Microscopy

1. Microscopy

2. Visible light is a radiation visible to human eye. It is a spectrum of wavesin a range between 380 nm to 740 nm • Fluorophores, fluorescent dyes (GFP) are chemicals. They can absorb light of a specific wave length

3. Example : Quanine(svKinin)– a chemical that gives a tonic characteristic bitter taste Kininabsorbslight ~300-350 nm long.During this absorption, electrons in a a chemical gets excited. That means that their energy increases.After a moment, these electrons go back to a stationary state (“relax”) and they “give back” the energy in a form of a visible light. This light is weaker than exciting light but also has a different length (emission spectra).For Kinin, is about 450 nm

4. Another Example : Cy3 Cy3 is chemical that is connectedto our detection oligonucleotides.Cy3absorbs light 550nm, emits 570 nm Cy5 649 nm and 670nm. In fluorescent microscopy, we exploit this property do “excite” Cy3 dyes that bind to our DNA balls, and we can see the light they emit as a single, bright spot.

5. Microscope has a set of filters, that let a very specific wave to go through it and hit the sample.During this process, dyes are exited and start to emit light.The filter is also designed to let this specific light go through itas well, this light reaches your eye or a camera

7. The basic task of the fluorescence microscope: • Illuminate the specimen with excitationlight • Separate the much weaker emission light from the brighter excitation light. • Only allow the emission light to reach the eye or other detector. • The background is dark, the fluorescent objects are bright