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Department of Molecular Physiology

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fluo-microscopy
Introduction

- Observing fluorescence
 
If a solution contains a fluorescent substance (a fluorochrome) it emits fluorescence when illuminated with light of appropriate colour (wavelength) - the excitation light. Fluorescence is best observed at a right angle to the axis of the excitation light because this avoids looking into the excitation lamp. In a fluorescence spectrometer, a light detector(PMT = photomultiplier tube) is ususally mounted at a right angle to the excitation light path. Excitation light and fluorescence can also be separated using optical filters.

- What is fluorescence?



The processes that lead from the absorption of excitation light by a fluorochrome to the emission of fluorescence can be illustrated by energy diagrams named after the polish physicist Alexander Jablonski. A Jablonski diagram depicts the various electronic energy levels of a fluorochrome molecule. Many fluorochromes have aromatic ring structures. Such molecules possess delocalized electrons in so-called pi-orbitals. Electrons in pi orbitals interact readily with the environment. Through absorption of a photon such an electron can be boosted into a higher orbital. Electrons in pi orbitals usually have anti-parallel spin, characterizing the singlet states S0, S1, and S2. In the dark, electrons sit in the resting state S0. When absorbing a photon of the right wavelength, the electrons reach the excited states S1 or S2. This is a very fast process, developing within 10-15 s. Excited electrons may hop from S2 to S1 without emitting anything (internal conversion). However, when returning to the ground state S0 (typically after a few nanoseconds), the energy difference is released as a fluorescence photon. The energy of the fluorescence photon is always lower than the energy of the absorbed excitation photon. Therefore, the wavelength of fluorescence light is always longer than that of the excitation light (Stokes shift). In some compounds electrons can reach a triplet state (T1) when jumping back from an excited singlet state. This process (intercombination) requires a spin reversalof the excited electron, and also the jump back to the ground state requires spin reversal. Such a process occurs with very low probability, and emission rates are very low (1 - 1000 per second). Compounds showing this property are phosphorescent. The low rates of interconversion and emission in these substances cause a slow decline of light emission, causing a persistent "glow" in the dark.



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