Application Note
Concentration and colocalization analysis of fluorescent EVs using the NanoQNTMaarten Danial1, Aron Opheij1, Sirin Korulu Koc2, Paolo Guazzi2
Dispertech BV, 2HansaBioMed Life Sciences Ltd.

1Introduction
Extracellular vesicles (EVs) including exosomes (alsoknown as small EVs), microvesicles, and apoptotic
bodies (collectively called large EVs) have been
identified in many biological fluids, including blood,
saliva, and urine. Being produced by many organisms
and cell types, EVs display different lipids, proteins,and nucleic acids, which play a vital role in cell-to-cellcommunication (Figure 1). EVs have demonstrated
tremendous potential in the fields of regenerative
medicine, therapy, and the diagnosis of diseases.

Figure 2. (a) The light-sheet illuminated sections in thehydrogel are imaged. (b) A Z-stack is reconstructed fromwhich the measurement volume can be determined. (c)The analysis is performed on the Z-stacks from which
particle concentration can be determined.

The hydrogel immobilization process also ensuresthat the fluorescent EVs remain in the field of view,
allowing fluorescent biomarker concentration
analysis and colocalization using up to 4 excitation
wavelengths in rapid succession.
We utilized the FluoEVs, produced by HansaBioMed
Life Sciences. FluoEVs are fluorescent EVs stably
mono-labeled expressing EGFP or mCherry as afusion protein with CD9 or CD63, or dual labeledexpressing both fusion proteins CD9-mCherry/CD63EGFP. In this application note we demonstrate thepotential of our NanoQNT to accurately determine EVparticle concentrations and perform colocalization ofdual-labeled EVs within minutes.

Figure 1. Schematic representation of an extracellular
vesicle and its lipids, proteins, and nucleic acids
components.

Colocalization of biomarkers with the NanoQNTThis application note demonstrates the potential of
the NanoQNT, a device developed by Dispertech thatutilizes light sheet fluorescence microscopy to rapidly
determine EV particle concentrations. The
measurements make use of user-defined sectioned
image analysis of fluorescent EVs immobilized in ahydrogel (Figure 2a). As the light sheet is moved
through the hydrogel with nanometer precision, anaccurate sample volume can be reconstructed fromthe sectioned images (Figure 2b). Subsequently, theZ-stack of images is analyzed from which theconcentration of EVs can be determined (Figure 2c).

Material and methods
FluoEVs were analyzed using nanoparticle tracking
analysis (NTA) in fluorescence mode (Zetaview
analyzer, Particle Metrix). Each sample of lyophilized
FluoEVs was reconstituted in water according to themanufacturer’s specifications to achieve aconcentration of 108 particles/mL and subsequently
diluted 10-fold with phosphate buffered saline (PBS)
to achieve a particle concentration of 107
particles/mL.
The NanoQNT (Dispertech, Amsterdam, TheNetherlands) was utilized to rapidly determine theconcentration of the FluoEVs based on fluorescent
EGFP or mCherry-labeled tetraspanins CD9 andCD63. The FluoEV sample was immobilized in an
acrylamide / bis-acrylamide–based hydrogel createdusing a 96-square well plate.

Two fluorescence channels were employed in theNanoQNT to obtain the excitation of EGFP at 488 nm,mCherry at 561 nm, or both EGFP and mCherry at488 and 561 nm, respectively. The resulting stacks ofimages where fluorescent EVs are revealed as spots
were analyzed by custom-made NanoQNT analysis
software.

Analysis of mono-labeled EVs
The results were obtained using the NanoQNT
analysis software, which algorithmically determines
the particle concentration based on image analysis,
where each fluorescent EV appeared as a bright spot
on the image taken by a camera.
Table 1. Expected concentrations based on NTA versus
determined concentrations using the NanoQNT.
EV sample
CD9-EGFP
CD63-EGFPCD63-mCherry

Expected conc.
(particles/mL)
1×108
1×107
1×108
1×107
1×108
1×107

NanoQNT conc.
(particles/mL)
1.9×108
1.5×1072.0×108
2.9×107
6.0×107
5.6×106

As presented in Table 1, the EV particle
concentrations of the EGFP or mCherry-labeled
FluoEVs were within the expected concentration
range as determined by NTA.

Analysis and colocalization of dual-labeled EVs
Analysis of the stack of images derived from the duallabeled EVs consisting of CD9-mCherry and CD63EGFP was also performed. Figure 2 shows apredominant overlap between fluorescent spots in theEGFP channel (Figure 2a) and in the mCherrychannel (Figure 2b). However, not all spots exhibit
colocalization , suggesting that a subset of EVs only
has one fluorescently labeled tetraspanin.
The asymmetry of the scattering plot shown in Figure
2c, confirms the difference in fluorescence intensity
between the two channels. Further analysis of theEVs demonstrates the concentration differences
between the two channels and particles with dual
labeling (Table 2).
Table 2. Analysis of the dual-labeled (CD9-mCherry/CD63EGFP) EVs using the NanoQNT.a
Measured dual
Measured
Measuredlabeled conc.
EGFP conc.
mCherry conc.
(particles/mL)(particles/mL)(particles/mL)
8.75×107
1.75×108
1.15×108a Total expected particle conc.: 108 particles/mL.

Figure 2. Colocalization analysis of CD9-mCherry/CD63-EGFP dual labeled EVs showing (a) the EGFP channel usinga laser operating at 488 nm and (b) the mCherry channel using a laser operating at 561 nm. (c) A scattering plot showing
the fluorescence intensity from the EGFP and mCherry channels.

Conclusions•••
1Dispertech BV

Science Park 400
1098XH Amsterdam
Netherlands
www.dispertech.com

Accurate analysis of EV particle concentrations within minutes.
Colocalization analysis of dual-labeled EVs.
Population analysis and quantitation of dual-labeled EVs.
2HansaBioMed Life-Sciences Ltd.

Mäealuse 2/1
12618 Tallinn
Estonia
www.hansabiomed.eu