Abstract
Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.
Highlights
A fundamental principle in light microscopy is to uncover the composition of matter by sending photons with defined properties onto a piece of matter and detecting the scattered photons
Popular methods to improve the image quality by post-processing such as image deconvolution[16,17,18] often remain unsatisfying because the point-spread function (PSF) at the camera is different for each position inside the 3D object, which is because fluorescence photons are scattered differently at each position
We demonstrate the principle of separating weakly and strongly scattered fluorescence photons using identical Arabidopsis Thaliana roots, which we image in four different illumination and detection modes
Summary
A fundamental principle in light microscopy is to uncover the composition of matter by sending photons with defined properties onto a piece of matter and detecting the scattered photons. A suitable microscopical method to tackle the problem of light scattering in both the illumination and detection path is light-sheet based microscopy (LSM)[2,3] Besides advantages such as high acquisition speed, effective sectioning, high contrast and low phototoxicity, this technique is fascinating because it allows to observe how the illumination light propagates from the side through selected planes of the object. To the refractive index inhomogeneities of the object, which would otherwise lead to unwanted beam deflections and distortions[13] This holographic shaping of the illumination beam requires coherent light, which is characterized by its unique phase dependency between all photons incident onto the object.
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