Limits...
Fast imaging of live organisms with sculpted light sheets.

Chmielewski AK, Kyrsting A, Mahou P, Wayland MT, Muresan L, Evers JF, Kaminski CF - Sci Rep (2015)

Bottom Line: A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively.This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution.Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK [2] Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK.

ABSTRACT
Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

No MeSH data available.


Related in: MedlinePlus

Principle of light sheet adjustment with tunable lenses.(a) and (b): diagrams explaining how variation of the tuneable lenses foci f1 and f2 changes the magnification, D2/D1, thus changing lateral (dx) and axial (dz) extent of the beam focus. Below are measured beam profiles for 0.13 NA and 0.3 NA illumination beams, respectively. (c) and (d) show how displacement of the foci f1, f2 changes beam convergence/divergence thus translating the illumination focus axially. Below are measured beams (0.3 NA) with positions displaced by 150 μm. All scale bars 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403519&req=5

f1: Principle of light sheet adjustment with tunable lenses.(a) and (b): diagrams explaining how variation of the tuneable lenses foci f1 and f2 changes the magnification, D2/D1, thus changing lateral (dx) and axial (dz) extent of the beam focus. Below are measured beam profiles for 0.13 NA and 0.3 NA illumination beams, respectively. (c) and (d) show how displacement of the foci f1, f2 changes beam convergence/divergence thus translating the illumination focus axially. Below are measured beams (0.3 NA) with positions displaced by 150 μm. All scale bars 30 μm.

Mentions: An overview of the custom built dSLM used in this work is given in the Supplementary Fig. S1 (for detailed description see Methods section). A key feature of the system is a telescope featuring two electrically tuneable lenses through which the laser beam generating the light sheet is passed before entering the excitation objective. The telescope permits the continuous variation of the excitation beam diameter and divergence at the back aperture of the excitation objective, to result in corresponding changes of light-sheet thickness and position of the focal point. Changing the beam diameter at the back aperture of the illumination objective varies the numerical aperture, NA, in the excitation path and hence light sheet extension/thickness (see diagrams in Figure 1a, b). Changing the divergence mimics the quadratic defocus phase resulting in the translation of the beam focus (diagrams in Figure 1c, d).


Fast imaging of live organisms with sculpted light sheets.

Chmielewski AK, Kyrsting A, Mahou P, Wayland MT, Muresan L, Evers JF, Kaminski CF - Sci Rep (2015)

Principle of light sheet adjustment with tunable lenses.(a) and (b): diagrams explaining how variation of the tuneable lenses foci f1 and f2 changes the magnification, D2/D1, thus changing lateral (dx) and axial (dz) extent of the beam focus. Below are measured beam profiles for 0.13 NA and 0.3 NA illumination beams, respectively. (c) and (d) show how displacement of the foci f1, f2 changes beam convergence/divergence thus translating the illumination focus axially. Below are measured beams (0.3 NA) with positions displaced by 150 μm. All scale bars 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403519&req=5

f1: Principle of light sheet adjustment with tunable lenses.(a) and (b): diagrams explaining how variation of the tuneable lenses foci f1 and f2 changes the magnification, D2/D1, thus changing lateral (dx) and axial (dz) extent of the beam focus. Below are measured beam profiles for 0.13 NA and 0.3 NA illumination beams, respectively. (c) and (d) show how displacement of the foci f1, f2 changes beam convergence/divergence thus translating the illumination focus axially. Below are measured beams (0.3 NA) with positions displaced by 150 μm. All scale bars 30 μm.
Mentions: An overview of the custom built dSLM used in this work is given in the Supplementary Fig. S1 (for detailed description see Methods section). A key feature of the system is a telescope featuring two electrically tuneable lenses through which the laser beam generating the light sheet is passed before entering the excitation objective. The telescope permits the continuous variation of the excitation beam diameter and divergence at the back aperture of the excitation objective, to result in corresponding changes of light-sheet thickness and position of the focal point. Changing the beam diameter at the back aperture of the illumination objective varies the numerical aperture, NA, in the excitation path and hence light sheet extension/thickness (see diagrams in Figure 1a, b). Changing the divergence mimics the quadratic defocus phase resulting in the translation of the beam focus (diagrams in Figure 1c, d).

Bottom Line: A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively.This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution.Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK [2] Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK.

ABSTRACT
Light-sheet microscopy is an increasingly popular technique in the life sciences due to its fast 3D imaging capability of fluorescent samples with low photo toxicity compared to confocal methods. In this work we present a new, fast, flexible and simple to implement method to optimize the illumination light-sheet to the requirement at hand. A telescope composed of two electrically tuneable lenses enables us to define thickness and position of the light-sheet independently but accurately within milliseconds, and therefore optimize image quality of the features of interest interactively. We demonstrated the practical benefit of this technique by 1) assembling large field of views from tiled single exposure each with individually optimized illumination settings; 2) sculpting the light-sheet to trace complex sample shapes within single exposures. This technique proved compatible with confocal line scanning detection, further improving image contrast and resolution. Finally, we determined the effect of light-sheet optimization in the context of scattering tissue, devising procedures for balancing image quality, field of view and acquisition speed.

No MeSH data available.


Related in: MedlinePlus