Interferometric differentiation between resonant Coherent Anti-Stokes Raman Scattering and nonresonant four-wave-mixing processes

A major impediment of using Coherent Anti-Stokes Raman Scattering to identify biological molecules is that the illumination levels required to produce a measurable signal often also produce significant nonresonant background from the medium, especially from water, that is not specific to the resonance being investigated. We present a method of using nonlinear interferometry to measure the temporal shape of the anti-Stokes signal to differentiate which components are resonant and nonresonant. This method is easily adaptable to most existing pulsed CARS illumination methods and should allow for distinguishing resonant CARS when using higher energy pulses. By examining the differences between signals produced by acetone and water, we show that the resonant and nonresonant signals can be clearly differentiated.Comment: 8 pages, 4 figure

In-vivo two-photon imaging of the honey bee antennal lobe

Due to the honey bee's importance as a simple neural model, there is a great need for new functional imaging modalities. Herein we report on the use of two-photon microscopy for in-vivo functional and morphological imaging of the honey bee's olfactory system focusing on its primary centers, the antennal lobes (ALs). Our imaging platform allows for simultaneously obtaining both morphological measurements of the AL and in-vivo calcium recording of neural activities. By applying external odor stimuli to the bee's antennas, we were able to record the characteristic odor response maps. Compared to previous works where conventional fluorescence microscopy is used, our approach offers all the typical advantages of multi-photon imaging, providing substantial enhancement in both spatial and temporal resolutions while minimizing photo-damages and autofluorescence contribution with a four-fold improvement in the functional signal. Moreover, the multi-photon associated extended penetration depth allows for functional imaging within profound glomeruli.Comment: 3 pages, 3 figure

Saúde Perinatal e Medicina Translacional

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Automated motion artifact removal for intravital microscopy, without a priori information

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber

Mapping Molecular Agents Distributions in Whole Mice Hearts Using Born-Normalized Optical Projection Tomography

To date there is a lack of tools to map the spatio-temporal dynamics of diverse cells in experimental heart models. Conventional histology is labor intensive with limited coverage, whereas many imaging techniques do not have sufficiently high enough spatial resolution to map cell distributions. We have designed and built a high resolution, dual channel Born-normalized near-infrared fluorescence optical projection tomography system to quantitatively and spatially resolve molecular agents distribution within whole murine heart. We validated the use of the system in a mouse model of monocytes/macrophages recruitment during myocardial infarction. While acquired, data were processed and reconstructed in real time. Tomographic analysis and visualization of the key inflammatory components were obtained via a mathematical formalism based on left ventricular modeling. We observed extensive monocyte recruitment within and around the infarcted areas and discovered that monocytes were also extensively recruited into non-ischemic myocardium, beyond that of injured tissue, such as the septum

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins)

Broadband Faraday Isolator

Driving on an analogy with the technique of composite pulses in quantum physics, we propose a broadband Faraday rotator and thus a broadband optical isolator, which is composed of sequences of ordinary Faraday rotators and achromatic quarter-wave plates rotated at the predetermined angles.Comment: submitted to JOSA A, comments are welcom

Fluorescence microscopy tensor imaging representations for large-scale dataset analysis

Understanding complex biological systems requires the system-wide characterization of cellular and molecular features. Recent advances in optical imaging technologies and chemical tissue clearing have facilitated the acquisition of whole-organ imaging datasets, but automated tools for their quantitative analysis and visualization are still lacking. We have here developed a visualization technique capable of providing whole-organ tensor imaging representations of local regional descriptors based on fluorescence data acquisition. This method enables rapid, multiscale, analysis and virtualization of large-volume, high-resolution complex biological data while generating 3D tractographic representations. Using the murine heart as a model, our method allowed us to analyze and interrogate the cardiac microvasculature and the tissue resident macrophage distribution and better infer and delineate the underlying structural network in unprecedented detail