Universal guidelines for the conversion of proteins and dyes into functional nanothermometers

In the last decade, technological advances in chemistry and photonics have enabled real‐time measurement of temperature at the nanoscale. Nanothermometers, probes specifically designed to relay these nanoscale temperature changes, provide a high degree of temperature, temporal, and spatial resolution and precision. Several different approaches have been proposed, including microthermocouples, luminescence and fluorescence polarization anisotropy‐based nanothermometers. Anisotropy‐based nanothermometers excel in terms of biocompatibility because they can be built from endogenous proteins conjugated to dyes, minimizing any system perturbation. Moreover, the resulting fluorescent proteins can retain their native structure and activity while performing the temperature measurement, allowing precise temperature recordings from the native environment or during an enzymatic reaction in any given experimental system. To facilitate the future use of these nanothermometers in research, here we present a theoretical model that predicts the optimal sensitivity for anisotropy‐based thermometers starting with any protein or dye, based on protein size and dye fluorescence lifetime. Using this model, most proteins and dyes can be converted to nanothermometers. The utilization of these nanothermometers by a broad spectrum of disciplines within the scientific community will bring new knowledge and understanding that today remains unavailable with current techniques.     

Noninvasive detection of nanoscale structural changes in cornea associated with cross‐linking treatment

Corneal cross‐linking (CXL) using ultraviolet‐A (UVA) irradiation with a riboflavin photosensitizer has grown from an interesting concept to a practical clinical treatment for corneal ectatic diseases globally, such as keratoconus. To characterize the corneal structural changes, existing methods such as X‐ray microscopy, transmission electron microscopy, histology and optical coherence tomography (OCT) have been used. However, these methods have various drawbacks such as invasive detection, the impossibility for in vivo measurement, or limited resolution and sensitivity to structural alterations. Here, we report the application of oversampling nanosensitive OCT for probing the corneal structural alterations. The results indicate that the spatial period increases slightly after 30 minutes riboflavin instillation but decreases significantly after 30 minutes UVA irradiation following the Dresden protocol. The proposed noninvasive method can be implemented using existing OCT systems, without any additional components, for detecting nanoscale changes with the potential to assist diagnostic assessment during CXL treatment, and possibly to be a real‐time monitoring tool in clinics.     

Photon emission and changes in fluorescent properties of bone after laser irradiation

Laser scalpels used in medical surgery concentrate light energy, heating the tissues. Recently, we reported thermoluminescence emission from laser-treated soft tissues. Here we investigated the thermo-optical effects caused by a laser operating at 808 nm on animal bones (beef ribs) through luminescence and fluorescence imaging, thermal imaging and scanning electron microscopy. Laser-induced artificial lesions emitted luminescence peaking around 650 nm, with a half-life of almost 1 hour. As concerns fluorescence, 24 hours after laser treatment we observed an increase of the emission and a shift from 500 (untreated) to 580 nm (treated). Recrystallization observed by SEM indicates that the temperature in the artificial lesions is over 600°C. We can conclude that laser treatment induces specific luminescent and fluorescent emissions due to heating of the bone and modification of its components. Monitoring these emissions could help prevent tissue overheating and its potential damages during laser-assisted medical procedures.     

Dynamic imaging of zebrafish heart with multi-planar light sheet microscopy

Light sheet fluorescence microscopy has become a research hotspot in biomedicine because of low phototoxicity, high speed, and high resolution. However, the conventional methods to acquire three-dimensional spatial information are mainly based on scanning, which inevitably increases photodamage and is not real-time. Here, we propose a method to generate controllable multi-planar illumination with a dielectric isosceles triangular array and a design of multi-planar light sheet fluorescence microscopy system. We carry out experiments of three-dimensional illumination beam measurement, volumetric imaging of fluorescent microspheres, and dynamic in vivo imaging of zebrafish heart to evaluate the performance of this system. In addition, we apply this system to study the effects of bisphenol fluorene on the heart shape and heart-beating rate of zebrafish. Our experiment results indicate that the multi-planar light sheet microscopy system provides a novel and feasible method for three-dimensional selected plane imaging and low-phototoxicity in vivo imaging.     

Inside Cover: A novel in vivo Cerenkov luminescence image‐guided surgery on primary and metastatic colorectal cancer (J. Biophotonics 3/2020)

This study presents a novel intraoperative in vivo imaging approach which harnessed Cerenkov luminescence (CL) to detect primary and metastatic colorectal cancer (CRC) using clinically approved radiopharmaceuticals. In the mice and swine experiments, the proposed approach effectively improved the effect of CRC surgery. The approach is believed to be promising for utilizing CL in open surgery. Further details can be found in the article by Zeyu Zhang, Yawei Qu, Yu Cao et al. ( e201960152 )

     

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.     

Indocyanine green-enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA-approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG-RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15-fold at 24 h post-injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.     

Inside Cover

A novel 0.9 mm-diameter intravascular photoacoustic catheter with coaxial excitation and detection was developed to overcome the limitation of imaging range. A miniature ring-shaped ultrasound transducer with a 0.18 mm-diameter orifice in the center was successfully fabricated. The results demonstrated that the coaxial catheter exhibited much better photoacoustic/ultrasound imaging performance from the intima to the adventitia. Further details can be found in the article by Riqiang Lin, Qi Zhang, Shengmiao Lv, Jiaming Zhang, Xiatian Wang, Dongliang Shi, Xiaojing Gong, and Kwok-ho La ( e202200269 ).

     

Front Cover

The first post-pandemic face-to-face international conference in Biophotonics took place in Porto in April 2022. High quality and inspiring presentations have been delivered by leading scientists and internationally recognized professionals, standing on the forefront of Biophotonics research and representing broadness of the field. A selected number of these presentations resulted in the papers that are presented in this special issue. The cover images were selected from Hoffer et al., Lomanovskaya et al., Li et al., and Burton et al. (clockwise).

     

Front cover

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuouswave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application. Further details can be found in the article by Limin Wu, Yuye Wang, Haibin Li, Zelong Wang, Meilan Ge, Degang Xu, and Jianquan Yao ( e202100245 ).

     

Contrast‐enhanced optical coherence tomography for melanoma detection: An in vitro study

Optical coherence tomography (OCT), with a high‐spatial resolution (<10 microns), intermediate penetration depth (~1.5 mm) and volumetric imaging capability is a great candidate to be used as a diagnostic‐assistant modality in dermatology. At this time, the accuracy of OCT for melanoma detection is lower than anticipated. In this letter, we studied for the first time, the use of a novel contrast agent consist of ultra‐small nanoparticles conjugated to a melanoma biomarker to improve the accuracy of OCT for differentiation of melanoma cells from nonmelanoma cells, in vitro. We call this approach SMall nanoparticle Aggregation‐enhanced Radiomics of Tumor (SMART)‐OCT imaging. This initial proof of concept study is the first step toward the broad utilization of this method for high accuracy all types of tumor detection applications.     

Colorimetric recognition of aromatic amino acid enantiomers by gluconic acid-capped gold nanoparticles

In this work, we prepared gold nanoparticles (AuNPs) by employing gluconic acid (GlcA) as reducing-cum-stabilizing agent. The proposed GlcA-AuNPs successfully worked as a colorimetric sensor for visual chiral recognition of aromatic amino acid enantiomers, namely tyrosine (d/l-Tyr), phenylalanine (d/l-Phe), and tryptophan (d/l-Trp). After adding L-types to GlcA-AuNPs solution, the color of the mixture changed from red to purple (or gray), while no obvious color change occurred on the addition of D-types. The effect can be detected by naked eyes. The particles have been characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, zeta potential, the dynamic light scattering analysis as well as UV–Vis spectroscopy. This assay can be used to determine the enantiomeric excess of l-Trp in the range from 0 to + 100%. The method has advantages in simplicity, sensitivity, fast response, and low cost.

     

Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay

Lateral flow (LF) immunoassays (i.e., immunochromatographic assays) have traditionally been applied to analytes that do not require very high analytical sensitivity or quantitative results. The selection of potential analytes is often limited by the performance characteristics of the assay technology. Analytes with more demanding sensitivity requirements call for reporter systems enabling high analytical sensitivity. In this study, we systematically compared the performance of fluorescent europium(III) [Eu(III)] chelate dyed polystyrene nanoparticles and colloidal gold particles in lateral flow assays. The effect of time-resolved measurement mode was also studied. Because binder molecules used in immunoassays might not behave similarly when conjugated to different reporter particles, two model assays were constructed to provide reliable technical comparison of the two reporter systems. The comparative experiment demonstrated that the fluorescent nanoparticles yielded 7- and 300-fold better sensitivity compared with colloidal gold in the two test systems, respectively. Although the two reporter particles may induce variable effects using individual binders, overall the high specific activity of Eu(III) nanoparticles has superior potential over colloidal gold particles for the development of robust high-sensitivity bioaffinity assays.     

Cover Image

Probing amyloid formation of intrinsically disordered proteins (IDPs) in aqueous solutions with ionic liquids (ILs) could help identify the IL–IDP interactions causing the process in this media. Takekiyo et al. report that ethylammonium nitrate (EAN) initiates the formation of intermolecular β-sheet structure – amyloid-like aggregates – of α-synuclein. The cations and anions of EAN function as charges to mask side chains, leading to the formation of a polyproline II helix that assists the formation of α-synuclein aggregates. The present results could help develop promoting agents for amyloid aggregates in biomaterials science. (DOI: 10.1002/bip.23352)

     

Preclinical mouse model of optical coherence tomography for subcortical brain imaging without dissection

The purpose of this study was to investigate the feasibility of using optical coherence tomography (OCT) to identify internal brain lesions, specifically intracerebral hemorrhage, without dissection. Mice with artificially injected brain hematomas were used to test the OCT system, and the recorded images were compared with microscopic images of the same mouse brains after hematoxylin and eosin staining. The intracranial structures surrounding the hematomas were clearly visualized by the OCT system without dissection. These images reflect the ability of OCT to determine the extent of a lesion in several planes. OCT is a useful technology, and these findings could be used as a starting point for future research in intraoperative imaging.     

Raman spectroscopy‐based detection of RNA viruses in saliva: A preliminary report

Several non‐invasive Raman spectroscopy‐based assays have been reported for rapid and sensitive detection of pathogens. We developed a novel statistical model for the detection of RNA viruses in saliva, based on an unbiased selection of a set of 65 Raman spectral features that mostly attribute to the RNA moieties, with a prediction accuracy of 91.6% (92.5% sensitivity and 88.8% specificity). Furthermore, to minimize variability and automate the downstream analysis of the Raman spectra, we developed a GUI‐based analytical tool “RNA Virus Detector (RVD).” This conceptual framework to detect RNA viruses in saliva could form the basis for field application of Raman Spectroscopy in managing viral outbreaks, such as the ongoing COVID‐19 pandemic. ( http://www.actrec.gov.in/pi-webpages/AmitDutt/RVD/RVD.html ).     

Near-infrared emissive polymer-coated IR-820 nanoparticles assisted photothermal therapy for cervical cancer cells

Photothermal therapy (PTT) has attracted wide attention due to its noninvasiveness and its thermal ablation ability. As photothermal agents are crucial factor in PTT, those with the characteristics of biocompatibility, non-toxicity and high photothermal stability have attracted great interest. In this work, new indocyanine green (IR-820) was utilized as a photothermal agent and near-infrared (NIR) fluorescence imaging nanoprobe. To improve the biocompatibility, poly(styrene-co-maleic anhydride) (PSMA) was utilized to encapsulate the IR-820 molecules to form novel IR-820@PSMA nanoparticles (NPs). Then, the optical and thermal properties of IR-820@PSMA NPs were studied in detail. The IR-820@PSMA NPs showed excellent photothermal stability and biocompatibility. The cellular uptaking ability of the IR-820@PSMA NPs was further confirmed in HeLa cells by the NIR fluorescent confocal microscopic imaging technique. The IR-820@PSMA NPs assisted PTT of living HeLa cells was conducted under 793 nm laser excitation, and a high PTT efficiency of 73.3% was obtained.     

Colocalization of cellular nanostructure using confocal fluorescence and partial wave spectroscopy

A new multimodal confocal microscope has been developed, which includes a parallel Partial Wave Spectroscopic (PWS) microscopy path. This combination of modalities allows molecular‐specific sensing of nanoscale intracellular structure using fluorescent labels. Combining molecular specificity and sensitivity to nanoscale structure allows localization of nanostructural intracellular changes, which is critical for understanding the mechanisms of diseases such as cancer. To demonstrate the capabilities of this multimodal instrument, we imaged HeLa cells treated with valinomycin, a potassium ionophore that uncouples oxidative phosphorylation. Colocalization of fluorescence images of the nuclei (Hoechst 33342) and mitochondria (anti‐mitochondria conjugated to Alexa Fluor 488) with PWS measurements allowed us to detect a significant decrease in nuclear nanoscale heterogeneity (Σ), while no significant change in Σ was observed at mitochondrial sites. In addition, application of the new multimodal imaging approach was demonstrated on human buccal samples prepared using a cancer screening protocol. These images demonstrate that nanoscale intracellular structure can be studied in healthy and diseased cells at molecular‐specific sites.