0 OH CH COH l 800 Peak intensity HOW quotOH OCH HO l CH2 ItDGluwu Research at Fisk x unpublished M Guo et al Intensity au 600 400 l l l l l l l 800 900 1000 1100 1200 1300 1400 Wavenumber cm1 V nter 2008 EADBIM 289 CELST Results for Quantitative Analy3is of r quot1 oil 3 DGlucose Concentration Lower limit for SERS Raman Intensity vs Concentration Area vs Concentration 180903 100E06 E 160E03 a 55 to t39 S 140E03 S A 100E05 12 120E03 g a i E o 3 in 08 100E03 g 18 um 6 c 5 800E02 t m 2 5 600E02 lt E 400E02 100E03 100E 04 100E 03 100E02 100E01 100E00 100E 04 100E 03 100E02 100E 01 100E00 Concentration mglmL Concentration mglmL Correlation between Raman intensity area and aDglucose concentration Research at Fisk University 41 V nter 2008 EADBIM 289 AFM How Does It Work Incoming Segmented Photo 39 1e error Laser Beam B Cantilever Substrate Flexible Cantilever Sample Piezoelectric quot Scanner A C Atomic Force Microscope AFM Winter 2008 EADBIM 289 Beam Position Photo diode Diode L aser Lens Beam Path Cantilever Sample Sample Stage Piezoelectric Feedback Scanner loop XY Scan A FIGURE 2 A Schematic Illustration of theAFM Set up V nter 2008 EADBIM 289 AFM Schematic Illustration SE1 th39lwww xintpk SUM X2 300 quotMIN WD ll 5mm FM m mm gmmmg mm mng SM V nter 2008 EADBIM 289 IF quotSK JNIVERSITY IFIISK UNIVERSITY XY 39 adjustme V V nter 2008 EADBIM 289 V nter 2008 EADBIM 289 CBLST I SK 3 AFM mage of HOPG Scan range 16 nm Scan rate 100 Hz Sampleline 256 V nter 2008 EADBIM 289 STM on highlyoriented pyrolytic graphite HOPG proving STM mode and atomic resolution The image on the left is Fourier ltered but the one on the right is un ltered raw data VCCCO V nter 2008 EADBIM 289 WET DNA in fluid Live Cell Imaging Mouse fibroblast cell imaged live in buffer solution Image size 40 microns V nter 2008 EADBIM 289 lElS lg ForceDistance curve s3 What the Force Distance curve is AFM as a Force Sensing Instrument g lt oz Force Sensitivity g SAMPLEPC SmO 103914N picoNewtons w Laser oz Displacement Sensitivity 001 nm 01 Angstroms oz Time Resolution milliseconds seconds oz Contact Area Cantilever quotm2 ll t i Veeco CPII Training Module 5 Force Spectroscopy Page 50 V nter 2008 EADBIM 289 eT Molecular Recognition V nter 2008 EADBIM 289 Nanolithography with AFM Nanolithography with the Nanoplot package Scratching on a polycarbonate substrate Image size 10 pm V nter 2008 EADBIM 289 AFM image of noble metal nanostructures for SERS edge lengths of the triangles are about 80nm University of Bath UK A llie C Durkan Wl Milne and ME Welland quotSurface Enhanced Raman Spectroscopy as a probe for local modification of carbon lmsquot Phys Rev B 66 045412 2002 V nter 2008 EADBIM 289 GK sT Chemical Imaging with a Raman AtomicForce Microscope A Dr Tom Pike Imperial College UK httpwwweeicacukwtpRAFMhtml Locally Enhanced Raman Spectrum ft A IntensityNVavenumber Laser Tiprr 1 J h l Sampb Substrate V nter 2008 EADBIM 289


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