Fluorescence Microscopy

1. Fluorescence Microscopy G.A.M.E.S. 2008 Bioengineering Camp

2. Light travels through many important parts of the microscope. • Light of one wavelength comes from microscope light • Travels through excitation filter • Reflects off dichromic mirror (down) • Hits sample • Sample emits light (up) • Travels through dichromic mirror • Travels through emission filter • Is seen by eye • (page 6)

3. The fluorescence microscope diagram shows these light paths.

4. Actin is stained by rhodamine phalloidin. • Reminder: rhodamine phalloidin is two different molecules that work together. • Rhodamine – fluorescence • Phalloidin – attaches to actin • Green light makes rhodamine molecules move around; they release red light at a lower energy (longer wavelength) • What you can expect to see • Black background with red stress fibers visible • No green

5. Photobleaching makes the fluorescence fade. • If any fluorescent sample is left exposed too long to light, the fluorescence will fade. • Protect sample from light after adding rhodamine phalloidin – cover with aluminum box. • Results will not be as good after photobleaching.

6. GFP makes transfected cells glow green. • Reminder: GFP is made by cells that have taken up the new DNA. • Just like rhodamine, the GFP molecules move around when specific light is used. • Shine on blue, glow green.

7. Multi-colored fluorescence images are hard to make. • Must change the color of light from the microscope • Must layer each picture – no single picture looks like this --> • Blue is the nucleus • Red is actin • Green is golgi • Pink is mitochondria

8. Emitted light is always a different color than the original one. GFP (Transfection) Actin Staining Purple Green Orange Blue Yellow Red