Building up spectral libraries for mapping erythrocytes by hyperspectral dark field microscopy

Abstract

Abstract. BACKGROUND:Red blood cells (RBC) are obtained by non-invasive methods and widely used for diagnostic tests of health status. Hyperspectral Dark Field Microscopy (HDFM) is a promising technique for nanoscale bio imaging and spectral analysis without additional sample preparation. OBJECTIVE: Develop a protocol for human RBC characterization by HDFM, checking the feasibility of a reference spectral library that can image and afford a new comprehensive descriptor of RBC status. METHOD: A step-by-step protocol for HDFM measurement of human RBC was for the first time established using 5 µl of EDTA-treated whole blood from healthy adults (n = 30). Hyperspectral characteristics of solutions/suspensions at biological concentrations of phospholipids, hemoglobin, spectrin, cholesterol and protoporphyrin IX, as the most relevant RBC components, were also determined. RESULTS: A library made of 8 end-member spectra and classification of their spectral distribution carried out by Single Angle Mapper (SAM) were determined, furnishing a comprehensive mapping and descriptor of healthy human RBC. The spectra of single components allowed some of the RBC spectral bands to be attributed. CONCLUSIONS: This work reports for the first time the hyperspectral optical imaging of the human RBC by a library made of 8 scattering spectra, whose spectral signatures are compared with those of the main RBC molecular components. The percent distribution of the spectral end-members was also achieved, thus giving for the first time the HDFM mapping of human healthy RBCs. The protocol developed herein allows the clinical potential of hyperspectral imaging to be developed for the use of RBC mapping in health and disease.

High predictive values of RBC membrane-based diagnostics by biophotonics in an integrated approach for Autism Spectrum Disorders

“…3 the HDFM spectra registered for phospholipids and protoporphyrin IX, which are important components of the RBC membrane and sub-membrane regions, are shown. The acquisition of these spectra was carried out as previously reported 18 (see also details in the Supplementary Information). In Supplementary Fig.…”

“…Using spectral classification map and their percentage distribution, created by the spectral mapping software, the HDFM spectral features are precisely described, therefore changes occurring in the samples are reflected in changes of HDFM spectral distribution 15 , 16 . We focused on HDFM for the comprehensive and advanced imaging of complex biological scenarios such as human red blood cell (RBC) membrane 17 , and first published the healthy adult RBC imaging by a library of 8 spectral end-members with their typical distribution map 18 . In the RBC membrane and sub-membrane regions, cytoskeleton proteins, phospholipids and membrane-bound hemoglobin are the main constituents.…”

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

“…CellSave (Veridex, USA) and Streck (Streck, USA) tubes have been used to stabilize tumor cells in blood but analytical reports have shown that they lead to the loss of 40 to 60% of tumor cells spiked in blood [98]. In fact, blood cell membranes remain intact for up to 5 hours [99] after blood collection and degrade rapidly and dramatically after that (Dr Carla Ferreri, personal communication). For this reason, the ISET ® system manufacturer’s guidelines indicate that blood has to be treated by the ISET ® System within 5 hours after blood collection.…”

Fatty Acids in Membranes as Homeostatic, Metabolic and Nutritional Biomarkers: Recent Advancements in Analytics and Diagnostics

“…Mean values obtained for unselected RBCs and mature RBCs are summarized in Table 1 and reported as percent intervals of the fatty acid families. Further research is ongoing on mature RBC membranes and lipidome characterization, also using innovative biophotonic methodologies, recently discovered in our laboratories [47]. …”