Label-free detection and size estimation of combustion-derived carbonaceous particles in a microfluidic approach

Detection and size estimation of combustion-derived carbonaceous particles (CDCPs) are important to understand their toxicity. Size determination of individual nano- and microparticles (NMPs) based on scattered light is a straightforward method. However, detection and sizing of CDCPs in biological samples based on scattering alone are not possible due to the compositional heterogeneity of NMPs present in biological samples. Label-free identification of CDCPs based on unique white light (WL) emission, using femtosecond (fs) pulsed near-infrared (NIR) lasers, has emerged as a reliable method even in complex biological samples. However, size estimation of CDCPs in biological samples using label-free techniques is still lacking. Here we report the development of a dual-channel multiphoton flow cytometry (DCMPFC) setup for label-free identification and size-determination of CDCPs in suspensions. Scattering intensity calibration with reference polystyrene (PS) nanoparticles (NPs) and Mie Theory allow us to determine the sizes of CDCPs in aqueous suspensions. Further, the relationship between particle sizes and WL emission intensity was determined, and the sizes of CDCPs in urine samples could also be estimated. This approach is believed to open new opportunities for the quantification and size determination of CDCPs, originating from exposure to air pollution, in liquid biopsies. This is an important step in determining the CDCP exposure of individual persons.


Nanoparticle tracking analysis (NTA)
The hydrodynamic diameters of particles suspended in ultrapure water were also measured using nanoparticles tracking analysis (NTA) (NanoSight, UK). The measurements were performed on the same sample multiple times for repeatability.

Scanning electron microscopy (SEM) characterization
The dried nanoparticles were observed using a scanning electron microscope (FEI Quanta 250 FEG) with an acceleration voltage of 20 kV.    Figure S6: For very small CDCPs, scattering peaks can be seen slightly above the background in NIRC, but no peaks visible in VISC which could be due to the very low WL emission intensity. The binwidth is 100 µs. The threshold is is set at mean+5SD from measurements on blank MQ.
SD: standard deviation S10 Figure S7: Cross-correlation between VISC and NIRC shows strong peaks a t=0 time lag for all 4 CDCPs used in this study.

Calculations for actual count rate of APDs
After arrival of each photon pulse, an APD stays unresponsive for a specific amount of time which is called APD dead time. APD deadtime can underestimate the number of detected photons at very high photon count rates. Therefore at high photon counts, the count rate needs to be adjusted based on the theoretical calculations from the manufacturer's data. 1 Based on the dead time of the APDs, a linearity correction factor for each APD is provided by the manufacturer. The linearity correction factors and photon detection efficiency of APDs (at specific wavelengths) can be used to calculate the actual photon count rates for each APD. The typical deadtime for SPCM-AQRH-10 is 24 ns and the dark counts are 1500 cps as specified by the manufacturer's data. The photon detection efficiency is around 55% at 550 nm for VISC APD and around 65% at 780 nm for NIRC APD. saturating. For both APDs, a 5 th order polynomial fitting was used for the correlation of the linearity correction factor with the detected photon counts. The coefficients from the fitted data were used to estimate actual photon counts. This data was used for further photon burst identification for VISC and NIRC for our measurements. At the low count rates, the correction factor is almost equal to 1 as shown in Figure S14, however with an increase in the observed count rates the correction factor can go as high as 6.

Calculations for scattering cross-section and scattering intensity based on Mie Theory 2 :
The maximum projection angle of the objective with respect to the optical axis; Where is the refractive index of the medium.
The integration boundaries of the azimuthal angle ( and ) can be expressed as ; Figure S15: The polar coordinate system and the variables used to calculate the scattering intensity from a spherical particle illuminated using a fs-pulsed NIR laser polarized in xz plane. For sidescatter light detection using NIRC, the numerical aperture of the objective determines the acceptance angle α.
The scattering cross-section (in nm 2 ) is given by 3 ; Whereas S1 and S2 are the amplitude scattering matrix elements and = 2 is the wavenumber.
The values of the Mie scattering cross-section (in nm 2 ) for PS NPs can be calculated using the MieConScat software. 4 Based on the scattering cross-section and the measured median scattering intensity from NIRC-A of PS NPs, we estimated a scaling factor through linear regression to relate measured median scattering intensity (I) from PS NPs to their theoretical cross-section in nm 2 ; 10 ( ) = 10 ( ) . ( 5) Where C is the scaling factor that relates the median measured scattering intensity (counts/bin) to the scattering cross-section (nm 2 ) of PS NPs. The same scaling factor can be used to estimate the scattering intensity of CDCPs of different sizes.