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by: Immanuel Brakus PhD


Immanuel Brakus PhD
GPA 3.66


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Class Notes
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This 30 page Class Notes was uploaded by Immanuel Brakus PhD on Monday October 19, 2015. The Class Notes belongs to ECSE 4963 at Rensselaer Polytechnic Institute taught by Staff in Fall. Since its upload, it has received 28 views. For similar materials see /class/224778/ecse-4963-rensselaer-polytechnic-institute in ELECTRICAL AND COMPUTER ENGINEERING at Rensselaer Polytechnic Institute.





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Date Created: 10/19/15
ECSE4963 Introduction to Subsurface Sensing and Imaging Systems Lecture 21 Optical Imaging Kai Thomenius1 amp Badri Roysam2 1Chief Technologist Imaging Technologies General Electric Global Research Center 2Professor Rensselaer Polytechnic Institute GE Global Research Center for SubSurface Imaging amp Sensing Recap We have discussed several macrolevel imaging modalities Ultrasound Acoustic Xray CT absorption of Xrays MRI nuclear spins PET SPECT radioactive emissions They sense different properties of the imaged matter Advantageous to combine them Sensor Fusion Register the images and analyze them jointly Today Optical Imaging Basics Molecular Imaging Contrast Agents L D 3 3 E 393 I I I I I I II I I 39L sh 01 nanny Hzi 1am Freq l O m L I I I I I I I u I I1 I I I I I I 1 O a ha 5 Optical Imaging Systems Electromagnetic Spectrum Gam ma Rays iiiRays Ultravinietquot i i I infra red Microwaves TM AMi iFNi Radio Waves IUI Al 4L 1411 I II I IngrLgAI AIAII l LAIAJJ 1045 inquot2 10 s 1945 10 3 1a 10 mi 10 1 Waveia myth meters Decreasing Waveixengitii increasing Frequency Use light waves to perform imaging Ultraviolet 400nm through infrared 1 000nm Why bother Organic molecules of biological and industrial interest interact strongly with light in the optical region of the EM spectrum The Optical Band Visible Light Spectrum an Ultraviniet infrared r if V i W d nm sannm Baum Tamrm midspectrum wavelength 550nm The human Visual system can see color Color is associated with substances Color Vision allows us to distinguish substances E g some brightly colored berries are poisonous Molecular Imaging Aside Sense of smell is a molecular sensing capability Molecules 101 Molecules are built of multiple atoms Sizes range from 2 to several billion atoms Some molecules of interest Water Carbohydrates eg glucose Proteins RNA ribo nucleic acid DNA deoxy ribo nucleic acid Usually we re interested in selected portions of RNA and DNA molecules Organic and inorganic contrast agents Fluorochromes Fluorescent Proteins Nanocrystals aka Quantum Dots These are arti cial structures with sizes in the lOnm range that respond in the optical band LightMatter Interactions Many ways by which light and matter interact Each of these modes of interaction can be exploited to build imaging systems Common types of interactions Absorption Emission Scattering Change in speed phase Dispersion Re ection Refraction photon Light Absorption a Molecular View 2 hv1 D1313 J lt3tij Hm Every molecule has a characteristic set of discrete energy levels Energy levels are associated with molecular phenomena such as rotations vibrations and bending When a molecule is hit with a photon it can absorb the energy of the photon and start to vibrate and or rotate It transitions to a new state Transitions between some states are more probable than others Some transitions are forbidden quantum mechanical constraints The set of states and transition probabilities are a molecule s observable signature Molar Extinction Coefficie 0t cm1 I39M Light Absorption a Molecular View Absorption can only occur when E2 E photon 11 1 ltL E l 1000000E 39 100000 ug j M I quot ql wi l 10000 liHb HR 1000 39 lif HJH 39 E H000 L a 7 Z 4 kitquotf 100 2L0 400 000 000 1000 Wavelength rim AE hv 2 E2 E1 If the energy of the probing photon is mismatched to the energy difference between the quantum states the material is transparent to the photon By varying the excitation wavelength one gets an absorption spectrum example hemoglobin Hb and oxygenated hemoglobin HbOZ Broader Picture Wavelength Range Type of interaction Comment 10m 1meter Change of nuclear spin Nuclear Magnetic Resonance Nucleons absorbemit based on their spin Radio magnetic component of EM Property Frequency wave more important 1 m 1 cm Change of electron spin Electron Spin Resonance Electrons absorbemit based in their spin Radio magnetic component of EM Property Frequency wave more important 1cm 100um Change of orientation rotation Mostly rotational effects Microwaves electric component of EM wave more important 100nm 1um Change of con guration Mostly Vibrations rotations and bending of molecules While Infrared electric component of EM it still remains in its electronic ground state The molecule wave more important must be asymmetric Vibrations need more energy than rotations 20 um or shorter 1um 10nm Change of electron Changes in electronic states of atoms in the molecule Visible distribution electric produce changes in electric dipoles of the atoms that ultraviolev component of EM wave interact with the applied wave more important 10nm 7 100pm Change of electron distribution At these shorter wavelengths photons can actually disrupt the Xray absorbing molecule by photodissociation or even produce photoionization of individual atoms 1000angstrom photons will photoionize electrons in the outer shells whereas 100angstrom or shorter photons will photoionize electrons in the inner shells 100pm and smaller gamma rays Change of nuclear configuration Mostly passes through E2 Ex E 1 Absarptiun E Excitation E a 3 E Q m am 400 emission Emission Fluorescence Emis sian 6W 7 Wavelength nm 112 Opposite of absorption Can be coherent Raman or incoherent Fluorescence Typically there is some loss in the molecule so the emitted energy is lower than the absorbed energy Energy transfers Within the molecule The difference v2 v1 is called the Stokes Shift Important The emission spectrum is characteristic of the molecule and is independent of the excitation wavelength A stable part oflhe overall molecular signalure Common Patterns of Absorption and Emission Luminescence Emission Without absorption E g re y protein named Lucifer Fluorescence Immediate emission following absorption Phosphorescence Delayedslow emission following absorption Factors Affecting Emission Quantum ef ciency of the molecule photons emitted photons absorbed Strengthintensity of the excitation Strengthintensity of the excitation Emission Molar Concentration of uorophore If 77gtltI gtlt1 e8AC Molar absorptiVity Excitation Eluor 6SC 1VICe intensity Path 1ntenSIty Quantum length Ef ciency photons emitted To get a stronger signal it helps to look for more efficient emitters ways to put more molecules and minimize absorption of the emission A Molecule s Overall Signature Every molecule has a characteristic set of energy levels and a set of allowed transitions between them Some transitions are more probable than others quantum mechanical constraints The set of states and transition probabilities are a molecule s externally observable signature This signature is in uenced by the molecule s environment and the state of activation of the molecule One problem often some important molecules of interest are either not abundant or have a weak signal or a signal in an inconvenient part of the spectrum Molecular Contrast Agents A contrast agent is a small molecule or a nanocrystal quantum dot that can Attach itself selectively to just the molecules of interest Interact strongly with light in a known manner Allows us to infer the presence of the molecules of interest by proxy Absorbance DAPI An Organic Fluorophore DAPI bound to DNA Fluovescence emission 300 550 400 450 500 550 600 Wavelength nm 0 5mm 0 Nmogen Oxygen O Carhnn a Aw 4t i39 0 at v5 A v vdlamldlnoZphenyllndale DAPI A typical organic uorophore Used commonly to label DNA hilequot Liohl 51 w sible Wuvelcng scornn l quotmam 700nm Engineered Fluorophores Quantum Dots Syntheticengineered uors 7 Made in Troy Evident Technologies Troy NY 7 High quantum yield 7 Long lived compared to organic Semiconductor CdSe nanocrystals 7 roughly llOnm in size 7 Coated to enhance uorescence properties and reduce toxicity Wellbehaved Spetra 7 Emission peak depends only on their size 7 Broad absorption spectrum 7 Strong absorption Quantum Dot Spectra EviTau Ahsnrmiun Spectra Emissiun mhilrary Unils Arbitrary Unils V Conjugated Quantum Dots We can buy quantum dots that come attached with antibodies on their surface These are proteinbased molecules that attach to molecules of interest in a highly selective manner Usually one can buymake an antibody for just about any known protein Maj or advantages We can choose the emission color Stable wellbehaved uor Antibody From Evident Technologies Inc Troy NY Cool and Important Fact 1 We can often use more than one contrast agent at once This is known as multiplexing Need to make sure that spectra are nonoverlapping Reveals multiple things at once in their relative spatial context Things we re interested in Structural molecules that indicate structures of interest Functional molecules that indicate molecular activities of interest By observing structural and functional molecules simultaneously we can image biological processes in their spatial and temporal context Color What is What it labeled Shows Cyan DAPI Cell Nuclei Red GFAP Astrocytes marker Green Laminin Blood marker Vessels Yellow IBAl Microglia Magenta Nissl Endoplas mic Reticulum of Cells 7 2 Courtesy Dr W Shain Wadsworth Center Albany NY Individual Channels I n 71quot M Vas As New Auchanneb w gf h 1 1 11 Cool and Important Fact 2 We can genetically modify cellsanimals so that they generate molecules such as proteins that come attached with contrast agents Known as Fluorescent Proteins This eld started with the discovery of a naturally uorescent protein in jelly sh Green Fluorescent Proteins GFP Now this has grown into a multibillion industry that sells 100 s of highperformance uorescent proteins covering the entire spectrum Allows us to image speci c molecules in living systems Example Seeing the Cytoskeleton of a Living Cell Basic Idea Attach a GFP to each of the octubulin protein molecules red balls below Tubul n Filament http WWWolympus uoviewcomapplicationsgfpintrohtInl Multiplexing GFP A powerful Combination Dendirite 1 Courtesy Gary Banker OHSU The Purpose of Imaging Structure of specimens The detailed structure anatomy of objects as revealed by physical and chemical properties transmissivity re ectivity phase molecular signatures Relative arrangement of structures Function of speci c parts of specimens The location of speci c biochemical substances within cells and tissue The activities of these entities in space and time such as movement and signal transduction Biochemical reactions Dynamics of structure and function How structures change in time How activities evolve over time Combined structural and functional imaging over time allows us to image biologicalother processes in their 3D spatial context Tells us where the function is happening relative to the specimen anatomy Tells us how an activity evolving in time its dynamics Summary We have discussed Basics of Optical Imaging Basics of MoleculeLight Interaction Organic molecules of major interest can be imaged with high speci city Our interest in organic molecules is a major Next Class Modern Optical Microscopy Homework Lecture 21 1 Study the spectra of Hemoglobin on Slide 8 Propose a design for a system that measures the oxygen level of blood by optical means The device could be wrapped around a nger Hint Choose speci c parts of the spectrum at which you would like to make measurements and construct a formula that generates a stable and reliable measure of oxygenenation Use internet search tools to answer the following questions What is the role of hemoglobin and where does this molecule reside o What kind of molecule is it and just how big is it Why is it useful to measure blood oxygen levels name some clinical applications 2 With reference to the formula in Slide 13 Why can t we get a stronger signal by just increasing the excitation inde nitely Instructor Contact Information Badri Roysam Professor of Electrical Computer amp Systems Engineering Of ce JEC 7010 Rensselaer Polytechnic Institute 110 8th Street Troy New York 12180 Phone 518 2768067 Fax 518 27662612433 Email roysamecserpiedu Website httpWWWrpieduroysab NetMeeting ID for offcampus students 1281136180 Secretary Laraine Michaelides JEC 7012 518 276 8525 michalrpiedu Pensselaer why not change the world Instructor Contact Information Kai E Thomenius Chief Technologist Ultrasound amp Biomedical Of ce KW C300A GE Global Research Imaging Technologies Niskayuna New York 12309 Phone 518 3877233 Fax 518 3876170 Email thomenngcrdgecom thomenius ecseIpiedu Secretary Laraine Michaelides JEC 7012 518 276 525 michal gpiedu Rensselaer GE Global Research m M mquot m mum


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