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Light sheet microscopy for single molecule tracking in living tissue.

Ritter JG, Veith R, Veenendaal A, Siebrasse JP, Kubitscheck U - PLoS ONE (2010)

Bottom Line: By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample.Thereby we could directly observe and track small and large tracer molecules in aqueous solution.Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms Universität, Bonn, Germany.

ABSTRACT
Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 microm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

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Single molecule visualization and tracking in solution.(a) Standard epi-illumination fluorescence image of single ATTO647-labelled dextran molecules (MW 500 kDa) diffusing in transport buffer acquired at an exposure time of 10 ms. Scale bar, 5 µm (b) The same sample was imaged upon light sheet illumination demonstrating the striking contrast improvement (see Movie S1). Scale bar, 5 µm. (c) Intensity profile along a vertical line through the central pixels of the two brightest molecules shown in (a) and (b) illustrating the contrast improvement. (d) MSDs as a function of time for different molecules; dextran 500 kDa (boxes), ovalbumin (circles), and a 30mer oligonucleotide (triangles). Movies were recorded for all three species with 483 Hz. The data were fitted by lines to determine the diffusion coefficients. The error bars represent the standard deviation. The results were summarized in Table 2. (see DiscussionS1).
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pone-0011639-g003: Single molecule visualization and tracking in solution.(a) Standard epi-illumination fluorescence image of single ATTO647-labelled dextran molecules (MW 500 kDa) diffusing in transport buffer acquired at an exposure time of 10 ms. Scale bar, 5 µm (b) The same sample was imaged upon light sheet illumination demonstrating the striking contrast improvement (see Movie S1). Scale bar, 5 µm. (c) Intensity profile along a vertical line through the central pixels of the two brightest molecules shown in (a) and (b) illustrating the contrast improvement. (d) MSDs as a function of time for different molecules; dextran 500 kDa (boxes), ovalbumin (circles), and a 30mer oligonucleotide (triangles). Movies were recorded for all three species with 483 Hz. The data were fitted by lines to determine the diffusion coefficients. The error bars represent the standard deviation. The results were summarized in Table 2. (see DiscussionS1).

Mentions: For a quantitative comparison between LSFM and standard epi-illumination we compared the image contrast in image sequences of single, fluorescently labeled 500 kDa dextran molecules recorded at almost 100 Hz (Fig. 3 and Movie S1). As expected the contrast was clearly superior (0.97) when the light sheet illumination was used versus epi-illumination (0.37) for the given tracer concentration (Materials and MethodsS1). The signal-to-noise-ratio (SNR) increased by a factor of 4 yielding a significantly improved localization precision [25].


Light sheet microscopy for single molecule tracking in living tissue.

Ritter JG, Veith R, Veenendaal A, Siebrasse JP, Kubitscheck U - PLoS ONE (2010)

Single molecule visualization and tracking in solution.(a) Standard epi-illumination fluorescence image of single ATTO647-labelled dextran molecules (MW 500 kDa) diffusing in transport buffer acquired at an exposure time of 10 ms. Scale bar, 5 µm (b) The same sample was imaged upon light sheet illumination demonstrating the striking contrast improvement (see Movie S1). Scale bar, 5 µm. (c) Intensity profile along a vertical line through the central pixels of the two brightest molecules shown in (a) and (b) illustrating the contrast improvement. (d) MSDs as a function of time for different molecules; dextran 500 kDa (boxes), ovalbumin (circles), and a 30mer oligonucleotide (triangles). Movies were recorded for all three species with 483 Hz. The data were fitted by lines to determine the diffusion coefficients. The error bars represent the standard deviation. The results were summarized in Table 2. (see DiscussionS1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2909143&req=5

pone-0011639-g003: Single molecule visualization and tracking in solution.(a) Standard epi-illumination fluorescence image of single ATTO647-labelled dextran molecules (MW 500 kDa) diffusing in transport buffer acquired at an exposure time of 10 ms. Scale bar, 5 µm (b) The same sample was imaged upon light sheet illumination demonstrating the striking contrast improvement (see Movie S1). Scale bar, 5 µm. (c) Intensity profile along a vertical line through the central pixels of the two brightest molecules shown in (a) and (b) illustrating the contrast improvement. (d) MSDs as a function of time for different molecules; dextran 500 kDa (boxes), ovalbumin (circles), and a 30mer oligonucleotide (triangles). Movies were recorded for all three species with 483 Hz. The data were fitted by lines to determine the diffusion coefficients. The error bars represent the standard deviation. The results were summarized in Table 2. (see DiscussionS1).
Mentions: For a quantitative comparison between LSFM and standard epi-illumination we compared the image contrast in image sequences of single, fluorescently labeled 500 kDa dextran molecules recorded at almost 100 Hz (Fig. 3 and Movie S1). As expected the contrast was clearly superior (0.97) when the light sheet illumination was used versus epi-illumination (0.37) for the given tracer concentration (Materials and MethodsS1). The signal-to-noise-ratio (SNR) increased by a factor of 4 yielding a significantly improved localization precision [25].

Bottom Line: By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample.Thereby we could directly observe and track small and large tracer molecules in aqueous solution.Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms Universität, Bonn, Germany.

ABSTRACT
Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 microm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.

Show MeSH
Related in: MedlinePlus