Biology subjects

Curcio, C. A.

Publications and source records attributed to Curcio, C. A..

3 recordsLinked to original sources

bioRxiv2021

Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies

Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge cross-links between formaldehyde and amine/ thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artefactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared to fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.

biochemistry↗

bioRxiv2020

Fluorescence Lifetimes and Spectra of RPE and sub-RPE Deposits in Histology of Normal and AMD Eyes

PurposeTo investigate autofluorescence lifetimes as well as spectral characteristics of drusen and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). MethodFluorescence lifetimes and spectra of five eyes with AMD and nine control eyes were analyzed in cryosections by means of two-photon excited fluorescence at 960 nm. Spectra were detected at 490 - 647nm. Lifetime was measured using time-correlated single photon counting in two spectral channels: 500-550nm and 550-700nm. The fluorescence decays over time were approximated by a series of three exponential functions and the amplitude-weighted mean fluorescence lifetime {tau}m was determined. Results196 sub-RPE deposits were identified (AMD n=76, healthy n=120) and 230 RPE sites recorded. The peak emission wavelength of drusen was significantly green-shifted compared to RPE (peak at 570nm vs. 610nm), but not different between patients and controls. Drusen showed considerably longer {tau}m than RPE: (ch1: 581 {+/-} 163 ps vs. 177 {+/-} 25 ps, ch2: 541 {+/-} 125 ps vs. 285 {+/-} 31 ps, p < 0.001). Drusen found in AMD eyes had longer lifetimes than drusen of controls (ch1: 650 {+/-} 167 ps vs. 537 {+/-} 145 ps, ch2: 600 {+/-} 125 ps vs. 504 {+/-} 111 ps, p < 0.001). In addition, drusen in AMD eyes showed a more homogenous fluorescence distribution and more drusen were larger than 63{micro}m than in control eyes. ConclusionsEx vivo fluorescence imaging of drusen in cross-sections enables the separation of their autofluorescence from that of over- or underlying structures. Our analysis showed considerable variability of drusen lifetimes but not spectra. This indicates that drusen consist of a variety of different fluorophores or expose the same fluorophores to different microenvironments. Changes in drusen lifetimes could be related to AMD progression.

cell biology↗

bioRxiv2020

Lipid Landscape of the Human Retina and Supporting Tissues Revealed by High Resolution Imaging Mass Spectrometry

The human retina evolved to facilitate complex visual tasks. It supports vision at light levels ranging from starlight to sunlight, and its supporting tissues and vasculature regulate plasma-delivered lipophilic essentials for vision, including retinoids (vitamin A derivatives). The human retina is of particular interest because of its unique anatomic specializations for high-acuity and color vision that are also vulnerable to prevalent blinding diseases. The retinas exquisite cellular architecture is composed of numerous cell types that are aligned horizontally, giving rise to structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. Suitable for retinal investigations, MALDI imaging mass spectrometry (IMS) technologies are now capable of providing images at low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach combined with a recently developed method of high accuracy multi-image registration was used to define the localization of lipids in human retina tissue at laminar, cellular, and sub-cellular levels. Data acquired by IMS combined with autofluorescence and bright-field microscopy of human retina sections in macular and peripheral regions indicate differences in distributions and abundances of lipid species across and within single cell types. Of note is localization of signals within specific layers of macula, localization within different compartments of photoreceptors and RPE, complementarity of signals between macular retina and non-macular RPE, and evidence that lipids differing by a single double bond can have markedly different distributions.

biochemistry↗