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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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Related in: MedlinePlus

Dimensions of the light sheet.The red light sheet (λ = 638 nm) was formed in a solution with a 100 µM concentration of ATTO647N. It was imaged with a 10× NA 0.3 objective by a slow-scan CCD-camera (Materials and MethodsS1). Lateral (a) and axial (b) extension of the light sheet. Scale bars, 100 µm. (c) Full-width-at-half-maximum (FWHM) values of the light sheet along the illumination (x-) axis. Light sheet geometries of all excitation wavelengths were summarized in Table 1.
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pone-0011639-g002: Dimensions of the light sheet.The red light sheet (λ = 638 nm) was formed in a solution with a 100 µM concentration of ATTO647N. It was imaged with a 10× NA 0.3 objective by a slow-scan CCD-camera (Materials and MethodsS1). Lateral (a) and axial (b) extension of the light sheet. Scale bars, 100 µm. (c) Full-width-at-half-maximum (FWHM) values of the light sheet along the illumination (x-) axis. Light sheet geometries of all excitation wavelengths were summarized in Table 1.

Mentions: The actual light sheet dimensions defined the field of view and optical sectioning capability of the microscope. To measure the light sheet dimensions a aqueous solution of ATTO647N was filled into the sample chamber at a concentration of 100 µM. Its illumination directly revealed the 3D extensions of the light sheet, which were imaged and quantified (Fig. 2). The minimal FWHM along the illumination axis was 19.7±0.1 µm. Upon turning the elliptical illumination beam by 90° the thickness of the light sheet could directly be visualized and analyzed in a similar manner. Its axial width was 3.0±0.1 µm FWHM for an excitation wavelength of 638 nm. The extension of the usable light sheet along the illumination axis can be defined by twice the Rayleigh length of the Gaussian beam, so that the field of view for optimal contrast was approximately 84 µm×20 µm. Similarly, for excitation wavelengths of 488 nm and 532 nm the axial FWHM values were determined as 2.9±0.1 µm and 3.0±0.1 µm, respectively (Table 1). The optical sectioning thickness was comparable for all three wavelengths as it was expected for an achromatic illumination.


Light sheet microscopy for single molecule tracking in living tissue.

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

Dimensions of the light sheet.The red light sheet (λ = 638 nm) was formed in a solution with a 100 µM concentration of ATTO647N. It was imaged with a 10× NA 0.3 objective by a slow-scan CCD-camera (Materials and MethodsS1). Lateral (a) and axial (b) extension of the light sheet. Scale bars, 100 µm. (c) Full-width-at-half-maximum (FWHM) values of the light sheet along the illumination (x-) axis. Light sheet geometries of all excitation wavelengths were summarized in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011639-g002: Dimensions of the light sheet.The red light sheet (λ = 638 nm) was formed in a solution with a 100 µM concentration of ATTO647N. It was imaged with a 10× NA 0.3 objective by a slow-scan CCD-camera (Materials and MethodsS1). Lateral (a) and axial (b) extension of the light sheet. Scale bars, 100 µm. (c) Full-width-at-half-maximum (FWHM) values of the light sheet along the illumination (x-) axis. Light sheet geometries of all excitation wavelengths were summarized in Table 1.
Mentions: The actual light sheet dimensions defined the field of view and optical sectioning capability of the microscope. To measure the light sheet dimensions a aqueous solution of ATTO647N was filled into the sample chamber at a concentration of 100 µM. Its illumination directly revealed the 3D extensions of the light sheet, which were imaged and quantified (Fig. 2). The minimal FWHM along the illumination axis was 19.7±0.1 µm. Upon turning the elliptical illumination beam by 90° the thickness of the light sheet could directly be visualized and analyzed in a similar manner. Its axial width was 3.0±0.1 µm FWHM for an excitation wavelength of 638 nm. The extension of the usable light sheet along the illumination axis can be defined by twice the Rayleigh length of the Gaussian beam, so that the field of view for optimal contrast was approximately 84 µm×20 µm. Similarly, for excitation wavelengths of 488 nm and 532 nm the axial FWHM values were determined as 2.9±0.1 µm and 3.0±0.1 µm, respectively (Table 1). The optical sectioning thickness was comparable for all three wavelengths as it was expected for an achromatic illumination.

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