This was a class presentation I made at BITS Pilani as part of an advanced course on Microbiology

1. Fluorescence Microscopy Aalap Tripathy, 2004P3PS208

2. Then… Now…

10. Zeiss Axio Imager Z1 Objective lenses: Filtersets: Camera: Software: ImageJ Imaris 4.2 Monchrome Phase contrast Oil Imaging-Workstation: Color

16. Cutaway diagram of a modern epi-fluorescence microscope

17. Working of the Fluorescence Microscope 1. Light source – epi-fluorescence lamphouse 2. Light of a specific wavelength (or defined band of wavelengths), is produced by passing multispectral light from an arc-discharge lamp through a wavelength selective excitation filter 3. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also termed a dichroic ) mirror or beamsplitter through the microscope objective to bathe the specimen with intense light

18. Working of the Fluorescence Microscope 4. If the specimen fluoresces, the emission light gathered by the objective passes back through the dichromatic mirror 5. It is Filtered by a barrier (or emission ) filter, which blocks the unwanted excitation wavelengths

20. The “cube”

21. Working in greater detail 1. Excitation light travels along the illuminator perpendicular to the optical axis of the microscope 2. The light then impinges upon the excitation filter where selection of the desired band and blockage of unwanted wavelength occurs.

22. 3. Fluorescence emission produced by the illuminated specimen is gathered by the objective 4. Because the emitted light consists of longer wavelengths than the excitation illumination, it is able to pass through the dichromatic mirror and upward to the observation tubes or electronic detector. Working in greater detail

26. Total Internal Reflection in Prism Same Principle used in Dichromatic beam splitter

27. Modern fluorescence microscopes are capable of accommodating between four and six fluorescence cubes. This is where the “turret’s” come into picture. The “cube” A specific combination of excitation filter, emission filter and dichroic mirror are needed

32. The radiation collides with the atoms in the specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. Principle of Fluorescence 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing.

34. Visualizing The Cytoskeleton using Fluorescence Microscopy An Example of Fluorescent Dyes

36. Let us test the effects of different drugs on the cytoskeleton and cell shape Nocodazole prevents microtubule polymerization. Nocodazole Taxol binds and stabilizes microtubules, Taxol Latrunculin prevents actin polymerization. Latrunculin TPA/PMA causes a dramatic rearrangement of actin filaments TPA/PMA

37. Visualizing the cytoskeleton using fluorescence microscopy 1) Prepare samples: Fixation - kills and immobilizes cells A. aldehydes - cross-link amino groups in proteins (formaldehyde, glutaraldehyde) B. alcohols - denature proteins, precipitate in place (methanol) Permeabilization - detergents make proteins accessible to staining reagents (Triton X100)

38. 2) Staining Actin - phalloidin covalently linked to rhodamine (red) - binds to filamentous actin only Microtubules - immunofluorescence 1 o ab: rabbit anti-tubulin; 2 o ab: fluorescein anti-rabbit

39. 3) Fluorescence microscopy excitation emission fluorescent molecule wavelength ex em intensity

40. Microtubules = green DNA = blue interphase mitosis

41. mitosis

42. Green Fluorescent Protein (GFP) An Example of tagging proteins

44. These transgenic mice express enhanced green fluorescent protein under the control of a chicken beta-actin promoter and cytomegalovirus enhancer

45. Why do this ?? developing transgenic mice to identify critical neuronal subpopulations and target them for electrophysiological recordings and biochemical analyses.

46. Some Pictures

48. Cotton A cross section of cotton stained with Rhodamine B. Mammalian Cells Fluorescence double-labeling of mammalian cells. The DNA in the cell nuclei are shown in blue. Cytoplasmic fiber structures (microfilaments) are shown in green. Photo: Petra Björk, Stockholm University

49. Researchers tag proteins with fluorophores to study the motion of these molecules. However, this creates an extremely complex motion picture (for example, in this image different colored particles move independently)

50. http://nobelprize.org/physics/educational/microscopes/fluorescence/fm.html Control of a fluorescence microscope

51. Figure 3: Problems with Fluorescence microscopy

54. Some Pictures

55. Parainfluenza

58. Influenza

63. Fluorescent antibody detection

66. Fluorescent Antibody Staining

72. Resources Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657. Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.