1Bioimage InformaticsLecture 3, Spring 2012Fundamentals of Light Microscopy (II)Practical Issues in Bioimage InformaticsLecture 3 January 23, 20122Outline• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 13• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 14(A) Bright-field(B) Phase(C) DIC(D) Dark-fieldContrast Generation in Light Microscopy• Two fundamental roles of any microscope - To provide adequate contrast- To provide adequate resolution. • Contrast generation– Transmitted light illumination vs reflected light illumination– Bright-field vs dark-field– Phase contrast– Fluorescent microscopy5Reflected Light vs Transmitted Lighthttp://www.olympusmicro.com/primer/java/lightpaths/ix70fluorescence/ix70.html6Bright-field vs Dark-field (I)• Under bright-field contrast, the specimen appears dark against a bright background. • Dark-field contrast is particularly useful when imaging thin filaments or small particles.7Phase Contrast & DIC (II)PhaseDIC8What is wrong with ALL the cell images?9Green Fluorescence ProteinJellyfish: Aequorea victoriahttp://gfp.conncoll.edu/GFP-1.htm10Fluorescence Microscopy (I)11Fluorescence Microscopy (II)Fluorophores are available at many different colorsRoger Y. Tsien, 2009 Constructing and Exploiting the Fluorescent Protein Paintbox (Nobel Lecture). Angew. Chem. Int. Ed. 48: 5612 – 5626.12Fluorescence Microscopy (III)http://micro.magnet.fsu.edu/primer/techniques/fluorescence/gallery/cells/index.htmlB. N. G. Giepmans, S. R. Adams, M. H.Ellisman & R. Y. Tsien 2006 Thefluorescent toolbox for assessing proteinlocation and function. Science. 312: 217-224.13Fluorescence Microscopy (IV)• There are four commonly used fluorescence modes- Widefield fluorescence microscopy (epifluorescence)- Confocal fluorescence microscopy- Total internal reflection fluorescence microscopy- Two photon fluorescence microscopy14Widefield vs Confocal Fluorescence Microscopyhttp://www.olympusfluoview.com/theory/confocalintro.html15Total Internal Reflection Microscopy (I)irrisinnsin nθiθr• Typical thickness of the evanescent layer is less than 200nm• Often used for imaging- membrane related cellular processes- single moleculesninr16Total Internal Reflection Microscopy (II)• TIRF is often used for imaging- membrane related cellular processes- single molecules17Two Photon Fluorescence Microscopy• Multi photon fluorescence for deep tissue imaging.First predicted by Maria Goeppert-Mayer in 1931 in her Ph.D. thesis; First observed in 1961. http://belfield.cos.ucf.edu/one%20vs%20two-photon%20excitation.html18Fluorescence Microscopy Summary• High specificity- Chemical fluorophores (dyes)- Fluorescent proteins• High sensitivity- Up to single molecules• Multiplexity: - Multiple colors (channels)19• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 120Practical Considerations• Photobleaching- Fluorophores gradually lose their ability of light emission.- This results in a continuous decrease in image intensity.• Phototoxicity- Constant illumination generates free radicals that cause cell death. - This places a fundamental limit on how many frame of images can be collected.21References on Fluorescence Microscopy• Lakowicz JR, Principles of fluorescence spectroscopy, Springer, 2006. • Herman B, Fluorescence microscopy, 2nded., Taylor & Francis, 1998.22• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 123Formats of Microscopy Images (I)• Most commercial microscope is controlled by some kinds of control software.- Metamorphhttp://www.moleculardevices.com/pages/software/metamorph.html- Nikon Elementhttp://www.nis-elements.com/- Micromanagerhttp://www.micro-manager.org/• Commercial software often uses proprietary image formats to save metadata. • Free viewing software is sometimes available.24Formats of Microscopy Images (II)• There are ~50 proprietary image formats.Swedlow et al, Bioimage informatics for experimental biology, Ann. Rev. Biophys. 2009, 38: 327-346. • TIFF is the most commonly used format for image analysis. http://partners.adobe.com/public/developer/tiff/index.html• For bioimages, bit depth is normally > 8• In general, image compression that changes pixel values should be avoided.25Free Software For Viewing High Bit-Depth Images• Irfanviewhttp://www.irfanview.com/• ImageJ- Web: http://rsbweb.nih.gov/ij/- Initially started at NIH ; Implemented using JAVA.- Provides bioimage view and analysis functions.- Many contributed plug-ins.26• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 127Demo: Basic Image Manipulation Functions• Image read: imread• Image write: imwrite• Image file information: imfinfo• Image pixel informaiton: impixelinfo28• Contrast generation in microscopy• Practical considerations in fluorescence microscopy• Useful software tools for microscopy images• Basic image analysis concepts• References• Reading assignment 129Image Processing vs Computer Vision• Image processing normally refers to transformation from images to images. - Image enhancement- Image restoration- Image compression- Morphological image processing-…• Computer vision aims to extract from images application-oriented information- Feature detection- Stereo vision- Robotic vision- Face recognition (HCI)-…30Some Related Journals• IEEE Trans. Image Processing• IEEE Trans. Pattern Analysis & Machine Intelligence (PAMI)• International Journal of Computer Vision (IJCV)• Computer Vision and Image Understanding• Pattern Recognition• IEEE Trans. Medical Imaging• Medical Image Analysis31Some Microscopy Journals• Journal of Microscopyhttp://www.wiley.com/bw/journal.asp?ref=0022-2720• Biophysical