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)