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Fluorescent Dyes for Optical Microscopy and Imaging

0707-4132-BC-ZE

Background

Photochemical process of ‘uncaging’ (transformation into the fluorescent state, Figure 1) entails an important toolbox in life sciences and biophysical chemistry. Initially invisible (‘caged’) dyes may be conjugated to a target, which upon illumination produces the corresponding fluorescent species that allows visualization of the spatial and temporal distribution of these dyes in the target. The chromophores available for such photoswitching often contain 2-nitrobenzyl groups and its derivatives, which upon photolysis produce highly reactive 2-nitrosobenzaldehyde, 2-nitrobenzophenone and their derivatives, all of which are known to be toxic to the target. Furthermore, photolysis produces compounds that are colored and interfere with optical measurements.

Figure 1. ‘Uncaging’ 1 using illumination from mercury arc lamp produces the fluorescent dye (Rhodamine Q) and 4,5-dimethoxy-2-nitrosobenzaldehyde (2).

Technology

The technology presents small ‘caging’ groups that are characterized by fast ‘uncaging’, high solubility in water and aqueous buffer, and exhibit high fluorescence quantum yields and photostability following ‘uncaging’. Low phototoxicity of the decomposition products upon ‘uncaging’ and no optical interference of these products make these dyes highly reliable for high resolution imaging of living cells.

Advantages

• Small ‘caging’ molecules: Compounds shown in Figure 2 have small functional groups) absorbing UV light. Compounds with general formula I contain 2-diazoketone fragments, while those of the type II contain 1,2,3-thiadiazole heterocycle, and III contains N-nitrosothioamide moiety. All these residues are rather small molecules and are photochemically active. Compounds I-III are solids and yellow in appearance, however, their solutions are non-fluorescent. ‘Uncaging’ generates colored and highly fluorescent derivatives of rhodamine and carbopyronine dyes. The chromophores (I-III) penetrate into living cells, and upon irradiation eliminate small molecules, such as nitrogen and nitrogen (I) oxide. ‘Uncaging’ doesn’t generate any toxic substance that many interfere with biochemical processes in cells or tissues.

Figure 2. Basic structure of the chromophores for high resolution imaging of living cells and tissues.

• Imaging subcellular structures: For imaging subcellular structure with the photoactive dyes PtK2 cells were incubated for 2 hours in a staining solution and were subsequently imaged using epiflourescence microscopy. Images of stained cells before and after photoactivation are presented in Figure 3.

Figure 3. Organic fluorophore in living and fixed eukaryotic cells. The cells are imaged before and after excitation using mercury lamp.

• Tracking of proteins and organelles: To determine whether the dynamics of proteins, organelles and other subcellular structures can be analyzed using the chromophores presented here, living mammalian PtK2 cells were stained. Illumination using circa 400 nm of light for 30 ms resulted in brightly fluorescent structures. These fluorescent structures are predominantly mitochondria and vesicles that are visible within the photoactive area. After, however, 330 ms of further irradiation, bright structures were visible in areas outside of the region of illumination. The fluorescent structures outside of the illuminated area are due to the motion of the fluorescent structures within the cells. These images demonstrate that the chromophores presented in this technology can be used for live cell tracking experiments (Figure 4).

Figure 4. Tracking of proteins and organelles with living cells. Prior to imaging, the mammalian PtK2 cells were incubated in the staining solution. No fluorescence signal was detected upon laser illumination at 561 nm (t=0). After 30 ms (t=30’’) of illumination with 405 nm laser light within the region of illumination (indicated by the dotted box), bright structures were visible. After 330 ms of further illumination (t=360’’), bright structures were visible within the photoactive areas. Panel B shows the close-up of t=363’’ to t=390’’. Movement of individual vesicles containing the photoactive dye is visible and can be traced within living cells.

Single molecule switching experiments: To successfully use a dye in single molecule switching experiments, it is essential that the dye can be switched between a dark and a bright state. Furthermore, to achieve high localization accuracy, it is necessary that a single molecule emits a large number of photons before being bleached or being switched off. The localization accuracy is proportional to the square root of the number of emitted photons. For single molecule switching experiments, the microtubule cytoskeleton in fixed mammalian cells was immunolabeled with the secondary antibodies bearing the residues of the chromophores. These cells were then imaged in a wide-field microscope using a 375 nm laser for ‘uncaging’. The measured localization of accuracy was circa 50 nm (Figure 5).

Figure 5. (A) Left side of a PtK2 mammalian cell is displayed as superposition of the intensity of 28000 frames acquired in this experiment, showing the resolution, which a conventional diffraction-limited image would achieve. (B) The right side of the same cell is displayed as histogram representation of the acquired data providing the diffraction-unlimited optical super-resolution.

Patent Information

PCT application filed with priority date 10.09.2009.

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