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Improving Multicolor Colocalization in Single-Vesicle Flow Cytometry with Vesicle Transit Time
Luca A Andronico1, Seung-Ryoung Jung2, Bryant S Fujimoto2
1Department of Women's and Children's Health (KBH), Karolinska Institutet, Solna 17177, Sweden.
Insights
A new analysis method, Scorr, improves multicolor colocalization for single-vesicle immunoprofiling. This technique enhances the accuracy and efficiency of analyzing extracellular vesicles (EVs) and nanobeads, overcoming limitations of previous cross-correlation analysis (Xcorr).
Area of Science:
- Biophysics
- Nanotechnology
- Biochemistry
Background:
- Extracellular vesicle (EV) immunophenotyping is crucial for understanding their biological roles.
- Accurate surface protein analysis of small EVs (30-40 nm) is challenging due to velocity variations in flow cytometry.
- Traditional cross-correlation analysis (Xcorr) can yield incorrect results for nanometer-sized vesicles.
Purpose of the Study:
- To introduce and validate an alternative cross-correlation analysis strategy (Scorr) for improved multicolor colocalization.
- To address the limitations of velocity distribution in flow-focused nanovesicles during immunophenotyping.
- To enhance the accuracy and efficiency of single-vesicle immunoprofiling.
Main Methods:
- Developed and implemented a novel cross-correlation analysis strategy (Scorr) utilizing particle transit time.
- Tested Scorr's performance using multicolor nanobeads and extracellular vesicles (EVs).
- Validated Scorr through experimental data and Monte Carlo simulations.
Main Results:
- Scorr significantly improved the efficiency and accuracy of multicolor colocalization compared to Xcorr.
- Monte Carlo simulations showed Scorr increased colocalized peaks by approximately 1.2-4.7 fold.
- Experimental results demonstrated a 1.3-2.5 fold increase for nanobeads and 1.2-2 fold for EVs.
Conclusions:
- Scorr offers a more reliable method for multicolor colocalization in single-vesicle immunoprofiling.
- The transit time-based approach effectively mitigates issues caused by particle velocity variations.
- This advancement facilitates more precise analysis of nanometer-sized vesicles and their surface proteins.
Abstract:
Immunophenotyping of vesicles, such as extracellular vesicles (EVs), is essential to understanding their origin and biological role. We previously described a custom-built flow analyzer that utilizes a gravity-driven flow, high numerical aperture objective, and micrometer-sized flow channels to reach the sensitivity needed for fast multidimensional analysis of the surface proteins of EVs, even down to the smallest EVs (e.g., ∼30-40 nm). It is difficult to flow focus small EVs, and thus, the transiting EVs exhibit a distribution in particle velocities due to the laminar flow. This distribution of vesicle velocities leads to potentially incorrect results when immunophenotyping nanometer-sized vesicles using cross-correlation analysis (Xcorr), as the order of appearance of the vesicles might not be the same at different spatially offset laser excitation regions. Here, we describe an alternative cross-correlation analysis strategy (Scorr), which uses information on particle transit time across the laser excitation beam width to improve multicolor colocalization in single-vesicle immunoprofiling. We tested the performance of the algorithm for colocalization analysis of multicolor nanobeads and EVs experimentally and via simulations and found that Scorr improved both the efficiency and accuracy of colocalization versus Xcorr. As shown from Monte Carlo simulations, Scorr provided an ∼1.2-4.7-fold increase in the number of colocalized peaks and ensured negligible colocalization of peaks. In silico results were in good agreement with experimental data, which showed an increase in colocalized peaks of ∼1.3-2.5-fold and ∼1.2-2-fold for multicolor beads and EVs, respectively.
