Front. Environ. Sci. Eng.

2022, 16(1): 8
https://doi.org/10.1007/s11783-021-1442-2

REVIEW ARTICLE

What have we known so far for fluorescence staining and

quantification of microplastics: A tutorial review
Shengdong Liu1, Enxiang Shang (✉)2, Jingnan Liu1, Yining Wang1, Nanthi Bolan3,4,5,
M.B. Kirkham6, Yang Li (✉)1
1 Key Laboratory of Water and Sediment Sciences of Ministry of Education, State Key Laboratory of Water Environment Simulation,
School of Environment, Beijing Normal University, Beijing 100875, China
2 College of Science and Technology, Hebei Agricultural University, Huanghua 061100, China
3 School of Agriculture and Environment, The University of Western Australia, Perth, WA 6001, Australia
4 The UWA Institute of Agriculture, The University of Western Australia, Perth, WA 6001, Australia
5 Global Innovative Centre for Advanced Nanomaterials, College of Engineering, Science and Environment,
University of Newcastle, Callaghan, NSW 2308, Australia
6 Department of Agronomy, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, USA*

HIGHLIGHTS GRAPHIC ABSTRACT

• Fluorescence staining provides a fast and easy
method to quantify microplastics.
• Factors that influence staining are summarized to
obtain an optimum staining effect.
• Natural organic matter can be stained by dye and
interfere with quantification.
• Fluorescence staining is applied in both field and
laboratory studies.
• Future work involves developing new dyes and
automated image-analysis methods.

ARTICLE INFO ABSTRACT

Article history: Understanding the fate and toxicity of microplastics (MPs, < 5 mm plastic particles) is limited by
Received 23 July 2021 quantification methods. This paper summarizes the methods in use and presents new ones. First, sampling
Revised 22 September 2021 and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality
control are reviewed. Then the promising and convenient staining procedures and quantification methods
Accepted 12 October 2021 for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including
Available online 15 November 2021 their physicochemical properties, are summarized to provide an optimal operation procedure. In general,
the digestion step is crucial to eliminate natural organic matter (NOM) to avoid interference in
quantification. Chloroform was reported to be the most appropriate solvent, and 10–20 μg/mL are
Keywords: recommended as optimal dye concentrations. In addition, a heating and cooling procedure is
Plastic particles recommended to maintain the fluorescence intensity of MPs for two months. After staining, a
Fluorescence dyes fluorescence microscope is usually used to characterize the morphology, mass, or number of MPs, but
Identification compositional analysis cannot be determined with it. These fluorescence staining methods have been
Concentration quantification implemented to study MP abundance, transport, and toxicity and have been combined with other chemical
characterization techniques, such as Fourier transform infrared spectroscopy and Raman spectroscopy.
More studies are needed to focus on the synthesis of novel dyes to avoid NOM’s interference. They need
to be combined with other spectroscopic techniques to characterize plastic composition and to develop
image-analysis methods. The stability of stained MPs needs to be improved.
© The Author(s) 2021. This article is published with open access at link.springer.com and journal.hep.
com.cn 2021

✉ Corresponding author
E-mail: [email protected] (E. Shang), [email protected] (Y. Li)
Special Issue—Microplastic and Nanoplastic Pollution: Characterization, Transport, Fate, and Remediation Strategies (Responsible Editors: Wen
Zhang, Melissa Pasquinelli & Yang Li)
2 Front. Environ. Sci. Eng. 2022, 16(1): 8

1 Introduction to develop a convenient and cheap quantification method

for MPs present in the environment.
Over the past decades, the production and consumption of The fluorescence staining and quantification method
plastic products has risen rapidly (Thompson et al., 2004). provides a fast, convenient, and cheap way to quantify
Due to the continued discharge of plastic products and their MPs (Maes et al., 2017). This technique uses a dye called
low degradation rates, they accumulate in environmental Nile Red (NR, 9-diethylamino-5-benzo[α]-phenoxazi-
matrixes, including water, sediments, soils, and the none), a lipid soluble fluorescence dye, to stain neutral
atmosphere (Barnes et al., 2009; Liu et al., 2021). Once lipids in biological samples (Greenspan and Fowler, 1985).
the plastic products are discharged into the natural Afterwards, NR is used to stain synthetic polymers (Jee
environment, large particles may be fractured, weathered, et al., 2009). Since 2016, the staining ability of MPs by NR
or degraded into microplastics (MPs, 0.1 μm–5 mm) and has drawn the attention of researchers (Shim et al., 2016;
nanoplastics (NPs, £0.1 μm) via biodegradation and Erni-Cassola et al., 2017; Maes et al., 2017). Maes et al.
physical and chemical weathering processes (Browne proposed the identification and quantification method for
et al., 2011; Cole et al., 2011; Duan et al., 2021). MPs using NR staining and fluorescence microscopy, and
Commercial products, such as personal care products and they got an average recovery rate of 96.6% for marine
industrial beads, contain ultrafine plastic particles, which MPs, which was cross-validated by FTIR (Maes et al.,
are regarded as the primary source of MPs and NPs (Alimi 2017). Recently, heating protocols have been developed to
et al., 2018). The loads of MPs in freshwater, seawater, enhance the staining effect, and programmed-image-
soil, and sediment are 10–5–10 items/L, 10–6–10 items/L, analysis software has been applied to quantify fluorescence
1–104 items/kg, and 1–103 items/kg, respectively (McCor- of MPs automatically (Shim et al., 2016; Erni-Cassola
mick et al., 2014; Shahul Hamid et al., 2018; Zhang and et al., 2017; Karakolis et al., 2019). The MP fluorescence
Liu, 2018; Chen et al., 2020; Zhang et al., 2020). Studies staining and quantification method has achieved a recovery
have shown that MPs pose a threat to human health and rate of over 90%, which has been validated by FTIR using
ecosystems, because MPs can be transported via food the same batch of laboratory samples (Shim et al., 2016;
chains (Carbery et al., 2018; Sun et al., 2019; Liu and Erni-Cassola et al., 2017; Cook et al., 2020; Karakolis
Wang, 2020). The increasing load of MPs has drawn the et al., 2019). Therefore, the fluorescence staining and
attention of researchers (Wu et al., 2017; Guo et al., 2020; quantification method for MPs provides a promising and
Wang et al., 2021). Thus, studying their abundance is convenient technique for researchers.
important to assess the impact of MPs on health and to To summarize research on MP characterization and
ensure the security of ecosystems. quantification, a literature review of papers published
A fundamental step to understand the distribution, fate, between January 2016 and March 2021 was conducted. A
and toxicity of MPs is the identification and quantification total of 1411 articles were investigated with the following
of MPs. A variety of approaches have been developed to keywords: microplastics, quantification, characterization,
detect and characterize MPs (Shim et al., 2017; Fu et al., detection, sampling, fluorescence, and NR. As shown in
2020). However, the advantages and limitations of these Fig. 1, 32% of the research focused on quantification of
methods vary. Manual counting of MPs by optical MPs, which was more than research in any other area.
microscopy is one of the most widely used methods to Currently, most reviews have concentrated on character-
quantify MPs, which has the advantage of convenience but ization of physicochemical properties, transport, and
is limited by the operator’s subjectivity (Fu et al., 2020). transformation of MPs, as well as sampling, separation,
Scanning electron microscopy (SEM), transmission elec- and digestion procedures of MPs (Hidalgo-Ruz et al.,
tron microscopy (TEM), and atomic force microscopy 2012; Fu et al., 2020), but the reviews have ignored the
(AFM) can provide clear and high-resolution images of fluorescence-quantification method for MPs.
MPs, but these methods need to be combined with Thus, we focus on providing a comprehensive overview
spectroscopy methods to characterize the chemical com- of the fluorescence staining method to quantify MPs in
position of MPs (Patchaiyappan et al., 2020). Analysis by aquatic environments. The objective of this review is to
Fourier transform infrared spectroscopy (FTIR) and identify the optimum protocols and best operating
Raman spectroscopy can characterize the specific chemical conditions for fluorescence staining and quantification of
bonds of MPs, but they are difficult to use to quantify MPs MPs. First, procedures dealing with sample collection,
(Fu et al., 2020). Gas chromatography-mass spectrometry separation, digestion, identification, and quantification are
(GC-MS) is also an alternative method for measuring the summarized. Next, the advantages and limitations of dye
concentrations of monomers or additives in MPs, but it is a staining and the main factors that influence the dying
destructive method (Shim et al., 2017). All the above effect, including the physicochemical properties of MPs
methods require expensive instruments, experienced and environmental conditions, are summarized. Then, the
operators, time-consuming pretreatment processes, and method itself for the identification and quantification of
complex data analysis, except for the manual counting stained MPs is reviewed. After discussing the application
method via optical microscopy. Therefore, it is necessary of fluorescence staining and quantification methods for
 Shengdong Liu et al. Fluorescence staining for characterization of microplastics 3

MPs being done in current studies, knowledge gaps and shorelines and bottoms of rivers, lakes, or seafloors, the top
future perspective regarding standardized protocols for MP 0–5 cm layer of the surface of beaches and sediments is
quantification are proposed. most commonly collected (Patchaiyappan et al., 2020).
Sediment cores are also collected to study the occurrence
and transport of MPs in aquatic environments. For biota
2 Fluorescence staining methods for MPs samples, fish, invertebrates, and bivalves are most
frequently collected. Biota samples are generally collected
2.1 Sampling and pretreatment processes of MPs from wild environments, but some are sampled from
commercial operations (Mai et al., 2018). Processes used
Sampling and pretreatment processes of MPs include the to separate the MPs from collected samples include
collection of samples from the environment and the sieving, filtration, and drying, as shown in Table 1. Sieving
extraction of MPs from the samples. Approaches for is generally considered as the first step in sample
sampling and extracting MPs from various environmental processing for water and sand or sediment samples, and
matrixes, such as freshwater and seawater (Hidalgo-Ruz a mesh of 3 mm or 5 mm is used.
et al., 2012; Karakolis et al., 2019), sand and sediments
(Nuelle et al., 2014; Besley et al., 2017), and organisms 2.1.2 Digestion and density separation processes of MPs
and tissues (Claessens et al., 2013; Avio et al., 2015) have
been critically reviewed (Hidalgo-Ruz et al., 2012; Mai The widely distributed NOM in the environment can lead
et al., 2018; Prata et al., 2019a). Therefore, we summarize to overestimation of the particle number and environment
just the basic steps for the sampling and pretreatment concentration of MPs. Thus, the digestion step is usually
processes of MPs. We emphasize the importance of followed by sieving to eliminate NOM (Mai et al., 2018).
digestion protocols in fluorescence staining for MPs, The most commonly applied digestion protocol is adding
which directly influence the staining effect and the H2O2 (30%) and Fe(II) solution to samples (Prata et al.,
accuracy of detection. 2019a). Then the mixture was heated at 75°C for 0.5–4 h.
This digestion method is recommended by US National
2.1.1 Sample collection processes Oceanic and Atmospheric Administration (NOAA)
(Hanke et al., 2013). Moreover, the procedures of digestion
In field studies, different methods and equipment are depend on the source of environmental samples, which
applied to collect samples in specific environmental contain different concentrations of NOM. For instance, the
matrixes, including water, sand, sediment, the atmosphere, digestion for water samples with low content of NOM by
and biota (Table 1) (Dowarah et al., 2020; Scircle et al., H2O2 (30%) is enough, while for sediment or sand samples
2020; Valine et al., 2020). For water samples, manta nets or containing high concentration of NOM requires H2O2
trawls are commonly used to collect large-sized MPs (30%) with Fe(II) solution (0.05 M) and to be heated at
(>100 μm) in surface waters, whereas pumping with filters 75°C (Fu et al., 2020). When dealing with biota samples,
(100 or 300 μm) is used as a complemental sampling the digestion protocol is extremely important because of
method for smaller sized MPs ( < 100 μm). For sediment the high biomass content, and an enzyme (such as
and sand samples, which are generally sampled from proteinase K, chitinase, and cellulase) is commonly

Fig. 1 The proportion of research papers investigating the quantification and characterization of microplastic.
4 Front. Environ. Sci. Eng. 2022, 16(1): 8

Table 1 Sampling and pretreatment processes of MPs for fluorescence staining

Sample collection process Sample pretreatment process
Sample origin Sampling Sampling Sampling Density Extraction Ref.
Sieving Digestion Filtration
location equipment details separation recovery
Fresh water Kinnickinnic Glass jar NA NA NA 0.05 M FeSO4 Polycarbonate NA Simmerman
River, USA 1L H2SO4 filter & Wasik,
30% H2O2 0.4 μm 2020
Fresh water Four rivers, Plankton Below NA 84.2 mg/L 10% KOH Strainer NA Valine
USA tow net river NaCl 200 µm et al.,
200-μm surface 2020
0.3-1 m
Sea water Mississippi Glass jar Below 25-μm NA 0.05 M Fe (II) Polycarbonate NA Scircle
Sound, USA 946 mL surface mesh 30% H2O2 filter et al.,
water 10 µm 2020
Beach sand Three beaches, NA Top of 5-mm CaCl2 H2O2 Mesh 89.5%- Tiwari
India beach sand mesh 1.34 g/cm3 38 μm 97.5% et al.,
3–4 cm 2019
Sediment South Metal Top layer 3-mm NaCl 0.05 M Fe (II) Vacuum NA Patchaiyappan
Andaman spoon of beach 5-mm 1.2 g/cm3 30% H2O2 filtration et al., 2020
beaches, 1 cm mesh
India
Biota Kinnickinnic D-shaped NA NA NA 0.05 M FeSO4 Steel sieve NA Simmerman
(Macroinvertebrates) River, USA kick net 3mL H2SO4 20 μm & Wasik,
600-μm 30% H2O2 2020
Biota Puducherry Bought NA NA NA 10% KOH Vacuum NA Dowarah
(bivalve) coastline, in fish filtration et al., 2020
India market 11 μm
Biota Forth Stainless- NA NA Super- Enzyme Vacuum NA Catarino
(mussels) River, steel saturated mixture filtration et al., 2018
UK wired NaCl (Corolase 0.8 μm
scrubber 7089)
Atmosphere Hamburg PE-funnel Above NA NA 15% v/v Vacuum NA Klein &
metropolitan PE bottle ground NaClO filtration Fischer,
area, level 5–13 μm 2019
Germany 100 cm

NA means it is not available in the references.

applied to eliminate the tissue (Prata et al., 2019a). In samples are retained on the filter and the pour size of filter
addition, there are other digestion methods, such as acid determines the retention particle size. The filters with pour
digestion, alkali digestion, oxidizing digestion, and enzy- sizes in the range between 0.2 μm and 55 μm have been
matic digestion (Prata et al., 2019a). Detailed digestion used in different studies based on their purpose. The
procedures for each method have been discussed in smaller pour size of filter they used, the smaller sized
published reviews (Prata et al., 2019a). plastic particles can be retained on the filter for further
After the digestion step, density-separation procedure is detection. In addition, considering the visual observation
conducted to separate specific MPs from water, soil, or of MPs, the filter should not exhibit a fluorescence signal
sediment samples. Based on the different densities of and interfere the detection result. For example, a study
plastics (0.8–1.6 g/cm3) and sediment (2.7 g/cm3), density- tested 6 types of filters and found that only glass fiber filter
separation methods have been developed to separate MPs (1.2 μm) and black polycarbonate filters (0.2 μm) are
and sediment or sand by mixing the samples in salt- appropriate for the detection of NR-stained MPs without
saturated solutions and afterwards collecting the super- introduction of fluorescence intensity from the filters (Prata
natant, which contains MPs (Rocha-Santos and Duarte, et al., 2019b). Some plastic particles directly identified by
2015). In the density-separation step, the most frequently visual observation cannot use the special filter membrane.
used salt is NaCl, while other salts such as CaCl2, NaI, and After filtration, drying procedures (generally at 60°C) are
ZnBr2 are also available (Prata et al., 2019a). Filtration is carried out, and they are indispensable as the final steps for
necessary after the digestion protocol, where all the sample processing.
 Shengdong Liu et al. Fluorescence staining for characterization of microplastics 5

2.1.3 Quality assurance and quality control in the sampling important to get accurate and reliable data and high
process recovery rates during the entire sampling processes.

Guaranteeing quality assurance and quality control (QA/ 2.2 Methods for fluorescence staining of MPs
QC) in the sampling process is essential to get reliable
findings concerning the abundance of MPs. For example, Fluorescence methods are distinguished from regular,
procedural blanks (only containing water) and spiked optical approaches to detect and quantify MPs, because
blanks (containing water with known composition and they require a staining process during pretreatment. The
number of MPs) should be analyzed during sample fluorescence staining methods for MPs can be categorized
collection and processing of samples (Hanke et al., 2013; into staining MPs on filter paper or in solution. The first
Catarino et al., 2018). In addition, the recovery rate of MPs dying method for MPs on filter paper includes collecting
throughout the whole sampling process should be reported the environmental samples, separating and extracting the
to reveal the actual level of MPs in the environment. MPs from the environmental samples, placing the MPs on
Determining the recovery rate is also beneficial, because it filter paper (usually a polycarbonate filter paper), and
allows comparison of the abundance with other studies adding the dye solution on the filter paper to stain the MPs
(Wiggin and Holland, 2019). Currently, the recovery rates for a period of time (2–4 h) (Shim et al., 2016; Erni-
of generally used sampling processes (sieving, digestion, Cassola et al., 2017). After the staining procedure, the
density separation, and filtering) for MPs can reach number or concentrations of the stained MPs are detected
83.3%–96.6% (Maes et al., 2017; Tamminga, 2017). In by a fluorescence microscope or spectrophotometer.
field sampling, non-plastic collection tools and storage The other method is staining MPs in solution (Cook
containers should be used to avoid cross contamination. et al., 2020; Karakolis et al., 2019), as shown in Fig. 2. The
During laboratory sampling, it is necessary to wear latex first step is preparation of the staining solution (Fig. 2a),
gloves and cotton clothes to avoid contamination from which is an organic solvent that dissolves the NR. The
airborne fibers, which are widely detected in the environ- most commonly used solvents include methanol, chloro-
ment via atmospheric fallout (Ziajahromi et al., 2017). form, acetone, and n-hexane (Tamminga, 2017). The
Overall, quality assurance and quality control protocols are second step is suspending MPs in the staining solution

Fig. 2 MP staining process in solution. (a) adding Nile Red in solvent, (b) staining in solution while heating and cooling, (c) vacuum
filtering, (d) Nile Red stained MPs.
6 Front. Environ. Sci. Eng. 2022, 16(1): 8

(Fig. 2b). The heating and cooling procedures may fibers are especially difficult to be stained compared with
enhance the intensity of the fluorescence signal and inhibit particles and fragments due to their irregular shapes
leaching of the dye (Lv et al., 2019). The third step is (Tamminga, 2017). Moreover, the color of MPs may
filtering the staining solution to get the fluorescence- disturb the staining effect and quantification. Most
stained MPs and washing them with deionized water to get commonly used plastics are white, which plays a minor
rid of the adsorbed dye (Fig. 2c). Then, the fluorescence- role in detection after staining. But certain polymer types,
stained MPs are obtained, and they usually show red or like PVC in black color, are difficult to be stained and
purple color when NR is used as dye (Fig. 2d). detected (Shim et al., 2016).
In early studies, the staining method on filter paper was The composition of MPs determines the affinity between
commonly applied and was used for most types of MPs and dyes. As a hydrophobic dye, NR preferentially
polymers (Shim et al., 2016; Erni-Cassola et al., 2017). combines with polymer materials such as PP and PE that
In recent years, staining in solution is an improved method have a low polarity. Fluorescence staining using lipophilic
due to stirring and the addition of heating or cooling dyes, such as NR, has proven to be effective in
procedures. Therefore, recent studies tend to stain MPs in quantification of small particles of PE, PP, PS, PC, PUR,
solution (Stanton et al., 2019; Konde et al., 2020). This and PEVA, because of their high hydrophobicity, whereas
staining method requires MPs to be distributed in a PVC, PA, and PES could not be detected after staining due
homogeneous solution. Thus, it may be difficult to use with to their low hydrophobicity (Shim et al., 2016). Among the
certain types of MPs such as PE, which tends to float on the physicochemical properties of MPs, the chemical compo-
surface of water due to its low density. To solve this sition of the polymer plays a primary role in the staining
problem, researchers have suspended a powder of PE in a effect, and it determines the affinity between MPs and
mixture of ultrapure water and dimethyl sulfoxide (v = 1:1) dyes. To note, the fluorescence staining has minor effect on
for better distribution in solution (Cook et al., 2020). In the aggregation of MPs. On the one hand, Raman spectrum
conclusion, to get a better staining effect, the staining and FTIR spectra measurements prove that the stained
method for MPs in solution is recommended, which has MPs have almost no alteration on composition compared
the advantages of flexible heating or stirring procedures. to pristine MPs, because only a few dye molecules are
added into MPs surface by diffusion (Lv et al., 2019). On
2.3 Factors influencing the staining effect the other hand, the Zeta potentials of pristine PET MPs and
NR stained PET MPs have been measured, their Zeta
The staining effect of MPs depends on many factors, such potentials are – 6.000.78 mV and – 7.640.81 mV,
as dye concentration, solvent type, and temperature respectively. This proves that staining process has a minor
(Wiggin and Holland, 2019). In this section, we compre- effect on the surface charge of MPs. Similarly, the Zeta
hensively discuss how these factors influence the staining potentials of stained PE, PP, and PVC MPs were also
effect to develop an optimum MP staining protocol. measured and no significant alteration of Zeta potentials
has been observed.
2.3.1 Physicochemical properties of MPs
2.3.2 Experimental parameters
The physicochemical properties of MPs, such as particle
size, shape, and composition, affect the staining process 1) NR concentration
(Wiggin and Holland, 2019). The recovery rate of
fluorescent-stained MPs decreases as the size of particle Fluorescence intensity of stained MPs is influenced by the
decreases. For example, studies have found that recovery concentration of NR, which has been applied in the range
rates of NR-stained MPs decreased from 82% to 49% of 0.1 μg/mL and 500 μg/mL in current studies (Rumin
when the size decreased from 500 to 1000 μm to 20–63 et al., 2015; Lv et al., 2019). For example, Lv et al. found
μm, respectively (Wiggin and Holland, 2019), which may that fluorescence intensity first increased (0.1–25 μg/mL
be because the smaller particles are more difficult to be NR concentration) and then decreased (25–100 μg/mL NR
detected under a microscope. Moreover, approximately concentration) with NR concentration (Lv et al., 2019),
95% of particles with sizes larger than 1 mm could be which was attributed to NR aggregation at high dye
detected after staining by NR, while only 71.7% of concentration that decreased fluorescence intensity (Rumin
particles smaller than 1 mm could be detected (Tamminga, et al., 2015). Similarly, the fluorescence intensity increased
2017), which was primarily because the small particles and then decreased with rising dye concentration (0.1–
were stained less strongly and exhibited a weaker 100 μg/mL) when selecting Fluorescein isophosphate
fluorescence intensity; therefore, they were more difficult (FITC) to stain MPs. The fluorescence intensity increased
to be detected than larger particles. with Safranine T dye concentration increasing from
In addition, the shapes of the MPs can influence the 0.1 μg/mL to 100 μg/mL, while it changed slightly until
staining effect, which are generally categorized as the dye concentration reached saturation at 100 μg/mL (Lv
particles, fragments, or fibers. Studies have indicated that et al., 2019). Moreover, a high NR concentration, such as
 Shengdong Liu et al. Fluorescence staining for characterization of microplastics 7

500 μg/mL, may lead to saturation of spectrometer surface, or the entrance of dye inside polymer molecules,
intensity. An obvious red shift in fluorescence spectra of depends on temperature. Fluorescence is exhibited only for
PVC could interfere with the fluorescence measurement a short period when MPs are stained at room temperature
and analysis (Konde et al., 2020). Thus, attention should be (25°C) without heating (Prata et al., 2020). For example,
paid to avoid using too high a NR concentration for after 2 months, 73.5% of NR-stained MPs without heating
staining. Conversely, low NR concentration ( < 1 μg/mL) were found to have lost fluorescence (Prata et al., 2020),
exhibits too weak fluorescence signals to detect (Konde which was attributed to the desorption of dye molecules
et al., 2020). Therefore, an appropriate NR concentration from the MP surface because the dyes were just adsorbed
between 10 μg/mL and 20 μg/mL is suggested in NR onto the surface. Studies have found that the heating
staining protocols, which gets both adequate fluorescence protocol promotes the staining effect resulting in both
intensity and minimum interference (Tamminga, 2017). higher fluorescence intensity and a more stable florescence
signal for a long period (over 2 months) (Lv et al., 2019).
2) Solvent Fluorescence intensity of MPs has shown an increasing
trend with increasing temperatures from 20°C to 50°C
NR is a hydrophobic dye with poor solubility and weak (Konde et al., 2020). Most types of MPs have their
fluorescence in water (Greenspan and Fowler, 1985). It strongest fluorescence intensity at 50°C, except for PVC
first needs to be dissolved in a solvent for staining. The which has its strongest fluorescence at 75°C (Lv et al.,
solvent has a significant impact on the NR staining effect. 2019). Furthermore, heating protocols have enabled
On the one hand, the solvent directly affects the recovery stained MPs to maintain a stable fluorescence intensity
rate of the fluorescent-stained MPs. On the other hand, NR over 2 months (Lv et al., 2019). Heating protocols have
contains a polar carboxyl function (-COOH) on its been applied in several studies and to obtain high recovery
aromatic rings; therefore, NR fluorescence spectra exhibit rates (> 95%) (Cook et al., 2020; Karakolis et al., 2019; Lv
dependency on the polarity of solvents due to the et al., 2019). The heating temperature of staining solutions
solvatochromism of NR (Rumin et al., 2015). Because of in previous studies have generally been set from 50°C to
the relatively polar nature of NR molecules when 75°C (Cook et al., 2020; Karakolis et al., 2019; Lv et al.,
compared to plastics, the partitioning of NR molecules 2019; Konde et al., 2020). The temperature should be
from the solvent to plastics can be made more feasible in lower than the melting point of some polymer materials
non-polar solvents, such as n-hexane, than in polar such as LDPE (85°C). PVC and PET are less hydrophobic
solvents (Shim et al., 2016). plastics and, thus, are difficult to be stained by NR. The use
The use of various solvents, such as chloroform, of a heating protocol enables PVC and PET to exhibit
acetone, n-hexane, and methanol, has been summarized strong fluorescence intensity (Lv et al., 2019). Overall, to
in the literature (Tamminga, 2017; Konde et al., 2020). obtain stronger fluorescence intensity and a more stable
Tamminga et al. investigated the influence of three staining effect for MPs, the protocol of heating staining
solvents, which were acetone, chloroform, and n-hexane, solutions is recommended. When the melting point of
on the staining effect of MPs by NR. Chloroform was the polymer materials is considered, 50°C–60°C would be an
most appropriate solvent for HDPE, LDPE, PP, and PVC, appropriate temperature range to use for NR staining
and the lowest recovery rate was 83.3% (Tamminga, protocols.
2017). Nevertheless, they found that some polymer
materials, such as cellulose acetone and PS, had the 2.4 Advantages of fluorescence staining for MPs
tendency to dissolve in chloroform or acetone (Tamminga,
2017). Konde et al. proposed a mixed solvent consisting of Fluorescence staining for quantification of MPs provides a
acetone and ethanol with v/v = 1:1 for the investigation of straightforward, quick, cheap, and convenient technique to
MP photoluminescence spectra, and the mixed solvent detect mass concentration of MPs and to investigate their
exhibited strong fluorescence intensity without PVC distribution in environmental samples. Spectral methods or
deformation (Konde et al., 2020). Overall, it is necessary chromatographic methods can cost up to hundreds of
to select an appropriate solvent to avoid leaching of the dollars for each batch of samples, while the cost of NR is
additive or monomer from MPs; to develop specific only $ 8.36 USD/g plastic with the additional need for
fluorescence spectra for each solvent; and to get reliable fluorescence microscopy (Karakolis et al., 2019).
detection results of fluorescence-stained MPs. A mixture of Fluorescence-staining methods also have been repre-
solvents, like acetone and ethanol, appears to offer a good sented as a time efficient and convenient way for
choice for staining. quantification of MPs. The staining protocol of MPs
could be as short as 30 min for one batch of samples, while
3) Temperature spectral methods take a longer time for detection of MPs.
For example, FTIR requires at least nine hours to scan one
Temperature has a significant impact on the staining effect, filter paper (Shim et al., 2017) including the requirement of
because the adsorption of the dye onto the NP or MP sample pretreatment time. The work experience required
8 Front. Environ. Sci. Eng. 2022, 16(1): 8

for quantification of MPs via fluorescence techniques is After confirming the fluorescent particles as MPs, these
much less than that needed for spectral (i.e. FTIR) or mass particles are counted either by manual work or automated
spectrometry methods (Rocha-Santos and Duarte, 2015). software (such as ImageJ) to obtain the number of
Quantification of MPs via fluorescence can be operated by particles. To be specific, the total number of low abundance
automated photo-analysis software. In contrast, the mass of MPs can be counted directly on the entire filter. While
spectrometry method, like GC-MS, requires well-trained counting the high abundance of MP samples, it is
operators as well as time-consuming pretreatment pro- commonly to count the total particles from 3 random
cesses (Rocha-Santos and Duarte, 2015). Overall, the fields of view and averaging these counts, then comparing
major advantages of fluorescence-staining methods are that the viewed area with the entire filter area and normalizing
they are time efficient and easy to use, and they are the observed numbers to the whole filter area (Simmerman
appropriate for the detection of the abundance of MPs in and Wasik, 2020).
bulk environmental samples. The influence of the excitation wavelength on the
In addition, NR staining protocols improve the count detection of stained MPs has been explored. Prata et al.
efficiencies of smaller-sized MPs compared with those tested NR-stained mixtures of polymers (PE, PP, PS, PVC,
taken with regular visual quantification methods. A study EPS, nylon) and organic matter and excited them under
showed that NR staining increased the detection number of light with multiple wavelengths (254, 365, 470, 495, 530,
MPs in every sample, and the greatest increase of the 625 nm) (Prata et al., 2020). The results showed that
numbers observed was in the smaller sized fraction ( < 124 excitation light at 254 nm had the advantage of a high
μm) (Wiggin and Holland, 2019). Furthermore, this study contrast with the background signals without interference
showed that the reported levels of MPs determined via the with organic matter. But PS, PVC, nylon, virgin HDPE,
NR staining and counting approach were higher than those and weathered PE could hardly be detected under the
determined by other methods worldwide, and this was 254 nm excitation wavelength. Most polymers (PE, PP,
likely due to the inclusion of smaller sized MPs after HDPE, PS, EPS) and NOM could be excited under 470 nm
dyeing samples from a highly urbanized aquatic environ- at the same time. Therefore, application of the 470 nm
ment (Los Angeles, California) (Wiggin and Holland, excitation wavelength requires a digestion step to remove
2019). Overall, smaller-sized MPs are more likely to be NOM (Prata et al., 2019b). In conclusion, the 254 nm
detected by the NR staining method, and it avoids excitation wavelength is suitable for limited types of
underestimation of MP abundance in the environment. polymers (PE, PP) with the advantage of less interference
However, there are challenges for detecting the small-sized by NOM. The excitation wavelength at 470 nm can excite
MPs, and the detection limit by the current fluorescence most types of polymers but it can excite NOM as well,
staining method for the size of MPs was down to 3 μm which requires digestion procedures to avoid interference
(Wiggin and Holland, 2019). Meanwhile, the detection with the NOM’s fluorescence signal.
limit of MPs mass concentration by fluorescence staining During the identification procedures, the fluorescence
methods lies in the range between 1 mg/L and 100 mg/L signals from MPs have different colors (Fu et al., 2020).
(Li et al., 2019). The NR-stained plastics appear orange color in a chloro-
form solvent when excited by blue or UV light (Maes et al.,
2017; Tamminga, 2017). In addition, MPs can fluoresce in
3 Fluorescence identification and quantifi- varied light ranges when stained by different dyes. For
cation methods for MPs example, iDye pink (pink dye), iDye blue (blue dye), and
Rit DyeMore Kentucky Sky (Kentucky dye) can fluoresce
3.1 Identification in the red range, the far red range, and the green and red
(both) ranges, respectively (Karakolis et al., 2019).
Basically, identifying MPs with fluorescence microscopes With the help of image-analysis techniques, the hydro-
includes the following steps: 1) the samples are placed phobic and hydrophilic characteristics of fluorescence
under the fluorescence microscope and are excited with the particles can be identified. Using this method, a simple
proper excitation wavelength; 2) the samples are observed “fluorescence index” can be calculated as (R + G)/R (‘R’
or photographed under the proper emission wavelength; and ‘G’ are the 8-bit color intensity values of red and
3) the fluorescence signal or images are detected and green, respectively). This index represents the “polarity” of
analyzed for identification. In detail, the particles were the polymer surface, and the larger the value of the index
identified as MPs by the following criteria: 1) no cellular or is, the higher hydrophobicity of the polymer particles is
organic structure are observed; 2) fiber particles are (Maes et al., 2017; Tamminga, 2017; Wiggin and Holland,
uniform in thickness throughout their whole length and 2019). Recently, an automated counting software (MP-
have no three-dimensional bending; 3) the colored VAT, Microplastics Visual Analysis Toll) has been
particles should present clear and homogeneous colors; developed, which can be applied to detect the sizes and
4) fiber particles have no segment; 5) particles do not shine shapes of stained MPs through their emitted light (Prata
(Nor and Obbard, 2014; Klein and Fischer, 2019). et al., 2019b).
 Shengdong Liu et al. Fluorescence staining for characterization of microplastics 9

3.2 Quantification investigate their aggregation and settling mechanisms in

sandy water. In this study, a fluorescence spectrophot-
The identification of MPs is commonly followed by a ometer was used to measure the fluorescence intensity of
quantification procedure. Identification can provide infor- MPs, and then the MP mass concentration was further
mation about the morphology and surface properties of calculated using a standard curve (Li et al., 2019). In
MPs, while quantification can show the number and mass conclusion, fluorescence MPs provide a fast method for
concentrations of MPs in samples, in order to study quantification of MP concentration, and the particles can
abundance and occurrence of MPs (Mai et al., 2018). easily be detected using their fluorescence intensity.
Procedures for quantification with fluorescence micro- The quantification methods used for field samples and
scopes involve the following steps: 1) the MPs are excited laboratory samples are different. In one study, field
under an excitation wavelength; 2) their fluorescence samples were first photographed under a certain excitation
signal is observed; and 3) the fluorescence particles are wavelength (Klein and Fischer, 2019). Then, images were
counted or selected MPs are picked up for further mass analyzed by software to count the number of fluorescence
weighing and quantifying (Prata et al., 2020). plastic particles. The size and particle circularity could also
In addition, using a fluorescence spectrophotometer to be determined by image-analysis software (Sfriso et al.,
determine the mass concentration of MPs in a water matrix 2020). As for laboratory samples, they can be detected
is also feasible, based on a standard curve that is developed easily and accurately by using a fluorescence spectro-
to show the relationship between fluorescence intensity photometer, because they have a known polymer type and
and MP concentration (Li et al., 2019). An example for few impurities (Li et al., 2019). Recently, fully- or semi-
quantification of stained PET using a fluorescence spectro- automated analytical methods have been applied to
photometer is shown in Fig. 3. This method is especially quantify MPs, which offer a promising way of MP
appropriate for simulated samples done under laboratory quantification in the future. Confocal microscopy also
conditions, which consist of pure polymers and little NOM has provided a straightforward way to observe stained
(Shim et al., 2016). For example, a study was done MPs. A study demonstrated that artificially generated MPs
with fluorescence polystyrene nanoplastics (PSNPs, are more recognizable under confocal microscopy than
100 nm) and polyethylene MPs (PEMPs, 1.0–1.2 mm) to field samples (Maxwell et al., 2020). Therefore, confocal

Fig. 3 Quantification of MPs using fluorescence spectrophotometer (a) fluorescence stained PET microplastic when being excited,
(b) fluorescence stained PET microplastic under bright-field, Scale bar = 650 μm, (c) correlation curve between fluorescence intensity and
mass concentration of PET microplastic.
10 Front. Environ. Sci. Eng. 2022, 16(1): 8

microscopy could be more appropriate in laboratory tions of MPs were 45–220 items/kg of dry sand (South
studies (Maxwell et al., 2020). The use of Laser Confocal Andaman beach) and in the other study they were 161.7–
Raman Spectroscopy in identifying and quantifying MPs 973.3 items/kg of dry sand (Girgaon Mumbai, Tuticorin
can increase the sensitivity and accuracy of analysis. This beach, Dhanushkodi beach) (Tiwari et al., 2019; Patch-
advanced technology can identify the physical properties aiyappan et al., 2020). Simmerman et al. examined the MP
and chemical compositions of MPs, with the advantages of levels in water and organisms in a cold-water stream in
high spectral coverage, high lateral resolution, specific western Wisconsin, USA. They found that the concentra-
fingerprint spectrum and low interference from organic tions of MPs stained by NR in water ranged from 545 to
matter/water/fluorescence background signals (Sobhani 3622 items/L and increased significantly from upstream to
et al., 2019). Thus, Laser Confocal Raman Spectroscopy downstream. The mean MP concentrations downstream of
has a promising future especially in the study of transport an urban area were 2–3 times those found upstream
and transformation of nanostructured materials in natural (Simmerman and Wasik, 2020), which may be because
waters. MPs move down stream with the water flow. Klein et al.
examined the MP abundance in atmospheric disposition
via fluorescence MP quantification. They determined that
4 Application of information from studies of the mean MP abundance was 275 items/m2/d. To
fluorescence-stained MPs characterize the chemical composition of MPs, Raman
spectroscopy was combined with fluorescence quantifica-
4.1 Quantification of MPs input in the environment tion, and polyethylene/ethylvinyl acetate copolymers were
found to dominate within the metropolitan area of
Microplastics can enter the terrestrial, aquatic, and atmo- Hamburg, Germany (Klein and Fischer, 2019). In conclu-
spheric environments directly through indiscriminate sion, dye stained MPs and the fluorescence quantification
disposal of plastic wastes and indirectly through applica- approach have been applied in field samples to examine the
tion of waste resources containing plastics such as abundance of MPs, which are generally coupled with
biosolids and composts (Bradney et al., 2019; Kumar spectroscopic methods to characterize the composition of
et al., 2020). The fluorescence-quantification method of MPs (Vermaire et al., 2017; Klein and Fischer, 2019;
MPs has been developed in recent years, and it has been Patchaiyappan et al., 2020).
validated to be a straightforward and cost-effective way to
quantify the input of MPs into environments through 4.3 Investigation of distribution of MPs in organisms
various waste sources (Lares et al., 2019). For example,
Lares et al. compared six different methods to detect MPs Assessment of the toxicity of MPs in organisms requires
in municipal wastewater and digested sludge samples, characterization of their distribution in organisms. Fluor-
spiked with seven different types of plastic particles and escence detection methods have been widely applied in
fiber, and they suggested that a staining method using Rose distribution studies of MPs, which may facilitate toxicity
Bengal could be useful in separating MPs from other assessments of marine organisms and evaluation of human
materials (Lares et al., 2019). Another study examined the health risks (Catarino et al., 2018; Maxwell et al., 2020).
occurrence of MPs in wild mussels via fluorescence Maxwell et al. reported a novel counterstaining method by
staining using NR. The results showed that the mean NR, Evans blue, and Calcofluor white dyes to detect MPs
concentration of MPs entering the body of Modiolus (a in terrestrial, invertebrate samples. The results could be
kind of mussel) was 0.0860.031 items/g wet weight used for investigation of MP ingestion by soil animals
(Catarino et al., 2018). The authors also reported that the (Maxwell et al., 2020). They studied the tissue distribution
ingestion of airborne fibers by humans during a meal can of PS-MPs in red tilapia (O. niloticus) during a 14-d
be up to 13731– 68415 items/y/person. In these studies, the exposure period. They found that the concentration of PS-
staining method was applied to separate MPs from other MPs in the fish gut was 171.1  1043.5  104 μg/kg, and
materials and to quantify the input of MPs into the the concentration of microplastics in organs were in the
environment. order of gut>gills>livers≈brain (shown in Fig. 4) (Ding
et al., 2018). Overall, fluorescence staining of MPs can be
4.2 Measurement of MPs abundance in the environment used to study the distribution and accumulation of MPs in
tissues of organisms, and the results can be helpful for the
The fluorescence-quantification method has been applied assessment of the toxicity of MPs to evaluate human health
to measure the concentrations of MPs in samples of fresh risk.
water, seawater, soil, beach sand, sediment, airborne MPs,
and biota (Vermaire et al., 2017; Gagné et al., 2019; Klein 4.4 Investigation of the environmental fate and transport of
and Fischer, 2019; Simmerman and Wasik, 2020). In two MPs
studies in India, the abundance of MPs in beach sand was
determined by NR-staining MPs. In one study, concentra- Fluorescence staining and quantification methods have
 Shengdong Liu et al. Fluorescence staining for characterization of microplastics 11

Fig. 4 Photographs of fish tissues under a bright-field microscope (top row) and representative fluorescence images of PS-MPs in
different fish tissues after 14 d of the exposure to 100 μg/L (bottom row), Scale bar = 100 μm. Graph was adapted from ref (Ding et al.,
2018) with permission.

been applied to determine the environmental fate of MPs. 5.1 Strengthen research on the synthesis of novel dye to
Results from simulated, laboratory experiments are avoid interference of organic matter
extremely useful. In simulated experiments done under
laboratory conditions, Li et al. studied the aggregation Organic matter is ubiquitous in natural aquatic, terrestrial,
behavior of MPs in suspended sediment (SS) using or biotic environments. Dyes can co-stain it along with
fluorescence PSNPs and PEMPs to quantify the concen- plastic particles, which interferes with the detection of MP
tration of MPs (Li et al., 2019). They found that PSNPs and fluorescence intensity and results in overestimation of the
SS formed heteroaggregates and settled in a water column abundance of plastic particles in samples, and, conse-
at a settling velocity of 0.010 m/s, which can affect the quently, their accumulation and toxicity is overstated
distribution and fate of PSNPs in aquatic environments. In (Stanton et al., 2019). Therefore, pre-purification of
contrast, the settling velocity for heteroaggregates of samples is required to remove organic matter. In a study
PEMPs and SS was low (~10–5 m/s), which indicated with benthic invertebrate samples, it was found that the use
that the effect of SS on the settling and distribution of of the NR staining method should take digestion protocols
PEMPs is negligible. In addition, Cook et al. used NR- into consideration, because chitin cannot be removed by
stained PEMPs to measure their longitudinal dispersion H2O2 digestion. It can get stained by NR and exhibits a
coefficients in laboratory flumes (Cook et al., 2020). They strong fluorescence intensity leading to false positives
calculated that the longitudinal dispersion coefficients (Sfriso et al., 2020). Thus, future studies should be done to
ranged from 0.0030 to 0.0690 m2/s for PE particles. Their develop counterstaining methods with novel dyes to
results were used to establish a model concerning the distinguish the fluorescence intensity of MPs from that of
transport of MPs in natural rivers. In summary, fluores- organic matter in order to avoid interfere in the quantifica-
cence staining and quantification methods facilitate a tion of plastic particles.
comprehensive understanding of the fate and transport of
MPs in aquatic and terrestrial environments. 5.2 Combine fluorescence staining with other chemical
methods to analyze MPs composition

5 Conclusions and future prospects The fluorescence-staining technique facilitates the quanti-
fication of the number, concentration, or abundance of
The novel fluorescence staining and quantification method MPs in various environmental matrixes. However, the
is straightforward, quick, cheap, and reliable for quantifi- fluorescence-staining method cannot stain all types of
cation of MP abundance and MP distribution in the polymers, because of the different affinity between dyes
environment, including water, soil, sediments, and organ- and plastics (Stanton et al., 2019). For example, NR can
isms (Duan et al., 2021; Liu et al., 2021; Wang et al., stain plastics like PP, PE, PS, PC, EPS, PU, and PEVA but
2021). The method is especially suitable for detection of cannot stain plastics like PVC, PA, and PES (Shim et al.,
MPs in large, bulk environmental samples and laboratory 2016). In addition, the NR-staining method does not
samples. However, challenges regarding detection of MPs provide information about the chemical bonds of the
and quantification of their concentrations remain, as detected MPs, and the method may be combined with
described in the following sections. spectroscopic techniques to determine the chemical
12 Front. Environ. Sci. Eng. 2022, 16(1): 8

composition of plastic particles (Erni-Cassola et al., 2017). developed to provide the mapping image of the sample
To better elucidate the transport, availability, and toxicity and high lateral resolution of optical images (Sobhani
of MPs, combinational analysis, in which fluorescence et al., 2019). However, how to improve the resolution of
staining is used with chemical identification methods like measurement when detecting nanoplastics and overcoming
FTIR and IR, is a current trend in studies concerning the nano- scale effects are huge challenges. Future work is
occurrence and abundance of MPs. suggested to develop detection methods of nanoplastics to
improve superior analytical algorithms and enhance the
5.3 Develop image-analysis methods for quantification of weak signal from nanoplastics.
stained MPs
Acknowledgements This study was supported by the National Key R&D
Developing fully- or semi-automated image-analysis Program of China (Grant No. 2017YFA0605001), the National Natural
Science Foundation of China (Grant Nos. 52170024, 21677015 and
protocols when using the fluorescence staining and 22006031), the Natural Science Foundation of Hebei Province (No.
quantification method has a broad future. Currently, B2019204315), and the Sponsored Research Overhead Fund (Grant No.
manual counting of the numbers of MPs under fluorescent 472120) from Kansas State University.
or optical microscopy is the most applied of the methods
Open Access This article is licensed under a Creative Commons
used for the quantification of the abundance and
Attribution 4.0 International License, which permits use, sharing, adaptation,
concentration of MPs. It has a low efficiency and may distribution and reproduction in any medium or format, as long as you give
cause error. Therefore, automated image analysis would be appropriate credit to the original author(s) and the source, provide a link to the
helpful and provide researchers with both accuracy and Creative Commons licence, and indicate if changes were made. The images
efficiency to save time and labor. The challenge for the or other third party material in this article are included in the article’s Creative
future of image-analysis methods includes the counting of Commons licence, unless indicated otherwise in a credit line to the material.
If material is not included in the article’s Creative Commons licence and your
complex aggregates, which are widely distributed in intended use is not permitted by statutory regulation or exceeds the permitted
aquatic and terrestrial environments. use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
5.4 Improve the stability of stained MPs

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