Label-Free Imaging of Carbon-Based Nanoparticle Pollutants in Biological Settings

Relevance and Applications

  • Fig. 1: Schematic representation of the label-free, non-incandescence related white-light emission of carbon-based particles in aqueous environments under femtosecond pulsed illumination.Fig. 1: Schematic representation of the label-free, non-incandescence related white-light emission of carbon-based particles in aqueous environments under femtosecond pulsed illumination.
  • Fig. 1: Schematic representation of the label-free, non-incandescence related white-light emission of carbon-based particles in aqueous environments under femtosecond pulsed illumination.
  • Fig. 2: Schematic representation of the label-free, pump-probe imaging of carbon-based particles in aqueous environments based on local heating and resulting slightly changes in refractive index. On the left, the pump beam (green) causes local heating and scattering of the probe beam (red). On the right, the probe beam propagation is undisturbed in absence of the pump beam.
  • Fig. 3: Imaging of the tubulin cytoskeleton of human lung fibroblast cells (MRC-5 cell lines, orange) in combination with the label-free, white-light detection (810 nm excitation, 150 fs, 80 MHz and 400-410 nm band-pass filter detection) of engulfed carbon black particles (150 nm aerodynamic diameter, cyan) after 4 hour exposure to 25 µg/cm² at 37°C. Scale bar: 25 µm.

Carbon-based nanoparticles are widely produced, both unintentionally like the environmental contaminant black carbon as well as intentionally such as carbon black or carbon nanotubes. Although detrimental environmental and health effects are generally reported for those materials, a direct, label-free approach for detecting them in aqueous environments, such as human tissue and liquid biopsies, was still lacking. Here, we present two novel methods for label-free and biocompatible detection of carbon-based particles in biological settings. Both techniques are prospective in application fields ranging from epidemiology and (nano)toxicology to material research.


Carbon-based (nano)materials include a group of airborne contaminants to which we are constantly exposed while breathing. Consequently, the recent discussion about car exhaust has partly focused also on carbonaceous particles as a threat for public health. Additionally, carbon nanoparticles are also a major risk in occupational settings wherein those materials are extensively produced and used. Hence, diverse research has been dedicated for unravelling the toxicological behavior of those materials. However, efficient research is hampered by the limited ability to adequately detect carbonaceous particles in relevant biological samples, which makes it hard to correlate observed adverse effects to the presence of those materials.

To date, information about carbon particulate pollution in air is obtained from fixed monitoring stations often based on absorption photometry in combination with dispersion models to estimate the residential exposure. This approach is biased towards the detection of larger, biologically less harmful microparticles and it further lacks the ability to take all exposure routes into account, particularly effects at the cellular level. Measurements in a biological setting could employ light and electron microscopy or radionuclide labeling strategies. The former lack sensitivity and especially specificity and the latter does not result in a physiological representation of carbon particle exposure and is ethically questionable as it requires the inhalation or study of a high concentration of labeled synthetic particles.

Therefore, the research activities of our teams were focused on the development of new detection methods that allow direct, label-free detection of carbon-based particles in aqueous environments.


White Is the New Black

Our firstly developed optical microscopy based method for the label-free and biocompatible detection of carbonaceous particles is based on the secondary emission of light: under femtosecond pulsed illumination with near-infrared light we found carbon-based particles to generate a bright, white-light signal (fig. 1) [1]. This observed white-light emission is attributed to multiphoton ionization of the spectrally broad and strong light absorbing particles followed by consecutive intraband transitions. A similar phenomenon has already been described for plasmonically active metal nanoparticles [2].

Hot Particle Detection

A different approach for label-free carbon-based particle detection includes the direct probing of the absorption characteristics of the particles by photothermal pump-probe microscopy (fig. 2) [3]. At first, pump photons are absorbed by the carbon particle causing it to heat up. Because of the local heating, the local refractive index of particle and surrounding medium changes slightly, resulting in a temporary change in scattering of a second probe beam. Rapid intensity modulation of the pump beam over time at radio frequencies causes a modulation of the probe beam propagation which can effectively be detected in the transmission channel. Technically, this requires two spatially overlapped picosecond laser lines of which one – the pump laser - is intensity modulated and the second – probe beam – is detected and translated into a ‘particle-present’ signal.

Application Examples
Both technologies will show their utility in the field of (nano)toxicology. The label-free signals of the carbon-based nanoparticles can be easily discriminated from the background and autofluorescence in biological samples (fig. 3), showing not only large aggregates but also smaller particles. The sensitive and specific detection is therefore superior over existing methods such as light microscopy. Our approaches allow additionally for three-dimensional, diffraction limited imaging and is scalable, for instance when screening samples in microfluidic devices. For bio-imaging, the signals of both techniques can conveniently be combined with almost all contrast-enhancing fluorophores. As a result, the particles can be unambiguously localized in their biological context and provide additional but essential information about e.g. targeted cellular organelles.

Recently, we have exploited and validated the white-light approach to detect and quantify the environmental contaminant black carbon in human urine samples [4]. The method can be successfully used to screen urine for the presence of carbonaceous particles without the need for sample pretreatment. Therefore, it can be employed as a diagnostic screening tool for the determination of the individual exposure to black carbon. This will lead to improved epidemiological studies, risk assessment and the unraveling of complex health effects related to ambient air pollution.

Beside their utilization in various research domains, the techniques can be applied to diverse carbon-based particles, including carbon black, black carbon, carbon nanotubes [5], carbon/graphene nanodots [6], graphene, fullerenes, etc. Hence, we anticipate that the reported technologies will have a major impact on future carbon-based research.

All in All

The presented methods can visualize carbon-based particles in a label-free and biocompatible manner with high sensitivity and specificity. The nature of the signals allow flexible detection schemes and are therefore generally applicable to various biological samples and carbon-based materials. As this is the very first time that aqueous environments can be screened for carbon-based particles, we anticipate the introduction of these approaches in many research domains.


We would like to thank all colleagues of KU Leuven and UHasselt as well as our collaborators for their contribution to our research. This work was supported by Fonds voor Wetenschappelijk Onderzoek (BE).

Hannelore Bové1, Christian Steuwe2, Marcel Ameloot1, Maarten Roeffaers2

1Biomedical Research Centre, Agoralaan Building C, Diepenbeek, Belgium
2Centre for Surface Chemistry and Catalysis, Leuven, Belgium

Prof. Dr. Maarten Roeffaers
Centre for Surface Chemistry and Catalysis
Leuven, Belgium

[1] Hannelore Bové, Christian Steuwe, Eduard Fron, Eli Slenders, Jan D’Haen, Yasuhiko Fujita, Hiroshi Uji-I, Martin vandeVen, Maarten Roeffaers, Marcel Ameloot: Biocompatible label-free detection of carbon black particles by femtosecond pulsed laser microscopy, Nano letters, 16(5): 3173-3178 (2016) doi: 10.1021/acs.nanolett.6b00502.
[2] Tobias Haug, Philippe Klemm, Sebastian Bange, John M. Lupton: Hot-electron intraband luminescence from single hot spots in noble-metal nanoparticle films, Physical Review Letters, 115(6): 067403 (2015) doi: 10.1103/PhysRevLett.115.067403.
[3] Christian Steuwe, Hannelore Bové, Jan Clerinx, Martin vandeVen, Eduard Fron, Tim Nawrot, Marcel Ameloot, Maarten Roeffaers: Rapid and label‐free optical detection of individual carbon air pollutant nanoparticulates in biomedical samples, Journal of Biophotonics, 11(5): e201700233 (2018) doi: 10.1002/jbio.201700233.
[4] Nelly Saenen, Hannelore Bové, Christian Steuwe, Maarten Roeffaers, Eline B Provost, Wouter Lefebvre, Charlotte Vanpoucke, Marcel Ameloot, Tim Nawrot: Children's Urinary Environmental Carbon Load: A Novel Marker Reflecting Residential Ambient Air Pollution Exposure?, American Journal of Respiratory and Critical Care Medicine, 196(7): 873-881 (2017) doi: 10.1164/rccm.201704-0797OC.
[5] Deniz Öner, Manosij Ghosh, Hannelore Bové, Matthieu Moisse, Bram Boeckx, Radu C Duca, Katrien Poels, Katrien Luyts, Eveline Putzeys, Kirsten Van Landuydt, Jeroen AJ Vanoirbeek, Marcel Ameloot, Diether Lambrechts, Lode Godderis, Peter HM Hoet: Differences in MWCNT- and SWCNT-induced DNA methylation alterations in association with the nuclear deposition, Particle and Fibre Toxicology, 15(11) (2018) doi: 10.1186/s12989-018-0244-6.
[6] Jing Liu, Ranhua Xiong, Toon Brans, Saskia Lippens, Eef Parthoens, Francesca C Zanacchi, Raffaella Magrassi, Santosh K Singh, Sreekumar Kurungot, Sabine Szunertis, Hannelore Bové, Marcel Ameloot, Juan C Fraire, Eline Teirlinck, Sangram K Samal, Riet De Rycke, Gaëlle Houthaeve, Stefaan C De Smedt, Rabah Boekherroub, Kevin Braeckmans: Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy, Light: Science & Applications, in press (2018) doi: 10.1038/s41377-018-0048-3.


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