Abstract
BackgroundLight 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.MethodsWe 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.ResultsWe 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.ConclusionOur 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.
Highlights
Light sheet microscopy became a popular tool allowing fast imaging with reduced out of focus light
Light sheet behind scotch tape We first performed wave-front shaping of the illumination behind scotch tape as a turbid medium
The intensity within the region of interest (ROI) could be enhanced 12 to 15 times compared to the average background intensity
Summary
Light sheet microscopy became a popular tool allowing fast imaging with reduced out of focus light. 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. Scattering of light severely compromises the image quality when turbid media such as thick tissues are observed using conventional fluorescence microscopes. Light sheet microscopy [29,30,31] 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. Traveling through scattering media damages this type of illumination pattern as well leading to a progressive widening of the illumination slice
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