New application for the identification and differentiation of microplastics based on fluorescence lifetime imaging microscopy (FLIM)

doi:10.1016/j.jece.2020.104769

Journal of Environmental Chemical Engineering

Volume 9, Issue 1, February 2021, 104769

https://doi.org/10.1016/j.jece.2020.104769 Get rights and content

Highlights

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FLIM measures fluorescence lifetime of plastics and thus can be used for identification and characterization of plastics
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FLIM offers additional morphological information as well as information about the size and surface of plastics
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Heat treatment causes changes in photon count and stable variations in fluorescence lifetime

Abstract

The problem of micro- and nanoplastic (short: plastics) pollution is an increasing global issue and therefore several detection methods for plastics, also investigating the chemical nature via spectroscopy and chromatography, have been developed over the years. A new approach for identification and characterization of plastics is fluorescence lifetime imaging microscopy (FLIM) – a microspectroscopic method to detect fluorescence lifetime (τ) of plastics. We tested whether FLIM can be employed for the identification and characterization of plastics. Six types of plastics (ABS, PA6, PET, PLA, PPE, PU), with and without prior heat treatment, were subjected to FLIM with excitation wavelengths of 470 nm and 440 nm. The results provided mean τ (intensity weighted) values of 3.850 (+- 0.033) ns for ABS, 8.143 (+- 0.060) ns for PPE, 3.519 (+-0.090) ns for PET of a bottle from Germany and 3.564 (+-0.126) ns for PET of a bottle from the USA. The combination of mean intensity weighted τ and mean amplitude weighted τ values allowed for the significant differentiation of 52 (94.55 %) of the 55 possible plastic comparisons. Moreover, FLIM showed the potential for the sub-micrometer range plastic characterization, phasor analysis and allows for visual 3D-sectioning of samples that could be important for identification and characterization of plastics in tissue and environmental samples.

Graphical abstract

Cryo-milling of various plastic types into microplastic particles and detection via FLIM

Introduction

The problem of micro- and nanoplastic (short: plastics) pollution is an increasing global issue. However, for the detection and differentiation of environmental plastics, simple, reliable and sensitive analytical tools are required to characterize and assess the distribution and potential impact of plastics contamination in the environment and its organisms.

The currently available methods, i.e. the destructive thermal desorption gas chromatography-mass spectrometry (TDS-GC–MS) [1,2] and pyrolization-gas chromatography-mass spectrometry (Py-GC/MS) [[3], [4], [5]], do not provide information about the number and morphology of the particles without prior analysis. The non-destructive Fourier Transform Infrared Spectrometer (FTIR) and Raman spectroscopy [6,7], provide chemical analysis of particles, but are limited by particle size (FTIR > 20 μm, μFTIR > 10 μm, Raman spectrometry >0.5 μm) [[8], [9], [10]], beyond being time-consuming and requiring a complicated sample preparation. Furthermore, FTIR can be disturbed by biological contamination, which would make direct analysis of biological or environmental samples challenging. During the measurement of spontaneous Raman scattering, autofluorescence of molecules can cause an interfering signal which appears as a spectral broadband background signal and thus covers the weaker Raman signals. For fluorescence, the cross section is σ = 10⁻¹⁶ cm² per molecule, compared to spontaneous Raman scattering, which ranges between 10^-26 and 10^-30 cm² per molecule and is therefore weaker [11]. Another new method, described by Peez et al. in 2018 [12], is the quantitative analysis of microplastics via ¹H NMR spectroscopy. While investment in a ¹H NMR spectroscopy instrument is cost intensive, ¹H NMR spectroscopy does not provide information regarding the morphology of the measured particles.

In previous work we discovered the potential of autofluorescence of plastics while working on a plastic particle sample extraction method [13]. With FLIM as a microspectroscopic method, it is possible to use this effect and to study the temporal resolution of fluorescence, which not only enables the detection of plastics, but also their characterization due to their specific fluorescence lifetime (τ). The fluorescence lifetime (τ) is the average time of how long a fluorophore remains in the excited state before emitting photons into the environment. This method is becoming more and more popular in the fields of (bio-)medical and biotechnical research and can be used as single and multiphoton FLIM, e.g. in fluorescence-guided surgery [14,15], insights to the cellular metabolism [[16], [17], [18], [19]], and 3D optical sectioning [20,21]. Further applications are the study of molecular interactions via Förster Resonance Energy Transfer (FRET) [[22], [23], [24], [25]] and use of FLIM-based sensors for monitoring microenvironmental parameters like pH, temperature and ion concentration [[26], [27], [28], [29]]. Fluorescence lifetime is a plastic type specific characteristic and is already employed in plastic detection and sorting during recycling processes [30,31]. The fluorescence decay can be fitted with an exponential function with one or multiple components, closely resembling the radioactive decay characterizing unstable isotopes of an element.

Thus, analysis of autofluorescence lifetime with FLIM technology should allow identification, differentiation and quantification of plastic particles as well as the determination of morphology and surface characteristic of individual particles. FLIM, as a non-destructive analysis method, offers the possibility of 3D representation of the particle in its surrounding environment thus potentially providing for a greater insight into microplastic-environment interactions. FLIM measures fluorescence lifetime as a (bio-)physical parameter and can be used as a measuring tool for fluorescence lifetime of materials or molecules, which can be sensitive to environmental changes. To determine the applicability FLIM for plastic particle detection at excitation wavelengths 470 nm and 440 nm, we generated microplastic particles from six different types of plastics and determined specific fluorescence lifetimes as well as the photon yield. Some of the latter plastics were also subjected to specific heat treatment to allow determination of the influence of heat treatment on the photon yield. Indeed, the photon yield determined using the two excitation wavelengths was sufficient to calculate three distinctive fluorescence lifetime components (τ₁, τ₂, τ₃). Differentiation of the microplastic species based on the specific fluorescence lifetimes was possible, thereby demonstrating the applicability of FLIM for non-destructive microplastic identification.

Section snippets

Sample material

Six different plastic types, i.e. polylactides (PLA) as filament from (Ultimaker PLA, Ultimaker B.V, Utrecht, Netherlands), polyphenylene oxide (PPE) as pellets from (Noryl, SABIC Europe Manufacturing, Geleen, Netherlands), polyamide 6 (PA6) as powder from (Ultramid, BASF SE, Ludwigshafen, Germany), acrylonitrile-butadiene-styrene copolymer (ABS) as pellets from (Kunststofftechnik Buzzi GmbH, Schiltach, Germany) and polyurethane (PU) from (Montageschaum B2, Soudal N.V., Leverkusen, Germany)

Excitation wavelength 440 nm

Selected values of the descriptive statistic for the five parameters, i.e. three fluorescence lifetimes (τ _1–3), the mean τ intensity weighted and amplitude weighted, which were employed to distinguish between plastic types (polymer) at 440 nm excitation are provided in Table 1, Table 2, Table 3, Table 4, Table 5, while the complete set of data obtained with the SP 8 FALCON are given in the Supporting Information (SI Table S4). The measurements carried out with this wavelength proved to be

Conclusion

This study investigates the fluorescence lifetime of different types of plastics (ABS, PA, PET etc.) and the influence of heat treatment on the fluorescence lifetime of selected plastics. A multiexponential emission decay was observed for the plastics and thus the determination of the fluorescence lifetimes 1–3 and the mean τ intensity weighted and amplitudes weighted. These generated fluorescence lifetimes could be used for a significant characterization of 94.55 % of the plastics.

Supporting Information

The Supporting Information is available free of charge.

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Implementation of cryogenic mill (Table S1) and thermal treatments (Table S2); Results of the one-way ANOVA (Table S3); Value tables of Fluorescence Lifetime Imaging microscopy measurements (Table S4 & S5); FLIM images with corresponding histograms and curve fittings (Figure S1-S15) (PDF)

CREDIT AUTHORS STATEMENTS

AM planned and carried out the experiments, did evaluations and wrote the manuscript. FW and HL helped with experimental planning and revising the manuscript. DR was deeply involved with experimental planning, data evaluation, writing and revising the manuscript.

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding sources

The author Adrian Monteleone is funded by the Ministry of Science, Research and Art Baden-Württemberg

Declaration of Competing Interest

There are no conflicts to declare

Acknowledgment

We would like to thank Leica Microsystems CMS + GmbH, Am Friedensplatz 3, 68165 Mannheim Germany for great support in giving us access to a Leica TCS SP8 FALCON system and for fruitful discussions. We would like to offer special thanks to Dr. Lars Kammermeier and Dr. Jens Peter Gabriel from the company Leica Microsystems for the discussion of the developed theories and the great support.

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New application for the identification and differentiation of microplastics based on fluorescence lifetime imaging microscopy (FLIM)

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Sample material

Excitation wavelength 440 nm

Conclusion

Supporting Information

CREDIT AUTHORS STATEMENTS

Author contributions

Funding sources

Declaration of Competing Interest

Acknowledgment

Chemosphere

Water Res.

Environ. Pollut.

Cell Rep.

Trends Biochem. Sci.

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Environ. Sci. Process. Impacts

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Environ. Sci. Technol.

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Anal. Methods

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Anal. Bioanal. Chem.

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The first application of quantitative 1H NMR spectroscopy as a simple and fast method of identification and quantification of microplastic particles (PE, PET, and PS)

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