Biomod/2014/Fluorescence

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Fluorescence

Molecules are known to have discrete levels of energy. Without any stimulation the molecules are in the so called ground state. If it is excited by irradiation of visible light, the molecule absorbs energy to reach higher – so called excited states – levels. These energy packages can be described as photons of different wavelengths. The molecule 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]

E = (h ∙ c)/λ

(1.1)



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 molecule is in the ground state S0, after absorption of energy the molecule gets excited to the first singlet-state S1 (light-blue). Every state is subdivided into different vibration-states, which could be reached by the molecule. The internal conversion describes radiationless transition (dashed grey) from a higher to a lower vibration-state. The spontaneous emission from S1 to S0 is known as the fluorescence (violet). A molecule could also 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 transition 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]





Figure 1: Jablonski-Diagramm with radiative transitions like absorption (light-blue), fluorescence (violet) and phosphorescence (orange) and radiationless transitions like internal conversion (dashed grey) and intersystem crossing (dashed red).

Figure 2 shows excitation (green) and emission spectrum (dashed green) of the dye Atto 532.




Figure 2: Excitation (green) and emission spectrum (dashed green) of the dye Atto 532.[3]

The emission spectrum is the mirror image of the excitation spectrum. This could be explained by the Stokes-Shift. The Stoke-Shift depends on two 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 determine 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 also 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.





Figure 3: Schematic of a widefield microscope. The sample is irradiated with laser light, while the excitation and the fluorescence are separated by a dichroic beamsplitter. A CCD camera is used for detection.[3]



Labeling and purification

As mica is not the ideal template for fluorescence microscopy, the Nanoscooter should be labeled as bright and stable as possible. Therefore, StreptAvidin coated yellow green fluorescent beads (40 nm diameter) were used. By incorporating of biotin bindings into the Nanoscooter the DNA origami can be labeled with the bead since StreptAvidin specifically binds to the biotin and thus, forms a very strong non-covalent interaction.[6]



Figure 4: Tetrameric structure of StreptAvidin with 2 bound biotins.[7]


To ensure that the fluorescent beads are bound to the Nanoscooter and are not only free fluorescent beads (which would look exactly the same on a fluorescence microscope), the Nanoscooter was labeled with red fluorophores (Atto647N) by so called external labeling.[8]

For this purpose, the Nanoscooter was folded and purified using the previously determined best folding conditions. Then, the anchor strands for Pt-nanoparticle binding were used to bind the complementary DNA strand labeled with the red dye. This could be easily done by incubating the purified Nanoscooter with 1 µL of the dye labeled counter strand (100 µM stock solution) in the standard folding buffer for 2 hours at 37 °C. With this method, we can achieve up to 12 red fluorescence labels per Nanoscooter. After purification by amicon ultrafiltration to remove the excess of dye labeled DNA strands, the Nanoscooter was incubated overnight at room temperature with the fluorescent beads as described by Wind et al.[9] (dilution 1:20000 from the 0.5% solids stock solution).

For the list of the master mixtures used and staple sequences see here.



Figure 5: Schematic illustration of the Nanoscooter colabeled with a fluorescent bead and red fluorophores.

Further purification proofed difficult since the fluorescent beads have the similar size to the DNA origami hence the standard filtering does not work. Moreover, the attempt to purify via gel electrophoresis failed (as illustrated in Figure 3): A very blurred fluorescent signal was received with UV illumination and therefore the DNA origami could not be extracted.





Figure 6: Gel electrophoresis of the DNA origami labeled with a fluorescent bead. The 1st lane shows the fluorescent beads only, 2nd-4th lane show Nanoscooter labeled with fluorescent beads and the 5th lane shows the scaffold p8064 as reference.



Sample preparations

First, the samples were prepared on standard microscope coverslips (#1.5, 0.17 mm thick) to check for correct fluorescence labeling. For this, the glass surface was coated with poly-L-lysin (PLL) (1:100 diluted with PBS) which enables electrostatic binding of the negatively charged DNA as it creates a positively charged surface.

Afterwards, the samples were prepared on the mica sheets as described in AFM sample preparations. Larger and thinner mica sheets had to be used for the fluorescence experiments since optical effects like reflection and aberration should be avoided as much as possible, so the mica sheets could not be effectively cleaved.





Fluorescence experiments

Since the sample could not easily be purified, the yield of colabeled spots was determined by fluorescence microscopy. Colocalization of red fluorophores and yellow green fluorescent beads was expected for successfully colabeled DNA origamis. First, the colocalization was measured for a simple rectangular DNA origami because this structure is well known and the colocalization was more likely to be observed. The fluorescence image is shown in Figure 7.





Figure 7: Fluorescence microscopy of colabeled rectangular DNA origamis (the colocated spots are framed by white circles).

It is obvious that the labeling worked out for the rectangular DNA origami, so the Nanoscooter was labeled using the same conditions, whereby the observed fluorescence image is shown in Figure 8.





Figure 8: Fluorescence microscopy of the colabeled Nanoscooter.

Unfortunately, the colocalization could not be observed for the Nanoscooter. We are positive that the red spots correspond to labeled Nanoscooter since the brightness and photophysical behavior was as expected. But because of the lack of colocalization, we conclude that the fluorescent beads have not bound to the Nanoscooter. A likely reason for this observation could be steric hindrance: As the biotins are incorporated using quite short linkers, they might not be accessible to the StreptAvidin coated fluorescent bead. By using a longer linker between the DNA origami construct and the biotin this problem could be solved in future applications.

Analogously to the AFM experiments, the floating of the rectangular DNA origami on mica should be observed by fluorescence microscopy after adding NaCl to the measurement buffer. We first immobilized the DNA origamis on the mica surface using MgCl2 as before. The resulting fluorescence image is shown in Figure 9.





Figure 9: Fluorescence microscopy image of yellow green fluorescent beads through a mica sheet.

Since mica sheets probably have a very heterogeneous structure, the resulting fluorescence image shows optical aberrations [10] (Figure 9) which might be caused by birefringence.[11] Unfortunately, this made fluorescence experiments on the applied inverse microscope impossible.

These problems could be solved by using a water dipping objective on an upright microscope: This way, the excitation and emission light do not pass the mica but are collected from above through the aqueous buffer solution.

All in all, the labeling of DNA origami structures with yellow green fluorescent beads was successful. Through a small modification the labeling with fluorescent beads should also work out for the Nanoscooter. Also our issues with the fluorescence experiments can easily be solved using a different kind of microscope. Even though we could not yet show the real time movement of our Nanoscooter on a fluorescence microscope, we can conclude that our approach was successful as the single components of the system work and our small issues should be easily solved.




[1]

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.

[2]

A. Jabłoński: Efficiency of anti-Stokes fluorescence in dyes, Nature, 1933, 131, 839-840.

[3]

Fluorophores.org – Database of Fluorescent Dyes, Properties and Applications, http://www.fluorophores.tugraz.at/substance/419, final request: 20.10.14.

[4]

J. R. Lakowicz: Principles of Fluorescene Spectroscopy, Nature, 2006, fourth edition.

[5]

Leica Mikrosysteme, http://www.leica microsystems.com/de/produkte/lichtmikroskope/biowissenschaften/fluoreszenzmikroskope/details/product/leica-sr-gsd/, final request: 20.10.14.

[6]

N. M. Green: Avidin, Adv. Protein Chem., 1975, 29, 85-113.

[7]

P. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, F. R. Salemme: Structural origins of high-affinity biotin binding to streptavidin, Adv. Science, 1989, 243, 85-113.

[8]

J. J. Schmied, M. Raab, C. Forthmann, E. Pibiri, B. Wünsch, T. Dammeyer and P. Tinnefeld: DNA origami–based standards for quantitative fluorescence microscopy, Nature protocols,2014, 243, 85-88.

[9]

R. Wang, C. Nuckolls, S. J. Wind: Assembly of Heterogeneous Functional Nanomaterials on DNA Origami Scaffolds, Angew. Chem. Int. Ed.,2012, 51, 1-4.

[10]

G. Elert - The Physics Hypertextbook, http://www.http://physics.info/, final request: 24.10.14.

[11]

Olympus Microscopy Resource Center, Olympus America Inc., http://www.http://physics.info/, final request: 24.10.14.

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