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Confocal Microcopy 1/11/2006 Interchromosomal Interactions Lecture 14: 1 Confocal Microcopy 1/11/2006 Resolution Number of Pixels Aspect Ratio 320x200 64,000 8:5 640x480 307,200 4:3 800x600 480,000 4:3 1024x768 786,43 4:3 1280x1024 1,310,720 5:4 1600x1200 1,920,000 4:3 Spatial Resolution The measure of how tightly the pixels in an image are packed is called the spatial resolution. Spatial resolution is measured in pixels per inch (ppi). However this is popularly refered to as dpi (dots per inch). For practical purposes the clarity of the image is decided by its spatial resolution (Actually all other factors are important) not the number of pixels in an image. The spatial resolution of computer monitors are generally 72dpi. Interchromosomal Interactions Lecture 14: 2 Confocal Microcopy 11/1/2006 The word laser is an acronym for light amplification by stimulated emission of radiation, although common usage today is to use the word as a noun -- laser -- rather than as an acronym -- LASER. A laser is a device that creates and amplifies a narrow, intense beam of coherent light. http://www.bell-labs.com/history/laser/laser_def.html. In 1917 Albert Einstein published an extraordinary piece of analysis which is generally accepted as the foundation of laser physics. This article, "Zur Quantentheorie der Strahlung" (On the Quantum Theory of Radiation), Physika Zeitschrift, Volume 18 (1917), pp 121-128, is also notable for first introducing the concept (but not the name) of the photon. In this article Einstein argues that in the interaction of matter and radiation there must be, in addition to the processes of absorption and spontaneous emission, a third process of stimulated emission. If stimulated emission exists then he can derive the Planck distribution for blackbody radiation and without it the same argument implies the empirically invalid Wien distribution. http://www.applet-magic.com/stimem.htm If a photon whose frequency corresponds to the energy difference between the excited and ground states strikes an excited atom, the atom is stimulated as it falls back to a lower energy state to emit a second photon of the same (or a proportional) frequency, in phase with and in the same direction as the bombarding photon. This process is called stimulated emission. The bombarding photon and the emitted photon may then each strike other excited atoms, stimulating further emission of photons, all of the same frequency and phase. This process produces a sudden burst of coherent radiation as all the atoms discharge in a rapid chain reaction. Interchromosomal Interactions Lecture 14: 3 Confocal Microcopy 11/1/2006 Laser: a narrow, intense beam of coherent light. http://www.fas.org/man/dod-101/navy/docs/laser/fundamentals.htm Interchromosomal interactions Lecture 14: 4 Confocal Microcopy 11/1/2006 Interchromosomal Interactions Lecture 14: 5 Confocal Microcopy 11/1/2006 And the atom A photon strikes emits a new an excited atom... photon just like the first one. one photon hits an excited atom and then we have two photons traveling together. When one of those finds another excited atom we get three photons, and so on and so on, but they are all exactly the same because they are being cloned by stimulated emission. http://www.colorado.edu/physics/2000/lasers/lasers2.html And the wave An takes the atom's electromagnetic extra energy to wave strikes an become a bigger excited atom... wave. Interchromosomal Interactions Lecture 14: 6 Confocal Microcopy 11/1/2006 BUT...in order to excite the atom we have to hit it with a photon to start with. So it takes two photons to get two photons. How is THAT ever going to get us extra photons? You're right. If we have to start with a photon of the correct color for each photon we eventually get we can't win. That was where things were in Einstein's day, and so they could not build lasers back then. Can you think of any way to get around this? Well, if we could get a whole bunch of the atoms excited without hitting them with photons, then just one photon would start a big chain reaction. Exactly. If we can get all the atoms to jump up into an excited state we can make a laser. We call this a Population Inversion. Well, it is not so easy, but we can do it by pumping electrical energy into our atoms in certain ways or shining different colored light at them. Both these processes stick the atoms into much higher energy levels, and under special conditions then they jump down and accumulate in the one excited energy level instead of going all the way to the ground state. Interchromosomal Interactions Lecture 14: 7 Confocal Microcopy 11/1/2006 we use mirrors to bounce the photons back and forth along one direction through the atoms. Resonance Cool! If the energy pump is high enough, eventually it builds up into a bigger and bigger wave or clump of photons. Wait...we have a leak. Some of the light is escaping to the right. That is so we can get the beam of laser light out! Interchromosomal Interactions Lecture 14: 8 Confocal Microcopy 11/1/2006 The laser diode is a light emitting diode with an optical cavity to amplify the light emitted from the energy band gap that exists in semiconductors. They can be tuned by varying the applied current, temperature or magnetic field. Gas lasers consist of a gas filled tube placed in the laser cavity. A voltage (the external pump source) is applied to the tube to excite the atoms in the gas to a population inversion. The light emitted from this type of laser is normally continuous wave (CW). One should note that if brewster angle windows are attached to the gas discharge tube, some laser radiation may be reflected out the side of the laser cavity. Large gas lasers known as gas dynamic lasers use a combustion chamber and supersonic nozzle for population inversion. Interchromosomal Interactions Lecture 14: 9 Confocal Microcopy 11/1/2006 Free electron lasers have the Dye lasers employ an active ability to generate wavelengths material in a liquid from the microwave to the X-ray suspension. The dye cell region. They operate by having an electron beam in an optical cavity contains the lasing medium. pass through a wiggler magnetic Many dyes or liquid field. The change in direction suspensions are toxic. exerted by the magnetic field on the electrons causes them to emit photons. Interchromosomal Interactions Lecture 14: 10 Confocal Microcopy 11/1/2006 Lasers used in the Confocal Microscope Lasers are used in confocal microscopes because they provide: 1) Single wavelength (very pure color) light and 2) very bright light. These usually non-pulsed gas lasers. 1. The argon ion laser which has two very strong lines at 488 nm and 514 nm. These are blue and blue-green wavelengths, respectively. The blue line at 488nm is nearly an ideal wavelength for exciting fluorescein and its derivatives. It also works well on some red-shifted forms of Green Fluorescent Protein (see GFP). Originally the 514nm line was used to excite rhodamine. This wavelength did excite rhodamine, but it was not useful for studies of double labeled specimens because this wavelength also excited fluorescein; sometimes even better than rhodamine. 2. A new mixed gas argon-krypton laser appeared to have the necessary line. This laser had strong lines at 488 nm (blue), 567 nm (yellow-green) and 647 nm (red) (an RYB laser). This laser had ideal wavelength characteristics for double-labeling experiments. The 567 nm line was far enough away from the excitation spectrum of fluorescein that the latter would not be excited, but excited rhodamine very well. Therefore fluorescein bleedthrough was no longer a problem. Furthermore, this laser had a red line far enough away from the rhodamine excitation spectrum so that a third fluorochrome such as allophycocyanin or Cy5 could be used and triple fluorescent probe experiments became possible. Bio_Rad confocal microscope uses this laser. Problems with the argon-krypton lasers: The smaller tube ArKr lasers began to fail after 100-200 hrs of use. All of the ArKr lasers begin to lose the red line at 647 nm after a short time. The life of all of the Ar-Kr laser tubes was also a problem. They did not last nearly as long as the argon ion lasers (MTBF: 2000-4000 hrs). Interchromosomal Interactions Lecture 14: 11 Confocal Microcopy 11/1/2006 3. One alternative was to use a small helium-neon laser. These could be manufactured to produce a line at either 543 nm or 633 nm. These were reasonable alternatives to the argon-krypton laser because while the lines produced were not exactly the same, the 543 nm green line and 633 red line were still in parts of the visible spectrum where there would not be overlapping of fluorochrome excitation and bleedthrough. Furthermore, helium-neon lasers were a proven technology. They last a long time ( up to 10,000 hrs or more) and have very low power consumption. 4. A fourth area of the electromagnetic spectrum of interest to confocal microscopists is the near UV range for excitation. Several useful biological fluorescent probes are excited in the near UV such as the DNA probes Hoescht 33258 and 33325 (bis-benzimide) and DAPI, and the calcium probes Indo-1 and Fura-2 and the antibody conjugate AMCA. These all emit a silvery white or light blue wavelength upon excitation. The laser used for this type of excitation is a much more powerful (up to five times) argon ion laser.
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