Computational Biology Part 20 Biological Imaging IBiological imagingChemical and molecular biological probes may be targeted within a specimenSlide 4Slide 5Image FormationDetector and image typesComparing types of imagingLight microscopyFluorescence MicroscopeLight sources in the objectSlide 12Point-spread functionRealistic Image of a Point SourceSlide 15Introduction to 3D Microscopy3D MicroscopySlide 18Real 3D image dataMicrotubules in a human cellReal 3D image of a point sourceSlide 22Slide 23Slide 24Confocal MicroscopyConfocal Microscope PrincipleSlide 27Slide 28Image FormatsSlide 30Common Image File FormatsSlide 32Slide 33How to use Images: OutlineFrom Images to KnowledgeImage DisplaySlide 37Image Display - LUT changeImage Display - Enhance contrastThresholdingSlide 41Ridler-Calvard MethodSlide 43Slide 44Slide 45Slide 46Slide 47Binary image operationsSlide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Basic Image Processing OperationsArithmetic OperationsSlide 58Kernel/Filter OperationsSlide 60Common Kernel Operations used in Image ProcessingSlide 62Examples of Kernel Operations using NIH ImageSlide 64Slide 65Slide 66Slide 67Slide 68Slide 69Slide 70Install a new plugin in ImageJKernel Operation : DeconvolutionSlide 73Image MathSlide 75Examples of Image Math using ImageJSlide 77Image Math vs. Arithmetic OperationsSummary: Basic Image Processing OperationsImage ProcessingObject finding (Particle analysis)Slide 82Slide 83Slide 84Slide 85Computational BiologyPart 20Biological Imaging IComputational BiologyPart 20Biological Imaging IE. Glory, G. Steven VanniE. Glory, G. Steven VanniMeel Velliste, Robert F. MurphyMeel Velliste, Robert F. MurphyCopyright Copyright  1998, 2000-2008. 1998, 2000-2008.All rights reserved.All rights reserved.Biological imagingBiological imagingSignificant advances in the fields of optics Significant advances in the fields of optics and electronics in the past two decades have and electronics in the past two decades have greatly increased the utility of imaging for greatly increased the utility of imaging for addressing biological questions.addressing biological questions.These advances permit These advances permit more diverse types of information to be extracted more diverse types of information to be extracted from biological specimens from biological specimens with greater accuracy with greater accuracy and under more demanding conditions.and under more demanding conditions.Imaging relies on generating a detectable signal Imaging relies on generating a detectable signal which can be used as a measure of a property of which can be used as a measure of a property of interest in the specimen. interest in the specimen. This property of interest is the initial signal, but it This property of interest is the initial signal, but it must be must be transducedtransduced or changed through several or changed through several forms before it becomes detectable.forms before it becomes detectable.Chemical and molecular biological probes Chemical and molecular biological probes may be targeted within a specimenmay be targeted within a specimenChemical and molecular biological probes Chemical and molecular biological probes may be targeted within a specimenmay be targeted within a specimenFor example: A protein may be modified so that For example: A protein may be modified so that when it enters a cell and bumps into another when it enters a cell and bumps into another protein involved in a specific activity, it fluoresces. protein involved in a specific activity, it fluoresces. The original activity was probably not detectable, The original activity was probably not detectable, but this newly generated fluorescence signal is but this newly generated fluorescence signal is detectable.detectable.Front-end of imaging system and detectorSpecimenSpecimenSpecimen may be difficult to see Specimen may be difficult to see except where labeled by probe.except where labeled by probe.Chemical and molecular biological probes Chemical and molecular biological probes may be targeted within a specimenmay be targeted within a specimenChemical and molecular biological probes Chemical and molecular biological probes may be targeted within a specimenmay be targeted within a specimenA A digital imagedigital image plane is acquired by recording a plane is acquired by recording a digital value proportional to the intensity of light digital value proportional to the intensity of light (or other form of energy) impinging on each (or other form of energy) impinging on each pixelpixel of a of a detectordetectorThis intensity usually corresponds to the amount This intensity usually corresponds to the amount of light emitted by or reflected from a of light emitted by or reflected from a corresponding point on a specimencorresponding point on a specimen0 0 0 2 1 0 00 0 0 1 0 0 00 3 7 8 3 0 00 6 8 8 8 2 00 2 8 8 8 4 00 0 4 8 8 3 0Projection of specimenonto dectector gridImageSpecimenImage Formation and AcquisitionImage Formation and AcquisitionPixelImage FormationImage FormationBiological images may be acquired via a Biological images may be acquired via a variety of imaging variety of imaging modesmodes or or modalitiesmodalitiesEach mode is a combination of an Each mode is a combination of an image image formation system formation system and a and a detectordetectorSampleImage formation systemDetectorWhile the examples so far have dealt with light microscope While the examples so far have dealt with light microscope images, we will now back up for a few minutes to consider many images, we will now back up for a few minutes to consider many different types of images before concentrating on light different types of images before concentrating on light microscopy.microscopy.Detector and image typesDetector and image typesIn general, images may be classified according to In general, images may be classified according to what is being detected:what is being detected:(Visible) light transmission, scattering or emission(Visible) light transmission, scattering or emissionsingle wavelength, 3 color, or full spectrumsingle wavelength, 3 color, or full spectrumElectron transmission or scatteringElectron transmission or scatteringX-ray transmissionX-ray transmissionRadioactive particle emissionRadioactive particle emissionMagnetic field perturbationMagnetic field perturbationPhysical displacement from “atomic force”Physical