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Conference paper2023Peer reviewed

Dual-Color Confocal Fluorescence Characterizations of Antibody Loading in Bioengineered Nanovesicles

Sanaee, M.; Ronquist, K.G.; Morrell, J.M.; Sandberg, E.; Widengren, J.; Gallo, K.


Confocal fluorescence microscopy is powerful for microscopic and nanoscopic analyses with a broad range of applications in biology and medicine, affording high specificity and sensitivites down to single-molecule level [1]. Here we apply a novel dual-color confocal fluorescence microscopy methodology based on coincident burst analysis [2] to quantitative study of the loading yields of bioengineered nanovesicles derived from human red blood cell (RBC) membranes loaded with fluorescently-tagged Antibody (Ab) or dUTP cargo molecules. We prove the successful loading of the RBC nanovesicles with both types of cargo molecules, assess their size statistics and provide quantifications of the loading at single-vesicle level. Fig. 1a shows the tagging scheme adopted for the experiment where Cellvue claret and Alexa488 dyes are respectively used to fluorescently label the outer membrane of the nanovesicles and the cargo molecules. The experimental setup is shown in Fig. 1b, consisting of an inverted microscope with dual excitations at λ1= 640 nm and λ2 = 485 nm. The fluorescence signals centered at λred= 720 nm and λgreen= 535 nm are single-photon-detected and post-selected in separate time gating windows with a delay of 25ns (Fig. 1c). After looking for coincident red-green bursts in the measured time traces (Fig. 1d), size histograms of the loaded vesicles are retrieved (Fig. 1e-f), obtaining average loading yields of 5% and 20% for Ab and dUTP cargos, respectively. Average values of 1.5 and 1.4 loaded Ab (dUTP) cargo molecules per nanovesicle are retrieved from the histograms of Fig. 1g (Fig. 1h). The average size of Ab-loaded nanovesicles is found to be slightly (≈10 nm in radius) larger than the one of dUTP-loaded ones.

Published in

Title: 2023 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC)
ISBN: 9798350345995
Publisher: Institute of Electrical and Electronics Engineers Inc.


2023 Conference on Lasers and Electro-Optics Europe and European Quantum Electronics Conference, CLEO/Europe-EQEC 2023

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      Medical Biotechnology

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