RES E A R C H Open Access
Dynamic light sheet generation and
fluorescence imaging behind turbid media
Jale Schneider and Christof M. Aegerter
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
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.
Keywords: Imaging through turbid media, Wave-front shaping, Phase modulation, Light sheet microscopy
Scattering of light severely compromises the image qual-
ity 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 devel-
Great technical developments in terms of multi-photon
microscopy , adaptive optics [2–4], wave-front shaping
[5–15], speckle(auto)correlation [16–18], time-reversal
 and optical phase conjugation [20–28]haveimproved
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 forma-
tion of variants of light sheet illumination behind scat-
tering 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 fluores-
cence from this plane is collected with a detection
objective placed perpendicular to the excitation. Never-
theless, traveling through scattering media damages this
type of illumination pattern as well leading to a progres-
sive widening of the illumination slice.
* Correspondence: firstname.lastname@example.org
Physik-Institut, University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Journal of the European Optical
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made.
Schneider and Aegerter Journal of the European Optical Society-Rapid Publications