Methods, Part 2

1. Methods, Part 2 February 9, 2012

2. Learning Outcomes • Discriminate between different types of microscopy, and justify their use for answering research questions. • Differentiate between conventional and confocal fluorescence microscopy. • Describe in writing how genes from different organisms can be modified, inserted into, and expressed in cells.

3. Microscopy • Resolution: The minimum distance between two objects that can be detected • Defined by The Abbe Equation: distance = _0.612 * λ_    NA • Visible light= 380-760 nm • Best resolution of visible light microscope: 200 nm • Electron microscopes use electron beam, wavelength 100,000 X shorter

4. Microscopy • Contrast: The ability to interfere with the illumination source • Bright field microscopy uses dyes (“stains”) to generate contrast • H&E is popular dye for medical diagnostics • Sample must be dead

5. Microscopy • Contrast: The ability to interfere with the illumination source • Phase contrast microscopy uses differences in diffraction to generate contrast • Image has grey background, black/white contrast • Colored filters can increase contrast a bit • No dyes used, cells can be alive when viewed • Movies of cells! Neuron in cell culture

6. Fluorescence Microscopy • By far the most frequently used microscopy technique in cell biology research • Contrast generated by fluorescent (visible) light emitted by target; background is black • Excitation wavelength is always shorter then emission wavelength • A “dichroic mirror” blocks excitation light, allows only emission light to reach observer • Can be used to visualize specific molecules

7. Fundamentals of Fluorescence Microscopy • A nice summary of fluorescence microscopy • An explanation of confocal fluorescence microscopy

9. Basics of recombinant DNA technology • Fundamental concept: Because DNA is structurally identical in all organisms, it is possible to combine DNA sequences from different organisms, and insert the combined DNA into any organism. • Isolating DNA from cells is easy. • Cutting DNA into pieces, according to DNA sequence, is easy. • Pasting the pieces together is easy, using DNA ligase. • Putting the hybrid DNA into cells (formerly called transfection) is easy but expensive.

10. Basics of recombinant DNA technology • Most often, genomic DNA is not combined together; instead, a DNA copy of a single gene, called complementary DNA (cDNA) is used instead. • cDNA is derived from mRNA, so the introns have already been removed from the gene, and the DNA is thus smaller and easier to use. • cDNA is frequently combined with a small circular piece of DNA, called a plasmid, before inserting it into a cell. The plasmid contains DNA sequences that help control when and where the cDNA gene will be expressed.

11. Basics of recombinant DNA technology • It is becoming quite common to build hybrid genes, which contain coding sequences for two entirely different proteins. The product of these genes is called a fusion protein. • One of the most popular classes of fusion proteins is genes fused to the gene that encodes Green Fluorescence Protein (aka GFP) or its derivatives. • GFP fusion proteins have revolutionized cell biology: you can use fluorescence microscopy to track specific proteins in living cells/tissues/organs/animals. • Those who discovered this gene and developed it for research were awarded the Nobel Prize in 2008.

12. All Hail, GFP

13. All Hail, GFP

14. All Hail, GFP

15. Fluorescent Histone Proteins in Living Cells

16. Sources • http://www.med.unc.edu/microscopy/services/light-microscopy • http://www.anatomy.wisc.edu/Dent/index_files/Page739.htm • http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Introduction-to-Fluorescence-Techniques.html • http://brainwindows.wordpress.com/category/gfp/ • http://www.olympusconfocal.com/applications/fpcolorpalette.html