Molecular Fluorescence

    Eitam Vinegrad, Instructor of the molecular fluorescence experiment

 

http://www.tau.ac.il/~lab3/OPTICFIBERS/office.jpg Multi-disciplinary building, room 210.

http://www.tau.ac.il/~lab3/OPTICFIBERS/email2.jpg eitamvin@post.tau.ac.il   

http://www.tau.ac.il/~lab3/OPTICFIBERS/tel2.jpg 03-6408628

 

         The experiment is performed at Lab 3, Shenkar Physics building.

 

Contents

  1. Introduction
  2. Topics of the experiment
  3. How to prepare for the experiment?
  4. The experimental schedule
  5. Literature
  6. Links

Introduction

The excitation - absorption and then re-radiation - emission of light by organic and inorganic specimens is typically the result of physical phenomena called fluorescence or phosphorescence. It was first the British scientist Sir George G. Stokes who described and nicknamed fluorescence in 1852, after observing that the mineral fluorspar emitted red light when illuminated by ultraviolet excitation. Stokes noted that fluorescence emission always occurred at a longer wavelength than that of the excitation light.

 

Early investigations in the 19th century showed that many specimens (including minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, flesh, and inorganic compounds) fluoresce naturally, when irradiated with ultraviolet light. This effect is called autofluorescence, as no external fluorochromes (fluorophores) are used in the process. Analytical properties of fluorescence were also noted: the emitted fluorescent signal is proportional to the concentration of the fluorophores. For example, small changes in ion concentrations in living cells can have significant physiological effects. Whereas absorbance measurements can reliably determine concentrations only as low as several tenths of a micromolar, fluorescence techniques can accurately measure concentrations one million times smaller pico and even femtomolar. However, it was not until the 1930s that the use of fluorochromes was initiated in biological investigations to actively stain tissue components, bacteria, and other pathogens.

 

Autofluorescence has been thoroughly exploited in the fields of botany, petrology, and the semiconductor industry. However, the study of animal tissues and pathogens is often complicated with either extremely faint or bright, nonspecific autofluorescence. Of far greater value for many studies are added fluorochromes which are excited by specific wavelengths of irradiating light and emit light of defined and useful intensity. Fluorochromes are stains that attach themselves to visible or sub-visible structures within cells, be that proteins, or intracellular organelles, are often highly specific in their attachment targeting, and have a significant quantum yield (the ratio of photon absorption to emission). This allowed the researchers to act on the cells with chemical and biological compounds, and then monitor the way cells react.

 

With the advances in synthesizing new fluorophores for new applications, the methods evolved from applications in biophysical research to applications in many fields of industry (for example food production, lighting), in medicine (early detection of cancer, surgery) and other fields.

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Topics of the experiment

  1. Instrument Calibration. 
  2. Beer-Lambert absorption law with fluorescent molecules solvated in water. 
  3. The fluorescence spectrum of fluorescent molecules solvated in water.
  4. Fluorescence polarization of fluorescent molecules in a viscous solution. 
  5. FRET, non radiative energy transfer.

 

 

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How to prepare for the experiment?

  1. Contact the Instructor.
  2. Read the experiment's instructions.
  3. Read the Preparation file.
  4. Make sure you understand the experimental technique.
  5. Understand the terms (in the preparation file).
  6. Read the Lab's safety instructions.

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The experimental schedule

  1. Written exam - Approximately one hour.
  2. Equipment overview and calibration.
  3. Absorption and Beer-Lambert law.
  4. Fluorescence spectra of fluorophores in solvents.
  5. Fluorescence anisotropy.
  6. FRET

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Literature

  1. On molecules and transitions. 
  2. Transition probabilities.
  3. Introduction to fluorescence. 
  4. Detailed description of fluorescence. 
  5. Fluorescence polarization.
  6. Preparation file.
  7. Experiment's instructions.
  8. Equipment descriptions.

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Links

  1. How to write a report

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