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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 tracking in the living salivary gland cell nucleus of the C.tentans larvae.(a) Scheme of the light sheet illumination of a salivary gland cell nucleus. The salivary glands are a living tissue with dimensions much larger than that of e.g. cell culture cells. Scale bar, 200 µm. (b) Typical image showing single diffusing BR mRNPs (red arrows) labelled with microinjected hrp36. The dashed line indicates the position of the nuclear envelope. Scale bar, 3 µm. (c) Time series of a moving mRNP particle marked by single ATTO647N-labelled hrp36 molecules inside the nucleus. The last panel shows the complete trajectory. Frame rate, 49.46 Hz; Scale bar 1 µm. (d) Normalized jump distance distribution of BR mRNP particles and microinjected 500 kDa dextran molecules (e) inside salivary gland cell nuclei for a time interval of 20 ms. The red line showed the complete fitting function and the dashed lines indicate the contributions of the single components (see Materials and MethodsS1). Fitting results were summarized in Table 3.
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pone-0011639-g004: Single molecule tracking in the living salivary gland cell nucleus of the C.tentans larvae.(a) Scheme of the light sheet illumination of a salivary gland cell nucleus. The salivary glands are a living tissue with dimensions much larger than that of e.g. cell culture cells. Scale bar, 200 µm. (b) Typical image showing single diffusing BR mRNPs (red arrows) labelled with microinjected hrp36. The dashed line indicates the position of the nuclear envelope. Scale bar, 3 µm. (c) Time series of a moving mRNP particle marked by single ATTO647N-labelled hrp36 molecules inside the nucleus. The last panel shows the complete trajectory. Frame rate, 49.46 Hz; Scale bar 1 µm. (d) Normalized jump distance distribution of BR mRNP particles and microinjected 500 kDa dextran molecules (e) inside salivary gland cell nuclei for a time interval of 20 ms. The red line showed the complete fitting function and the dashed lines indicate the contributions of the single components (see Materials and MethodsS1). Fitting results were summarized in Table 3.

Mentions: In a next step we employed LSFM for in vivo imaging within a 3D extended biological specimen with large dimensions compared to plain monolayer culture cells. As a suitable model system we chose the salivary gland cell nuclei of larvae of the dipteran C. tentans [27]–[29]. This system was well suited to demonstrate the imaging capabilities of the single molecule LSFM, because the salivary glands are an intact, large and living tissue. The salivary glands have a complex structure and dimensions of roughly 700 µm×2000 µm×250 µm as sketched in Fig. 4a. The gland cells contain large nuclei with diameters of 50–70 µm (Fig. 4a). Each gland cell nucleus contains four polytene chromosomes being roughly 10 µm in diameter. Each polytene chromosome is made up of 8000 to 16000 perfectly aligned chromatids [29], which form a distinct chromosome band structure. The remaining nucleoplasm is devoid of chromatin [30]. This is an ideal system to study the regulation of mRNA trafficking without the possibly retarding effect of chromatin (see Figure S1).


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 tracking in the living salivary gland cell nucleus of the C.tentans larvae.(a) Scheme of the light sheet illumination of a salivary gland cell nucleus. The salivary glands are a living tissue with dimensions much larger than that of e.g. cell culture cells. Scale bar, 200 µm. (b) Typical image showing single diffusing BR mRNPs (red arrows) labelled with microinjected hrp36. The dashed line indicates the position of the nuclear envelope. Scale bar, 3 µm. (c) Time series of a moving mRNP particle marked by single ATTO647N-labelled hrp36 molecules inside the nucleus. The last panel shows the complete trajectory. Frame rate, 49.46 Hz; Scale bar 1 µm. (d) Normalized jump distance distribution of BR mRNP particles and microinjected 500 kDa dextran molecules (e) inside salivary gland cell nuclei for a time interval of 20 ms. The red line showed the complete fitting function and the dashed lines indicate the contributions of the single components (see Materials and MethodsS1). Fitting results were summarized in Table 3.
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Related In: Results  -  Collection

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

pone-0011639-g004: Single molecule tracking in the living salivary gland cell nucleus of the C.tentans larvae.(a) Scheme of the light sheet illumination of a salivary gland cell nucleus. The salivary glands are a living tissue with dimensions much larger than that of e.g. cell culture cells. Scale bar, 200 µm. (b) Typical image showing single diffusing BR mRNPs (red arrows) labelled with microinjected hrp36. The dashed line indicates the position of the nuclear envelope. Scale bar, 3 µm. (c) Time series of a moving mRNP particle marked by single ATTO647N-labelled hrp36 molecules inside the nucleus. The last panel shows the complete trajectory. Frame rate, 49.46 Hz; Scale bar 1 µm. (d) Normalized jump distance distribution of BR mRNP particles and microinjected 500 kDa dextran molecules (e) inside salivary gland cell nuclei for a time interval of 20 ms. The red line showed the complete fitting function and the dashed lines indicate the contributions of the single components (see Materials and MethodsS1). Fitting results were summarized in Table 3.
Mentions: In a next step we employed LSFM for in vivo imaging within a 3D extended biological specimen with large dimensions compared to plain monolayer culture cells. As a suitable model system we chose the salivary gland cell nuclei of larvae of the dipteran C. tentans [27]–[29]. This system was well suited to demonstrate the imaging capabilities of the single molecule LSFM, because the salivary glands are an intact, large and living tissue. The salivary glands have a complex structure and dimensions of roughly 700 µm×2000 µm×250 µm as sketched in Fig. 4a. The gland cells contain large nuclei with diameters of 50–70 µm (Fig. 4a). Each gland cell nucleus contains four polytene chromosomes being roughly 10 µm in diameter. Each polytene chromosome is made up of 8000 to 16000 perfectly aligned chromatids [29], which form a distinct chromosome band structure. The remaining nucleoplasm is devoid of chromatin [30]. This is an ideal system to study the regulation of mRNA trafficking without the possibly retarding effect of chromatin (see Figure S1).

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