H. Nur Halipçi-Fatih University


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Scanning Laser confocal microscopy

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H. Nur Halipçi-Fatih University

  1. 1. SCANNING-LASER CONFOCAL MICROSCOPY (SLCM) Hatice Nur Halipçi 5003 0916 Fatih University-Molecular Biology and Genetic
  2. 2. OUTLINE <ul><li>Short History </li></ul><ul><li>General Definition </li></ul><ul><li>Principles of Confocal Microscopy </li></ul><ul><li>Types of Confocal Microscopes </li></ul><ul><li>Scanning Laser- Confocal Microscopy </li></ul><ul><li>Application Areas </li></ul><ul><li>Advantages </li></ul><ul><li>Disadvantages </li></ul>
  3. 3. HISTORY OF CONFOCAL and SLC MICROSCOPY <ul><li>The principle of confocal imaging was patented by Marvin Minsky in 1957 </li></ul><ul><li>In 1978 , Thomas and Christoph Cremer designed a laser scanning process </li></ul>
  4. 4. What is the Confocal Microscopy? <ul><li>is an optical imaging technique used to increase micrograph contrast and to reconstruct 3-D images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane </li></ul>confocal microscope
  5. 6. Why does it called“CONFOCAL?” <ul><li>In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information . Also the pinhole is con jugate to the focal point of the lens. – t hat’s why it’s called “ confocal ” </li></ul><ul><li>In a conventional fluorescence microscope, the entire specimen is flooded in light from a light source. So, all parts of the specimen throughout the optical path will be excited. </li></ul>
  6. 7. WHAT’S THE PRINCIBLE? <ul><li>The principle of confocal microscopy </li></ul><ul><li>is the elimination of out of focus light, </li></ul><ul><li>thus producing a high z-resolution image. </li></ul><ul><li>Confocal fluorescence microscopes achieve this, via two principal </li></ul><ul><li>mechanisms. </li></ul>
  7. 8. First , incident light is focused to a particular point within the specimen by passing it through a very small aperture, the first pinhole. The focusing helps to limit the excitation of fluorophores above and below the plane of focus.
  8. 9. Second , any emission that is above or below the plane of focus is blocked from reaching the detector by passing it through a second pinhole. The specimen is placed in the light-path at a conjugate focal plane such that movement in the vertical (z) direction keeps the focus at a fixed distance from the objective, and effectively scans in layers through the specimen.
  9. 10. WHAT’S THE FEATURE? <ul><li>The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as optical sectioning . . </li></ul><ul><li>Images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece. </li></ul>
  10. 11. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.
  11. 12. 1. Scanning laser confocal microscopes (CLSM) 2. Spinning disk confocal microscopes (SDCM) 3. Programmable a rray m icroscopes (PAM). Types of Confocal Microscopes
  13. 14. SCANNING: The science and techniques involved in the use of scanners LASER: an acronym for L ight A mplification by S timulated E mission of R adiation CONFOCAL: having the same focus   MICROSCOPY: research with the use of microscopes
  14. 15. S canning L aser Confocal M icroscopy ( S L C M ) is a valuable tool for obtaining high resolution images and 3-D reconstructions.
  15. 16. HOW DOES IT WORK? <ul><li>A laser is used to provide the excitation light (in order to get very high intensities). The laser light (blue) reflects off a dichroic mirror. From there, the laser hits two mirrors which are mounted on motors; these mirrors scan the laser across the sample. </li></ul>
  16. 17. … ..how does it work? <ul><li>Dye in the sample fluoresces, and the emitted light (green) gets descanned by the same mirrors that are used to scan the excitation light (blue) from the laser. The emitted light passes through the dichroic and is focused onto the pinhole. The light that passes through the pinhole is measured by a detector. </li></ul>
  17. 18. … ..how does it work? <ul><li>So, there never is a complete image of the sample -- at any given instant, only one point of the sample is observed. The detector is attached to a computer which builds up the image, one pixel at a time </li></ul>
  18. 19. <ul><li>Some of Microscopy Images </li></ul>Retina ganglion cell Rat tongue taste bud Human colon crypt visual interneurons of swallowtail butterfly
  19. 21. <ul><li>3-D IMAGES </li></ul>Mitosis Tobacco BY2 cells in telophase Tobacco leaf epidermal cells expressing neurocapsule Metaphase: tobacco BY2 chromosomes
  20. 22. <ul><li>In a laser scanning confocal microscope a laser beam passes a light source aperture </li></ul><ul><li>and then is focused by an objective lens into a small focal volume within a fluorescent specimen. </li></ul>Differences of Fluorescent and Confocal Microscopy <ul><li>Confocal microscopy is a form of fluorescent microscopy </li></ul><ul><li>BUT </li></ul><ul><li> it uses a computer, rather than projected through an eyepiece. </li></ul><ul><li> gives higher resolution </li></ul><ul><li> laser light source is used to excite the fluorophore resulting in enhanced contrast . </li></ul>
  21. 23. Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter Confocal Microscope
  22. 24. Conventional fluorescence microscope Laser scanning microscope specimen Arc lamp + excitation filter laser light source full field illumination full field detection point scan illumination point scan detection
  23. 25. Differences of non-confocal and confocal images
  24. 26. Rabbit Muscle Fiber (Magnification:100X)
  25. 27. <ul><li>Fava bean mitosis </li></ul>(Magnification:100X)
  26. 29. APPLICATIONS <ul><li>R esonance or flourescence energy transfer, </li></ul><ul><li>Stem cell research, </li></ul><ul><li>L ifetime imaging, </li></ul><ul><li>Total internal reflection, </li></ul><ul><li>Immuno-gold labeling, </li></ul><ul><li>DNA hybridization, </li></ul><ul><li>Membrane and ion probes, </li></ul><ul><li>Bioluminescent proteins, </li></ul><ul><li>and epitope tagging. </li></ul>
  27. 30. <ul><li>studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues </li></ul>… applications <ul><li>It is particularly useful for, </li></ul><ul><ul><li>evaluation of various eye diseases. Imaging, qualitative analysis and quantitification of endothelial cells of the cornea. </li></ul></ul>
  28. 31. Colocalization of Proteins <ul><li>It provides c olocalization of up to 4 different proteins </li></ul><ul><li>By using this function to analyze multi-color </li></ul><ul><li>specimens, it is possible to discover whether different labeled substances are </li></ul><ul><li>present in the same region </li></ul><ul><li>Decreased cross talk with multitracking feature </li></ul>… applications
  29. 32. -Advantages- Why is Confocal Microscopy Better? 1. More Color Possibilities Because the images are detected by a computer rather than by eye, it is possible to detect more color differences.
  30. 33. Why is Confocal Microscopy Better? 2. Less Cross Talk I f the fluorochromes have distinct excitation spectra, the fluorochromes can be excited sequentially using one excitation wavelength at a time. This is only possible with confocal systems that offer the multitracking feature.
  31. 34. Why is Confocal Microscopy Better? 3. Optical Sectioning of Objects Without Physical Contact Zebra fish embryo wholemount Neurons (green) Cell adhesion molecule (red)
  32. 35. Why is Confocal Microscopy Better ? 4. Three-Dimensional Reconstruction of Specimen 3D shadow projection Tight junctions (red) Cytoskeletal structures (green)
  33. 36. Animated 3-Dimensional Reconstruction Laser Scanning Microscopy LSM510 3D for LSM www.Zeiss.com
  34. 37. Why is Confocal Microscopy Better? 5. Improved Resolution Rat Cerebellum
  35. 38. Disadvantages Disadvantages <ul><li>Limited primarily to the limited number of excitation wavelengths available with common lasers, which occur over very narrow bands and are expensive to produce in the ultraviolet region. </li></ul><ul><li>The harmful nature of high-intensity laser irradiation to living cells and tissues. </li></ul><ul><li>The high cost of purchasing and operating multi-user confocal microscope systems. </li></ul>
  36. 39. <ul><li>Advantages: </li></ul><ul><li>+ More clear </li></ul><ul><li>+ Increase in effective resolution. </li></ul><ul><li>+ Bold net examination of samples </li></ul><ul><li>+ 3.D Reviews </li></ul><ul><li>+ Depth measurement </li></ul><ul><li>+ Advanced Signals </li></ul><ul><li>+ Electronic zoom </li></ul><ul><li>Disadvantages: </li></ul><ul><li>- Image control complicated </li></ul><ul><li>- Low tissue penetration </li></ul><ul><li>- Fading </li></ul><ul><li>- High-intensity laser can damage tissue </li></ul><ul><li>- Expensive! </li></ul><ul><li>- Too many educated people need it !!!! </li></ul>Briefly…
  37. 40. THE END
  38. 41. References 1)Confocal ımaging animation.htm 2)Confocal laser scanning microscopy-wikipedia the free enclopedia.htm 3)Green flouresence protein.htm 4)Nikon microscopy ıntroduction to confocal microscopy basic concepts.htm 5) http://www.loci.wisc.edu/optical-sectioning/confocal-imaging 6) http://www.crp-sante.lu/en/project/1209?template=description#immuno 7) http://doube.org/3dnuclei.html#animation 8) F:chotwo01.html 9) Fluoview1000_high.wmv 10) http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/cf_tut_part1-4.htm 11) http://www.centrallab.metu.edu.tr /