Phase-Retrieved Tomography enables imaging of a Tumor Spheroid in Mesoscopy Regime

Optical tomographic imaging of biological specimen bases its reliability on the combination of both accurate experimental measures and advanced computational techniques. In general, due to high scattering and absorption in most of the tissues, multi view geometries are required to reduce diffuse halo and blurring in the reconstructions. Scanning processes are used to acquire the data but they inevitably introduces perturbation, negating the assumption of aligned measures. Here we propose an innovative, registration free, imaging protocol implemented to image a human tumor spheroid at mesoscopic regime. The technique relies on the calculation of autocorrelation sinogram and object autocorrelation, finalizing the tomographic reconstruction via a three dimensional Gerchberg Saxton algorithm that retrieves the missing phase information. Our method is conceptually simple and focuses on single image acquisition, regardless of the specimen position in the camera plane. We demonstrate increased deep resolution abilities, not achievable with the current approaches, rendering the data alignment process obsolete.Comment: 21 pages, 5 figure

Laser induced strong-field ionization gas jet tomography

We introduce a novel in-situ strong field ionization tomography approach for characterizing the spatial density distribution of gas jets. We show that for typical intensities in high harmonic generation experiments, the strong field ionization mechanism used in our approach provides an improvement in the resolution close to factor of 2 (resolving about 8 times smaller voxel volume), when compared to linear/single-photon imaging modalities. We find, that while the depth of scan in linear tomography is limited by resolution loss due to the divergence of the driving laser beam, in the proposed approach the depth of focus is localized due to the inherent physical nature of strong-field interaction and discuss implications of these findings. We explore key aspects of the proposed method and compare it with commonly used single- and multi-photon imaging mechanisms. The proposed method will be particularly useful for strong field and attosecond science experiments.Comment: 8 pages, 3 figure

3D correlative single-cell imaging utilizing fluorescence and refractive index tomography

Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, quantifying the intracellular RI distribution and colocalizing it with fluorescence in 3D. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.Comment: 15 pages, 5 figure

Whole-brain vasculature reconstruction at the single capillary level

The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network

Quantitative performance characterization of three-dimensional noncontact fluorescence molecular tomography

© 2016 The Authors.Fluorescent proteins and dyes are routine tools for biological research to describe the behavior of genes, proteins, and cells, as well as more complex physiological dynamics such as vessel permeability and pharmacokinetics. The use of these probes in whole body in vivo imaging would allow extending the range and scope of current biomedical applications and would be of great interest. In order to comply with a wide variety of application demands, in vivo imaging platform requirements span from wide spectral coverage to precise quantification capabilities. Fluorescence molecular tomography (FMT) detects and reconstructs in three dimensions the distribution of a fluorophore in vivo. Noncontact FMT allows fast scanning of an excitation source and noninvasive measurement of emitted fluorescent light using a virtual array detector operating in free space. Here, a rigorous process is defined that fully characterizes the performance of a custom-built horizontal noncontact FMT setup. Dynamic range, sensitivity, and quantitative accuracy across the visible spectrum were evaluated using fluorophores with emissions between 520 and 660 nm. These results demonstrate that high-performance quantitative three-dimensional visible light FMT allowed the detection of challenging mesenteric lymph nodes in vivo and the comparison of spectrally distinct fluorescent reporters in cell culture