Resolution Number of Pixels Aspect Ratio 320x200 64,000 8:5 640x480 307,200 4:3

1. Lecture 14: 1 1/11/2006 Interchromosomal Interactions Confocal Microcopy 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.

2. Lecture 14: 2 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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.

3. Lecture 14: 3 11/1/2006 Interchromosomal Interactions Confocal Microcopy Laser: a narrow, intense beam of coherent light. http://www.fas.org/man/dod-101/navy/docs/laser/fundamentals.htm

4. Lecture 14: 4 11/1/2006 Interchromosomal interactions Confocal Microcopy

5. Lecture 14: 5 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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

6. Lecture 14: 6 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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.

7. Lecture 14: 7 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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!

8. Lecture 14: 8 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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.

9. Lecture 14: 9 11/1/2006 Interchromosomal Interactions Confocal Microcopy Free electron lasers have the ability to generate wavelengths from the microwave to the X-ray region. They operate by having an electron beam in an optical cavity pass through a wiggler magnetic field. The change in direction exerted by the magnetic field on the electrons causes them to emit photons. Dye lasers employ an active material in a liquid suspension. The dye cell contains the lasing medium. Many dyes or liquid suspensions are toxic.

10. Lecture 14: 10 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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).

11. Lecture 14: 11 11/1/2006 Interchromosomal Interactions Confocal Microcopy 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. Besides the strong 488 and 514 nm lines, the argon laser also emits a weaker line at 367-368 nm. In the UV laser, the 488 and 514 nm lines are blocked and only the UV line is allowed to come through.

12. Lecture 14: 12 11/1/2006 Interchromosomal nteractions Confocal Microcopy Three itical elements of modern confocal microscopy While the image that is seen with confocal filtering is all in-focus information, this creates another problem. Compared to a normal fluorescence microscope, the amount of light that is seen in the final image is greatly reduced by the pinhole, sometimes up to 90-95%. To compensate for this loss of light somewhat, two components have been incorporated into modern confocal microscopes. First, lasers are used as light sources instead of the conventional mercury arc lamps because they produce extremely bright light at very specific wavelengths for fluorochrome excitation. Second, highly sensitive photomultiplier-detectors (PMTs) were employed as imaging devices to pick up the reduced signal. The signal for detection in the original design of modern confocal microscopes is created by scanning a focussed laser beam across a square or rectangular field. A system of motorized scanner mirrors sequentially scans a horizontal beam across the specimen. A third technology that is incorporated into the confocal microscope is the modern microcomputer. The computer is used to control the microscope's scanner mirrors and motorized focusing mechanism as well as collect, store, and analyze the data. Data is stored in the form of digital images which may be observed on a computer video monitor or sent to a hardcopy output device such as a film graphics recorder or a video or digital color printer. Digital or computer imaging is a much different technology than straight photographic imaging. The computer allows the system to scan sequential planes in the Z-direction, store them, and create overlays of all the in-focus Z sections. This information can also be used to create three dimensional images, or movie rotations of well stained specimens.

13. Lecture 14: 13 11/1/2006 Interchromosomal Interactions Confocal Microcopy Fluorophores http://www.mcb.arizona.edu/IPC/spectra_page.htm 4',6-Diamidino-2-phenylindole (DAPI) For fluorescence microscopy, DAPI is excited with ultraviolet light. When bound to double-stranded DNA its absorption maximum is at 358 nm and its emission maximum is at 461 nm. (This emission is fairly broad, and appears blue/cyan.) DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emission shifts to around 400 nm when bound to RNA. DAPI as a useful stain for nuclear quantitation. Biotech Histochem. 1991;66(6):297-302. Tarnowski BI, Spinale FG, Nicholson JH. The EMBO Journal (2003) 22, 3971–3982

14. Lecture 14: 14 11/1/2006 Interchromosomal Interactions Confocal Microcopy propidium iodide Other dyes to quantitatively stain DNA: UV excited: Hoechst 33342, Hoechst 33258 457nm excited: Mithramycin 488nm excited: Propidium Iodide, 7Aminoactinomycin D, SYTOX Green, DRAQ5 633nm excited: TO-PRO-3 Iodide http://www.olympusmicro.com/primer/techniques/fluorescence/gallery/cells/cos7/cos7cellslarge.html A popular traditional probe that is useful in confocal and fluorescence microscopy is the phenanthridine derivative, propidium iodide, first synthesized as an anti-trypanosomal agent along with the closely related ethidium bromide. Propidium iodide binds to DNA in a manner similar to the acridines (via intercalation) to produce orange-red fluorescence centered at 617 nanometers. The positively charged fluorophore also has a high affinity for double-stranded RNA. Propidium has an absorption maximum at 536 nanometers, and can be excited by the 488-nanometer or 514-nanometer spectral lines of an argon-ion (or krypton-argon) laser, or the 543-nanometer line from a green helium-neon laser. The dye is often employed as a counterstain to highlight cell nuclei during double or triple labeling of multiple intracellular structures. Environmental factors can affect the fluorescence spectrum of propidium, especially when the dye is used with mounting media containing glycerol. The structurally similar ethidium bromide, which also binds to DNA by intercalation, produces more background staining and is therefore not as effective as propidium.

15. Lecture 14: 15 11/1/2006 Interchromosomal Interactions Confocal Microcopy TOTO-3 a b c d Red: in vivo synthesis Green: in vitro sythesis Yellow: overlap of in vivo And in vitro Blue: f g e Figure shows that HeLa cells in S phase, but not other phases, continue to synthesize DNA both in situ and in vitro. S-phase cells were prelabelled in vivo with bromodeoxyuridine (BrdU, a marker of DNA synthesis; red), permeabilized and labelled in vitro for 1 h at 37 °C using Digoxygenin-11-dUTP (Dig−dUTP, green). DNA was stained using TOTO-3 and appears blue in the three-channel merged image (Fig. 1d). Figure 1c is a merged image of Fig. 1a, b and shows that those nuclei that were replicating their DNA in vivo before permeabilization (as revealed by BrdU staining, Fig. 1a) were the same as those that were labelled in vitro (with Dig−dUTP, Fig. 1b), whereas cells that were unlabelled in vivo remained unlabelled in vitro (blue nuclei, Fig. 1d). At a higher magnification, newly replicated DNA showed the punctate pattern typical of incorporation into replication foci4, 5, 6. Individual foci replicating in vitro (Fig. 1f) coincided with those that incorporated BrdU in vivo (Fig. 1e), giving yellow foci in the merged image (Fig. 1g, h). The observation that no purely green replication foci were present in the merged image (Fig. 1g, h) supports the idea that only DNA elongation is occurring, and not the initiation of DNA replication. Nature Cell Biology 2, 244 - 245 (2000)

16. Lecture 14: 16 11/1/2006 Interchromosomal Interactions Confocal Microcopy Fluorescein isothiocyanates (FITC) Reverse painting of GM13139 chromosomes with the biotin-labelled MC1 probe revealed with avidin-FITC. The chromosomes were counterstained with propidium iodide. Fluorescein isothiocyanate (FITC) is a fluorochrome with an absorption maximum at 495 nm. Its excitation by 488-nm light leads to a fluorescence emission maximum around 520 nm. Advantages * Relatively high absorptivity * Excellent fluorescence quantum yield * Good water solubility * Excitation/emission maxima ~494 nm closely matches the 488 nm spectral line of the argon-ion laser, making it the predominant fluorophore for confocal laser scanning microscopy and flow cytometry applications * Extensive use in the past makes them very well characterized fluorophores * Availability of large quantities * Low cost per mg Reactive derivatives Fluorescein isothiocyanates (FITC) are still the predominantly used fluorescein derivatives. • Limitations of Fluoresceins • Unfortunately, fluorescein-based dyes and their conjugates have several drawbacks, including: • * A relatively high rate of photobleaching • pH-sensitive fluorescence ref (pKa ~6.4) that is significantly reduced below pH 7 • * A relatively broad fluorescence emission spectrum, limiting their utility in some multicolor applications • * A tendency toward quenching of their fluorescence on conjugation to biopolymers, particularly at high degrees of substitution.

17. Lecture 14: 17 11/1/2006 Interchromosomal Interactions Confocal Microcopy Rhodamine 6G a. and b.: Mitoses in the root meristem of Allium. 3a, c and e: rhodamine - phalloidine staining (specific for actin); 3b, d and f: labelling of the microtubuli by indirect immunofluorescence (b. preprophase band, d and e microtubuli as components of the nuclear spindle, microtubuli in plant cells. bar: 10µm

18. Lecture 14: 18 11/1/2006 Interchromosomal Interactions Confocal Microcopy Cy5-EDA-ATP Cy3-EDA-ATP

19. Lecture 14: 19 11/1/2006 Interchromosomal Interactions Confocal Microcopy 2D DiGE of wild type (Cy3-labelled) and mutant (Cy5-labelled) Erwinia carotovora samples. The central panel shows an overlay of the two images. If equal amounts of a protein are present in both samples the spots will appear yellow; if the protein is only present in the wild-type the spot will appear green; if the protein is only present in the mutant it will appear red. For each spot a Cy3:Cy5 ratio can be assigned reflecting the differing amounts of a given protein within the two samples. Proteins of BR treated and untreated Arabidopsis were labeled with Cy3 and Cy5 dyes and separate by two-dimensional gel electrophoresis. Cy3 and Cy5 labeled DNA used for microarray hybridization

20. Lecture 14: 20 11/1/2006 Interchromosomal Interactions Confocal Microcopy Cy3 conjugates can be excited maximally at 550 nm, with peak emission at 570 nm. For fluorescence microscopy, they can be visualized with traditional tetramethyl rhodamine (TRITC) filter sets since the excitation and emission spectra (Figure 2) are nearly identical to those of TRITC. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). Cy3 has been used with fluorescein for double labeling. However, the use of a narrow band-pass emission filter for fluorescein is recommended to minimize Cy3 fluorescence in the FITC filter set. Cy3 can also be paired with Cy5 for multiple labeling when using a confocal microscope. Cy5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm (Table 1 and Figure 2). They can be excited to about 98% of maximum with a krypton/argon laser (647 nm line) or to about 63% of maximum with a helium/neon laser (633 nm line). Cy5 can be used with a variety of other fluorophores for multiple labeling due to a wide separation of its emission from that of shorter wavelength-emitting fluorophores. A significant advantage of using Cy5 over other fluorophores is the low autofluorescence of biological specimens in this region of the spectrum. However, because of its emission maximum at 670 nm, Cy5 cannot be seen well by eye, and it cannot be excited optimally with a mercury lamp. Therefore, it is not recommended for use with conventional epifluorescence microscopes. It is most commonly visualized with a confocal microscope equipped with an appropriate laser for excitation and a far-red detector.

21. Lecture 13: 21 10/25/2006 Interchromosomal Interactions

22. Lecture 14: 22 11/1/2006 Interchromosomal Interactions Confocal Microcopy Figure 2. DNA FISH Reveals the Colocalization of the H Element and OR Genes. Figure 2. DNA FISH Reveals the Colocalization of the H Element and OR Genes (A) Cells expressing M50 were identified with an M50-specific antibody directed against the C terminus of the receptor (blue). A digoxygenin-labeled (DiG) DNA probe was used to detect the M50 locus and was visualized with FITC-conjugated anti-DIG antibody (green). A biotin-labeled DNA probe was used to detect the H enhancer locus and was visualized with rhodamine-conjugated neutravidin (red). The arrowhead points to the colocalization of the H and M50 signals in the nucleus.

23. Lecture 14: 23 11/1/2006 Interchromosomal Interactions Confocal Microcopy Figure 3. RNA and DNA FISH on sensory neuron nuclei reveal colocalization of H element with the transcriptionally active OR Allele. (A) Combined RNA and DNA FISH on nuclei from olfactory epithelium. DIG-labeled oligo-nucleotides complementary to the RNA of the olfactory receptor M50 were used for the detection of the nuclear transcripts of M50. The signal was visualized with FITC conjugated anti-DIG antibody (green). A biotin-labeled DNA probe was used for the detection of the M50 locus and was visualized with rhodamine-conjugated neutravidin (red). The nuclei were counterstained with TOTO-3 (blue).

24. Lecture 14: 24 11/1/2006 Interchromosomal Interactions Confocal Microcopy Interchromosomal associations between alternatively expressed loci Charalampos G. Spilianakis1, Maria D. Lalioti2*, Terrence Town1*, Gap Ryol Lee1 & Richard A. Flavell1,3 The T-helper-cell 1 and 2 (TH1 and TH2) pathways, defined by cytokines interferon-g (IFN-g) and interleukin-4 (IL-4), respectively, comprise two alternative CD4þ T-cell fates, with functional consequences for the host immune system. These cytokine genes are encoded on different chromosomes. The recently described TH2 locus control region (LCR) coordinately regulates the TH2 cytokine genes by participating in a complex between the LCR and promoters of the cytokine genes Il4, Il5 and Il13. Although they are spread over 120 kilobases, these elements are closely juxtaposed in the nucleus in a poised chromatin conformation. In addition to these intrachromosomal interactions, we now describe interchromosomal interactions between the promoter region of the IFN-g gene on chromosome 10 and the regulatory regions of the TH2 cytokine locus on chromosome 11.