Dynamic light sheet generation and fluorescence imaging behind turbid media

Journal of the European Optical Society-Rapid Publications, Feb 2018

Jale Schneider, Christof M. Aegerter

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Dynamic light sheet generation and fluorescence imaging behind turbid media

Schneider and Aegerter Journal of the European Optical Society-Rapid Publications Dynamic light sheet generation and fluorescence imaging behind turbid media Jale Schneider 0 Christof M. Aegerter 0 0 Physik-Institut, University of Zurich , Winterthurerstrasse 190, 8057 Zurich , Switzerland Background: Light sheet microscopy became a popular tool allowing fast imaging with reduced out of focus light. However, when light penetrates turbid media such as biological tissues, multiple scattering scrambles the illumination into a speckle pattern and severely challenges conventional fluorescence imaging with focused light or with a light sheet. In this article, we present generation of light sheet type illumination patterns despite scattering. Methods: We optimize the wave-front of the incoming light to transform the speckle pattern behind the scattering layer into a light sheet within the region of interest. We utilize a fast spatial light modulator for phase modulation and a genetic optimization algorithm. The light pattern behind the scattering layer is detected via a clear detection path and acts as a feedback signal for the algorithm. Results: We enabled homogenous light sheet illumination behind turbid media and enhanced the signal of fluorescent beads selectively at the desired focal plane up to eight times on average. The technique is capable to compensate the dynamic changes of the speckle pattern as well, as shown on samples consisting of living drosophila pupae. Conclusion: Our technique shows that not only single foci, but also a homogenous light sheet illumination can directly be created and maintained behind static and dynamic scattering media. To make the technique suitable for common biological settings, where the detection path is turbid as well, a fluorescent probe can be used to provide the feedback signal. Imaging through turbid media; Wave-front shaping; Phase modulation; Light sheet microscopy Background Scattering of light severely compromises the image quality when turbid media such as thick tissues are observed using conventional fluorescence microscopes. On the one hand, multiple scattering leads to a randomization of the illumination into a speckle pattern; on the other hand, the emitted fluorescence signal gets scrambled as well and cannot be traced back to its origin. Slicing, peeling, clearing etc. hence belong to common tasks of biologists who try to reduce the turbidity of their sample in order to unravel the happenings in tissues and developing animals. Great technical developments in terms of multi-photon microscopy [ 1 ], adaptive optics [ 2–4 ], wave-front shaping [ 5–15 ], speckle(auto)correlation [ 16–18 ], time-reversal [ 19 ] and optical phase conjugation [ 20–28 ] have improved microscopy in and/or behind turbid media to a great extent. However, the image quality, imaging speed and modalities are still subject to possible improvements. In this paper, we introduce direct and dynamic formation of variants of light sheet illumination behind scattering layers. Light sheet microscopy [ 29–31 ] combines the speed advantage of wide-field imaging with selective plane excitation to reduce out of focus fluorescence and has become a popular tool for biologists for fast three dimensional imaging. Light sheet microscopes illuminate only a thin slice of the sample and the emitted fluorescence from this plane is collected with a detection objective placed perpendicular to the excitation. Nevertheless, traveling through scattering media damages this type of illumination pattern as well leading to a progressive widening of the illumination slice. We use optical feedback based wave-front shaping to transform the speckle pattern behind a scattering layer into a light sheet within the region of interest. Hence, the setup employs three objective lenses: one objective lens for illumination; a second one facing the first on the opposite side of the sample to provide feedback signal for the optimization; and a third one placed perpendicular to the other two in order to detect the fluorescence signal coming from the excited slice of a fluorescently labelled sample. Figure 1 explains the setup as well as the problems faced for light sheet microscopy in turbid media in more detail. If one illuminates a sample (in our case a glass capillary filled with fluorescent beads) with a wide-field scheme, both detection objectives capture two perpendicular perspectives of the same scene at their respective focal planes. In this mode, the bright beads at the focal plane are heavily blurred with out of focus fluorescence. If the illumination is shaped into a light sheet, only a thin plane perpendicular to the optical axis of the detection objective 2 is excited. Hence, the detection objective 2 captures an image of clearly distinguishable beads from the sheet with significantly less out of focus signal. Detection objective 1 in that case faces a projection of the light sheet. If its focal (...truncated)


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Jale Schneider, Christof M. Aegerter. Dynamic light sheet generation and fluorescence imaging behind turbid media, Journal of the European Optical Society-Rapid Publications, 2018, pp. 7, Volume 14, Issue 1, DOI: 10.1186/s41476-018-0074-z