Biomod/2014/Fluorescence: Difference between revisions

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<h1> Team Nanoscooter Braunschweig </h1>

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</td><td> Fluorescence Electrons are known to have discrete levels of energy. Without any stimulation the electrons are in the so called ground state. If the electron is excited by irradiation of visible light, the electron absorbs energy to reach higher – so called excited states – levels. These energy package can be described as photons of different wavelengths. The electron can also relax back into the ground state while emitting a photon, this process is known as emission. The energy of a photon is described by the Planck´s law. [1] <table><colgroup>

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<tr><td><center>E = (h ∙ c)/λ </center></td><td><right>(1.1)</right></td></tr></table> This equation uses c and h which are the constants for speed of light in vacuum and the Planck constant. Further λ is used as wavelength. The spontaneous light emission is known as photoluminescence which could be subdivided into fluorescence and phosphorescence. Both terms are illustrated by the Jablonski diagram (see below). Without any photon absorption the electron is in the ground state S0, after absorption of energy the electron gets excited to the first singlet-state S1 (light-blue). Every state is subdivided into different vibration-states, which could be reached by the electron. The internal conversion describes radiationless crossing (dashed grey) from a higher to a lower vibration-state. The spontaneous emission from S1 to S0 is known as the fluorescence (violet). An electron could although relax from the singlet-state S1 to the triplet-state with a spin change from antiparallel to parallel, this is called intersystem crossing (dashed red). This radiationless crossing isn´t allowed by the selection rules, but occurs because of huge overlapping between S1 and T1. The phosphorescence describes emission under spin change from T1 to S0 (orange).[2] <div align="center"><img src="http://openwetware.org/images/thumb/8/83/Jablonski.png/800px-Jablonski.png" width="" height="" > </div> <div align="center">Figure 1: Jablonski-Diagramm with radiative crossings like absorption (light-blue), fluorescence (violet) and phosphorescence (orange) and radiationless crossing like internal conversion (dashed grey) and intersystem crossing (dashed red). </div> Figure 2 shows excitation (green) and emission spectrum (dashed green) of the dye Atto 532. <div align="center"><img src="http://openwetware.org/images/thumb/6/62/Atto_532.png/800px-Atto_532.png" width="" height="" > </div> <div align="center">Figure 2: Excitation (green) and emission spectrum (dashed green) of the dye Atto 532.[3] </div> The emission spectrum is the mirror image of the excitation spectrum. This could be explained by the Stokes-Shift. The Stoke-Shift depends on to different effects the variation of the vibronal states and the solvent relaxation. The first effect describes that an excitation not only changes the ground state to an excitation state but also changes the vibronal state. To determined fluorescence it is necessary that the lowest vibronal state of the singlet-state is reached. The internal conversion makes this possible. The lost energy results in a higher wavelength. The reorganization of a polar solvate in a polar solvent after excitation is described by the solvent relaxation. The solvent is aligned by the dipole moment μ of dissolved dyes. After an excitation the dipole moment could stabilize (μ ≤ μ*) or destabilize (μ > μ*) the dyes which results in a higher or lower energy and although in a lower or higher wavelength.[4] To observe the movement of the Nanoscooter on mica surfaces, we used a Leica GSDIM [5] (Ground State Depletion followed by Individual Molecule Return) microscope with 488 nm and 640 nm laser wavelength and CCD camera as a widefield microscope. <div align="center"><img src="http://openwetware.org/images/a/a4/Weitfeld.png" width="" height="" > </div> <div align="center">Figure 3: Schematic of a widefield microscope. The sample is irradiated with laser light, while the excitation- and the fluorescence-light are separated by a dichroic beamsplitter. A CCD camera is used for detection.[3] </div> <hr style="background-color:#be1e3c;"> <table><tr><td> [1] </td> <td>M. Planck: Über irreversible Strahlungsvorgänge, Sitzungsberichte der Königlich Preußischen Akademie der Wissenschaften zu Berlin, 1899, Erster Halbband (Berlin: Verl. d. Kgl. Akad. d. Wiss., 1899), 479-480.</td></tr> <tr><td> [2]</td> <td>A. Jabłoński: Efficiency of anti-Stokes fluorescence in dyes, Nature, 1933, 131, 839-840.</td></tr> <tr><td align="center" valign="top"> [3] </td> <td>Fluorophores.org – Database of Fluorescent Dyes, Properties and Applications, http://www.fluorophores.tugraz.at/substance/419, final request: 20.10.14. </td></tr> <tr><td align="center" valign="top"> [4]</td> <td>J. R. Lakowicz: Principles of Fluorescene Spectroscopy, Nature, 2006, Springer, fourth edition. </td></tr> <tr><td align="center" valign="top"> [5] </td> <td>Leica Mikrosysteme, http://www.leica microsystems.com/de/produkte/lichtmikroskope/biowissenschaften/fluoreszenzmikroskope/details/product/leica-sr-gsd/, final request: 20.10.14. </td></tr> </td></tr></table> </td> <td> </td>

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+</td><td>
+<br><br><font size="+2pt"><u>Fluorescence</u></font> <br><br>
+<p align="justify"; style="line-height:2em">
+<font size="3pt">Electrons are known to have discrete levels of energy. Without any stimulation the electrons are in the so called ground state. If the electron is excited by irradiation of visible light, the electron absorbs energy to reach higher – so called excited states – levels. These energy package can be described as photons of different wavelengths. The electron can also relax back into the ground state while emitting a photon, this process is known as emission. The energy of a photon is described by the Planck´s law. <sup>[1]</sup> <br><br>
+<table><colgroup>
+    <col width="600px">
+    <col width="200px">
+  </colgroup>
+<tr><td><p align="justify"; style="line-height:2em"><font size="3pt"><center>E =   (h ∙ c)/λ </center></font></p></td><td><p align="justify"; style="line-height:2em"><font size="3pt"><right>(1.1)</right></font></p></td></tr></table><br><br>
+<p align="justify"; style="line-height:2em">This equation uses c and h which are the constants for speed of light in vacuum and the Planck constant. Further λ is used as wavelength.<br><br>
+The spontaneous light emission is known as photoluminescence which could be subdivided into fluorescence and phosphorescence. Both terms are illustrated by the Jablonski diagram (see below). Without any photon absorption the electron is in the ground state S<sub>0</sub>, after absorption of energy the electron gets excited to the first singlet-state S<sub>1</sub> (light-blue). Every state is subdivided into different vibration-states, which could be reached by the electron. The internal conversion describes radiationless crossing (dashed grey) from a higher to a lower vibration-state. The spontaneous emission from S<sub>1</sub> to S<sub>0</sub> is known as the fluorescence (violet). An electron could although relax from the singlet-state S<sub>1</sub> to the triplet-state with a spin change from antiparallel to parallel, this is called intersystem crossing (dashed red). This radiationless crossing isn´t allowed by the selection rules, but occurs because of huge overlapping between S<sub>1</sub> and T<sub>1</sub>. The phosphorescence describes emission under spin change from T<sub>1</sub> to S<sub>0</sub> (orange).<sup>[2]</sup></font></p><br><br>
+<div align="center"><img src="http://openwetware.org/images/thumb/8/83/Jablonski.png/800px-Jablonski.png" width="" height="" ><br></div><br><i><font size="3 "><div align="center">Figure 1: Jablonski-Diagramm with radiative crossings like absorption (light-blue), fluorescence (violet) and  phosphorescence (orange) and radiationless crossing like internal conversion (dashed grey) and intersystem crossing (dashed red). </font></i></div><br>
+<p align="justify"; style="line-height:2em">
+<font size="3pt">Figure 2 shows excitation (green) and emission spectrum (dashed green) of the dye Atto 532.</font></p><br>
+<div align="center"><img src="http://openwetware.org/images/thumb/6/62/Atto_532.png/800px-Atto_532.png" width="" height="" ><br></div><br><i><font size="3 "><div align="center">Figure 2: Excitation (green) and emission spectrum (dashed green) of the dye Atto 532.<sup>[3]</sup> </font></i></div><br>
+<p align="justify"; style="line-height:2em">
+<font size="3pt">The emission spectrum is the mirror image of the excitation spectrum. This could be explained by the Stokes-Shift. The Stoke-Shift depends on to different effects the variation of the vibronal states and the solvent relaxation.
+The first effect describes that an excitation not only changes the ground state to an excitation state but also changes the vibronal state. To determined fluorescence it is necessary that the lowest vibronal state of the singlet-state is reached. The internal conversion makes this possible. The lost energy results in a higher wavelength.
+The reorganization of a polar solvate in a polar solvent after excitation is described by the solvent relaxation. The solvent is aligned by the dipole moment μ of dissolved dyes. After an excitation the dipole moment could stabilize (μ ≤ μ*) or destabilize (μ > μ*) the dyes which results in a higher or lower energy and although in a lower or higher wavelength.<sup>[4]</sup> <br><br>
+To observe the movement of the Nanoscooter on mica surfaces, we used a Leica GSDIM <sup>[5]</sup> <i>(Ground State Depletion followed by Individual Molecule Return)</i> microscope with 488 nm and 640 nm laser wavelength and CCD camera as a widefield microscope.
+</font></p><br><br>
+<div align="center"><img src="http://openwetware.org/images/a/a4/Weitfeld.png" width="" height="" ><br></div><br><i><font size="3 "><div align="center">Figure 3: Schematic of a widefield microscope. The sample is irradiated with laser light, while the excitation- and the fluorescence-light are separated by a dichroic beamsplitter. A CCD camera is used for detection.<sup>[3]</sup> </font></i></div><br>
+<br><br><hr style="background-color:#be1e3c;">
+<table><tr><td><font size="3pt"><p align="justify">
+[1]<br><br></font></td> <td><font size="3pt">M. Planck: <i>Über irreversible Strahlungsvorgänge</i>, Sitzungsberichte der Königlich Preußischen Akademie der Wissenschaften zu Berlin,<b> 1899</b>, <i>Erster Halbband (Berlin: Verl. d. Kgl. Akad. d. Wiss., 1899)</i>, 479-480.</font></td></tr>
+<tr><td><font size="3pt"><p align="justify">
+[2]</font></td> <td><font size="3pt">A. Jabłoński: <i>Efficiency of anti-Stokes fluorescence in dyes</i>, Nature,<b> 1933</b>, <i>131</i>, 839-840.</font></td></tr>
+<tr><td align="center" valign="top"><font size="3pt">
+[3]<br></font></td> <td><font size="3pt"><i>Fluorophores.org – Database of Fluorescent Dyes, Properties and Applications</i>, http://www.fluorophores.tugraz.at/substance/419, final request: 20.10.14.<br></font></td></tr>
+<tr><td align="center" valign="top"><font size="3pt">
+[4]</font></td> <td><font size="3pt">J. R. Lakowicz:  <i>Principles of Fluorescene Spectroscopy</i>, Nature,<b> 2006</b>, <i>Springer</i>, fourth edition. </font></td></tr>
+</font>
+<tr><td align="center" valign="top"><font size="3pt">
+[5]<br></font></td> <td><font size="3pt"><i>Leica Mikrosysteme</i>, http://www.leica microsystems.com/de/produkte/lichtmikroskope/biowissenschaften/fluoreszenzmikroskope/details/product/leica-sr-gsd/, final request: 20.10.14.<br></font></td></tr>
+</font><br></td></tr></table>
+</td>
+<td>
+</td>
+</tr>
+</table>
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Biomod/2014/Fluorescence: Difference between revisions

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