Single-Particle Mid-Infrared Photothermal Imaging Reveals Hidden Heterogeneity in Real-World Micro- and Nanoplastics.

Polyethylene terephthalate (PET) is a widely used thermoplastic whose high crystallinity poses a major barrier to upscaling enzymatic recycling. Although PETases with high activity and stability have been reported, no enzyme has been shown to directly depolymerize crystalline PET (cPET), and the molecular determinants limiting their efficacy remain challenging to characterize. Here, we integrate experimental conformational ratios of crystalline and amorphous PET chains with enhanced-sampling molecular dynamics simulations to map the free-energy landscape of a prototypical PETase bound to PET oligomers, revealing how structural equilibria translate to catalytic function. We find that productive enzyme-substrate catalytic configurations can be reached for both crystalline and amorphous PET chains. However, the formation of catalytic ensembles on cPET is strongly hindered by the enzyme shape and dynamics, with additional energetic costs required to separate crystalline chains to fit the active site, consistent with experimental data. The model highlights limitations of the active-site architectures typically found in α/β-hydrolase scaffolds used for PET depolymerization and indicates directions for their redesign to enable cPET degradation. Our approach showcases a general strategy to leverage the quantitative characterization of catalytic ensembles for interrogating substrate-enzyme interactions and informing molecular design.

Poly(Ethylene Terephthalate) (PET) is one of the most used polymers for packaging applications. Modifications induced by service conditions and the means to make this matter circular have to be understood to really close the loop (from bottle to bottle for example). Physico-chemical properties, crystalline organisation, and mechanical behaviour of virgin PET (vPET) are compared with those of recycled PET (rPET). Using different combined experimental methods (Calorimetry, Small Angle X-ray Scattering [SAXS], Atomic Force Microscopy [AFM], Dynamic Mechanical Analysis [DMA], and uniaxial tensile test), it has been proven that even if there is no change in the crystallinity of PET, the crystallisation process shows some differences (size and number of spherulites). The potential impact of these differences on local mechanical characterisation is explored and tends to demonstrate the development of a homogeneous microstructure, leading to well-controlled and relevant local mechanical property characterisation. The main contribution of the present study is a better understanding of crystallisation of PET and recycled PET during forming processes such as thermoforming or Injection Stretch Blow Moulding (ISBM), during which elongation at the point of breaking can depend on the microstructure conditioned by the crystallisation process.

Mid-infrared photothermal (MIP) microscopy overcomes the resolution and huge water background limits in conventional mid-infrared imaging by probing the mid-infrared absorption induced photothermal effect. However, to detect the subtle MIP signal, large probe power and lock-in detection are needed, which limit the imaging speed of current MIP systems. To overcome this limitation, we develop a single-pixel pump-probe camera that leverages the large well-depth capacity of photodiode to achieve high-speed wide-field MIP imaging. With compressive sensing applied, close to video-rate MIP imaging can be achieved, offering a powerful label-free chemical imaging tool to scrutinize the complex biological systems.

An inductor-capacitor (LC) non-resonant photodiode detector has been developed and optimized for the signal characteristics of mid-infrared photothermal (MIP) microscopy. The detector incorporates a large photosensitive area (diameter = 5mm), a flat frequency response, direct current-alternating current (DC-AC) separation, and a single-photodiode configuration. A gain of 2.7 × 104 is achieved for weak AC signals, with a wide-flat -3 dB frequency response ranging from 4.7kHz to 2.1MHz. Compared with that of built-in detector of a standard MIP instrument, the signal-to-noise ratio of MIP imaging is increased by ∼5 times. The detector is simple to operate, highly resistant to external noise, and fulfills the imaging requirements of MIP experiments.

Recently developed mid-infrared photothermal (MIP) microscopy (for a Review, Science Advances, 2021, 7: eabg1559) not only overcomes the diffraction limit in direct IR imaging, but also circumvents the limitations in AFM-IR. In MIP microscopy, a visible beam probes the thermal effects induced by an intensity-modulated infrared beam. The MIP signals are measured in scanner manner or in wide-field manner. Video rate MIP imaging of living cells and tissues has been reached through a single pulse digitization approach. In this presentation, I will discuss the principle, instrumentation, and applications of MIP microscopy.

Using a visible beam to probe the thermal effect induced by infrared absorption, mid-infrared photothermal (MIP) microscopy allows bond-selective chemical imaging at submicron spatial resolution. Current MIP microscopes cannot reach the high wavenumber region due to the limited tunability of the existing quantum cascade laser source. We extend the spectral range of MIP microscopy by difference frequency generation (DFG) from two chirped femtosecond pulses. Flexible wavelength tuning in both C-D and C-H regions was achieved with mid-infrared power up to 22.1 mW and spectral width of 29.3 cm-1. Distribution of fatty acid in live human lung cancer cells was revealed by MIP imaging of the C-D bond at 2192 cm-1.

Midinfrared photothermal (MIP) microscopy, also called optical photothermal infrared (O-PTIR) microscopy, is an emerging tool for bond-selective chemical imaging of living biological and material samples. In MIP microscopy, a visible probe beam detects the photothermal-based contrast induced by a vibrational absorption. With submicron spatial resolution, high spectral fidelity, and reduced water absorption background, MIP microscopy has overcome the limitations in infrared chemical imaging methods. In this review, we summarize the basic principle of MIP microscopy, the different origins of MIP contrasts, and recent technology development that pushed the resolution, speed, and sensitivity of MIP imaging to a new stage. We further emphasize its broad applications in life science and material characterization, and provide a perspective of future technical advances.

We report rapid and sensitive phenotyping of bacterial response to antibiotic treatment at single-cell resolution by a Raman-integrated optical mid-infrared photothermal (MIP) microscope. The MIP microscope successfully detected biochemical changes of bacteria in specific to the acting mechanism of erythromycin with 1 h incubation. Compared to Raman spectroscopy, MIP spectroscopy showed a much larger signal-to-noise ratio at the fingerprint region at an acquisition speed as fast as 1 s per spectrum. The high sensitivity of MIP enabled detection of metabolic changes at antibiotic concentrations below minimum inhibitory concentration (MIC). Meanwhile, the single-cell resolution of the technique allowed observation of heteroresistance within one bacterial population, which is of great clinical relevance. This study showcases characterizing antibiotic response as one of the many possibilities of applying MIP microscopy to single-cell biology.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals.Key points• PET hydrolases must be thermophilic, but their operation must be below 70 °C• Classical and state-of-the-art engineering approaches are useful for PET hydrolases• Enzyme activity on crystalline PET is most expected for future PET biorecyclingGraphical

Polyethylene terephthalate (PET) fibers are characterized by exceptional mechanical strength, and textiles blended with cotton fibers combine both comfort and durability, showcasing widespread use in daily applications. However, improper disposal of discarded polyester-cotton textiles has resulted in severe environmental pollution, necessitating urgent and effective mitigation strategies. Enzymatic recycling of textiles offers superior environmental benefits and holds greater potential for industrial applications than alternative recycling methods. This study aims to explore a large-scale solution for the treatment of waste textiles, particularly addressing the challenge of resource recovery from polyester-cotton blended fabrics. An innovative enzymatic depolymerization process has been developed to achieve the recovery of high-purity terephthalic acid monomers. Experiments were conducted on three different textile blends with polyester-to-cotton ratios of 65/35, 70/30, and 80/20, and the influences of different colors on the process were investigated. Initially, the textiles were pretreated through mechanical grinding, which was followed by depolymerization of cotton fibers with commercial cellulase. The crystallinity of PET in the textiles was reduced through a rapid heating and cooling process. Subsequently, the PET was depolymerized by the engineered PET hydrolase. The results demonstrated that after decolorization and separation of terephthalic acid (TPA) from the reaction system, the monomer recovery rates for the three textile blends (65/35, 70/30, and 80/20) reached 90%, 91%, and 92%, respectively. Characterization analysis by nuclear magnetic resonance (NMR) confirmed that the purity of the recovered TPA was greater than 99%. In conclusion, the fully enzymatic recycling process developed in this study shows considerable promise for large-scale industrial applications and is anticipated to significantly advance the adoption and development of enzymatic recycling technologies for PET in industrial processes.

Mid-infrared photothermal (MIP) microscopy is a valuabletool forsensitive and fast chemical imaging with high spatial resolution beyondthe mid-infrared diffraction limit. The highest sensitivity is usuallyachieved with heterodyne MIP employing photodetector point-scans andlock-in detection, while the fastest systems use camera-based widefieldMIP with pulsed probe light. One challenge is to simultaneously achievehigh sensitivity, spatial resolution, and speed in a large field ofview. Here, we present widefield mid-infrared photothermal heterodyne(WIPH) imaging, where a digital frequency-domain lock-in (DFdLi) filteris used for simultaneous multiharmonic demodulation of MIP signalsrecorded by individual camera pixels at frame rates up to 200 kHz.The DFdLi filter enables the use of continuous-wave probe light, which,in turn, eliminates the need for synchronization schemes and allowsmeasuring MIP decay curves. The WIPH approach is characterized byimaging potassium ferricyanide microparticles and applied to detectlipid droplets (alkyne-palmitic acid) in 3T3-L1 fibroblast cells,both in the cell-silent spectral region around 2100 cm–1 using an external-cavity quantum cascade laser. The system achievedup to 4000 WIPH images per second at a signal-to-noise ratio of 5.52and 1 μm spatial resolution in a 128 × 128 μm fieldof view. The technique opens up for real-time chemical imaging offast processes in biology, medicine, and material science.

Microplastic (MP) pollution is increasingly acknowledged as a critical environmental and public health issue. This study sought to establish a robust, clinically compatible method for detecting MP particles in deparaffinized formalin-fixed paraffin-embedded (FFPE) human colon tissue sections, using protocols compatible with routine clinical pathology. We employed mid-infrared photothermal (MIP) microscopy—also referred to as optical photothermal infrared (OPTIR) spectroscopy—as a non-destructive, high-resolution technique for chemical characterization and spatial mapping of polymer particles in intact FFPE samples. Following OPTIR analysis, identical sections underwent hematoxylin and eosin (H&E) staining to facilitate precise histopathological evaluation in defined regions of interest. Using this integrated workflow, we detected and localized polyethylene (PE), polystyrene (PS), and polyethylene terephthalate (PET) particles (21 PE particles, 1 PS particle, and 1 PET fiber) within distinct tissue areas. Subsequent histological assessment revealed characteristic inflammatory features near to these identified MP particles. To our knowledge, this represents the first demonstration of a diagnostic workflow that enables combined infrared spectroscopic and histopathological analysis of MPs in routinely processed human FFPE tissue. This approach offers a promising avenue to elucidate the role of microplastic accumulation in human disease and supports further investigation into potential mechanistic links between MP exposure and inflammatory processes in the colon.

Non-destructive evaluation of thermally induced structural transformation in PET using terahertz time-domain spectroscopy

The purpose of this study was to investigate the changes induced by a lypolytic enzyme on the surface properties of polyethylene terephthalate (PET). Changes in surface hydrophilicity were monitored by means of water contact angle (WCA) measurements. Fourier Transform Infrared spectroscopy (FTIR) in the Attenuated Total Reflectance mode (ATR) was used to investigate the structural and conformational changes of the ethylene glycol and benzene moieties of PET. Amorphous and crystalline PET membranes were used as substrate. The lipolytic enzyme displayed higher hydrolytic activity towards the amorphous PET substrate, as demonstrated by the decrease of the WCA values. Minor changes were observed on the crystalline PET membrane. The effect of enzyme adhesion was addressed by applying a protease after-treatment which was able to remove the residual enzyme protein adhering to the surface of PET, as demonstrated by the behavior of WCA values. Significant spectral changes were observed by FTIR-ATR analysis in the spectral regions characteristic of the crystalline and amorphous PET domains. The intensity of the crystalline marker bands increased while that of the amorphous ones decreased. Accordingly, the crystallinity indexes calculated as band intensity ratios (1,341/1,410 cm(-1) and 1,120/1,100 cm(-1)) increased. Finally, the free carboxyl groups formed at the surface of PET by enzyme hydrolysis were esterified with a fluorescent alkyl bromide, 2-(bromomethyl)naphthalene (BrNP). WCA measurements confirmed that the reaction proceeded effectively. The fluorescence results indicate that the enzymatically treated PET films are more reactive towards BrNP. FTIR analysis showed that the surface of BrNP-modified PET acquired a more crystalline character.

Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates