Search Results (34)

Background: Multiphoton tomography (MPT) is a femtosecond laser imaging technique that enables high-resolution virtual biopsies of human skin. It provides a non-invasive method for analyzing cellular metabolism, structural changes, and responses to cosmetic products, providing insights into cell–cosmetic interactions. This review explores the principles, historical development, and key applications of MPT in cosmetic research. Methods: The latest MPT device combines five modalities: (i) two-photon fluorescence: visualizes cells, elastin, and cosmetic ingredients; (ii) second harmonic generation (SHG): maps the collagen network; (iii) fluorescence lifetime imaging (FLIM): differentiates eumelanin from pheomelanin and evaluates the impact of cosmetics on cellular metabolic activity; (iv) reflectance confocal microscopy (RCM): images cell membranes and cosmetic particles; and (v) white LED imaging for dermoscopy. Results: MPT enables in-depth examination of extracellular matrix changes, cellular metabolism, and melanin production. It identifies skin responses to cosmetic products and tracks the intratissue distribution of sunscreen nanoparticles, nano- and microplastics, and other cosmetic components. Quantitative measurements, such as the elastin-to-collagen ratio, provide insights into anti-aging effects. Conclusions: MPT is a powerful in vivo imaging tool for the cosmetic industry. Its superior resolution and metabolic information facilitate the evaluation of product efficacy and support the development of personalized skincare solutions. Full article

In this study, thin mouse kidney sections from healthy mice and those infected leading to acute and chronic sepsis were examined with two-photon excited fluorescence lifetime imaging (2P-FLIM) using the endogenous fluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The results presented show that this approach is a powerful tool for investigating cell metabolism in thin tissue sections. An adapted measurement routine was established for these samples by performing a spectral scan, identifying a combination of two excitation wavelengths and two detection ranges suitable for detailed scan images of NADH and FAD. Selected positions in thin slices of the renal cortex of nine mice (three healthy, three with chronic sepsis, and three with acute sepsis) were studied using 2P-FLIM. In addition, overview images were obtained using two-photon excited fluorescence (2PEF) intensity. This study shows that healthy kidney slices differ considerably from those with acute sepsis with regard to their fluorescence lifetime signatures. The latter shows a difference in metabolism between the inner and outer cortex, indicating that outer cortical tubular cells switch their metabolism from oxidative phosphorylation to glycolysis in kidneys from mice with acute sepsis and back in later stages, as seen for mice with chronic infections. These findings suggest that 2P-FLIM could serve as a powerful tool for early-stage sepsis diagnosis and monitoring metabolic recovery during treatment. Full article

Understanding the deterioration processes in wooden artefacts is essential for accurately assessing their conservation status and developing effective preservation strategies. Advanced imaging techniques are currently being explored to study the impact of chemical changes on the structural and mechanical properties of wood. Nonlinear optical modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), combined with fluorescence lifetime imaging microscopy (FLIM), offer a promising non-destructive diagnostic method for evaluating lignocellulose-based materials. In this study, we employed a nonlinear multimodal approach to examine the effects of artificially induced delignification on samples of Norway spruce (Picea abies) and European beech (Fagus sylvatica) subjected to increasing treatment durations. The integration of SHG/TPEF imaging and multi-component fluorescence lifetime analysis enabled the detection of localized variations in nonlinear signals and τ-phase of key biopolymers within wood cell walls. This methodology provides a powerful tool for early detection of wood deterioration, facilitating proactive conservation efforts of wooden artefacts. Full article

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools. Full article

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC₁₈(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage. Full article

Bioinert materials such as the zirconium dioxide and aluminum oxide are widely used in surgery and dentistry due to the absence of cytotoxicity of the materials in relation to the surrounding cells of the body. However, little attention has been paid to the study of metabolic processes occurring at the implant–cell interface. The metabolic activity of mouse 3T3 fibroblasts incubated on yttrium-stabilized zirconium ceramics cured with aluminum oxide (ATZ) and stabilized zirconium ceramics (Y-TZP) was analyzed based on the ratio of the free/bound forms of cofactors NAD(P)H and FAD obtained using two-photon microscopy. The results show that fibroblasts incubated on ceramics demonstrate a shift towards the free form of NAD(P)H, which is observed during the glycolysis process, which, according to our assumptions, is related to the porosity of the surface of ceramic structures. Consequently, despite the high viability and good proliferation of fibroblasts assessed using an MTT test and a scanning electron microscope, the cells are in a state of hypoxia during incubation on ceramic structures. The FLIM results obtained in this work can be used as additional information for scientists who are interested in manufacturing osteoimplants. Full article

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior. Full article

Information on the penetration depth, pathways, metabolization, storage of vehicles, active pharmaceutical ingredients (APIs), and functional cosmetic ingredients (FCIs) of topically applied formulations or contaminants (substances) in skin is of great importance for understanding their interaction with skin targets, treatment efficacy, and risk assessment—a challenging task in dermatology, cosmetology, and pharmacy. Non-invasive methods for the qualitative and quantitative visualization of substances in skin in vivo are favored and limited to optical imaging and spectroscopic methods such as fluorescence/reflectance confocal laser scanning microscopy (CLSM); two-photon tomography (2PT) combined with autofluorescence (2PT-AF), fluorescence lifetime imaging (2PT-FLIM), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and reflectance confocal microscopy (2PT-RCM); three-photon tomography (3PT); confocal Raman micro-spectroscopy (CRM); surface-enhanced Raman scattering (SERS) micro-spectroscopy; stimulated Raman scattering (SRS) microscopy; and optical coherence tomography (OCT). This review summarizes the state of the art in the use of the CLSM, 2PT, 3PT, CRM, SERS, SRS, and OCT optical methods to study skin penetration in vivo non-invasively (302 references). The advantages, limitations, possibilities, and prospects of the reviewed optical methods are comprehensively discussed. The ex vivo studies discussed are potentially translatable into in vivo measurements. The requirements for the optical properties of substances to determine their penetration into skin by certain methods are highlighted. Full article

We study the low-temperature (T = 4.7 K) emission dynamics of a thin film of methylammonium lead bromide (MAPbBr${}_3$), prepared via the anti-solvent method. Using intensity-dependent (over 5 decades) hyperspectral microscopy under quasi-resonant (532 nm) continuous wave excitation, we revealed spatial inhomogeneities in the thin film emission. This was drastically different at the band-edge (∼550 nm, sharp peaks) than in the emission tail (∼568 nm, continuum of emission). We are able to observe regions of the film at the micrometer scale where emission is dominated by excitons, in between regions of trap emission. Varying the density of absorbed photons by the MAPbBr${}_3$ thin films, two-color fluorescence lifetime imaging microscopy unraveled the emission dynamics: a fast, resolution-limited (∼200 ps) monoexponential tangled with a stretched exponential decay. We associate the first to the relaxation of excitons and the latter to trap emission dynamics. The obtained stretching exponents can be interpreted as the result of a two-dimensional electron diffusion process: Förster resonant transfer mechanism. Furthermore, the non-vanishing fast monoexponential component even in the tail of the MAPbBr${}_3$ emission indicates the subsistence of localized excitons. Finally, we estimate the density of traps in MAPbBr${}_3$ thin films prepared using the anti-solvent method at n∼10${}^{17}$ cm${}^{-3}$. Full article

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79–97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board. Full article

Having access to fluorescence lifetime, researchers can reveal in-depth details about the microenvironment as well as the physico-chemical state of the molecule under investigation. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore reported in the literature. This could be the reason for the impression that inconsistent results are obtained depending on which detection and excitation scheme is used. To clarify this controversy, the two most common techniques for measuring fluorescence lifetimes in the time-domain and in the frequency-domain were implemented in one single microscopy setup and applied to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the two techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, quantitative guidance as to which technique is most suitable for which research task and how to perform the experiment properly to obtain consistent fluorescence lifetimes is discussed. Full article

Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality. Full article

An amorphous carbon film with embedded detonation nanodiamond (DND) particles (a-C:ND) was produced by magnetron sputtering of nanodiamond powder. An Ag film was deposited on the carbon structure by radiofrequency magnetron sputtering. The silver film was irradiated with a 150 eV Ar⁺ to form plasmonic-active nanoparticles (NP) on the surface of the a-C:ND. The structure of the obtained a-C:ND and a-C:ND/Ag structures were studied by scanning and transmission electron microscopy, electron energy-loss spectroscopy, UV–Visible absorption spectroscopy, Raman spectroscopy, and fluorescence lifetime imaging at two-photon excitation. The analysis revealed 76% of sp³-carbon and a good dispersion of diamond nanoparticles in the a-C. Surface-enhanced Raman scattering (SERS) was applied to investigate the a-C:ND/Ag structure, allowing for the observation of SERS from the sp²-carbon species and the absence of significant a-C:ND damage after Ar⁺ irradiation of the Ag overlayer. A plasmonic-metal-enhanced luminescence was observed at one- and two-photon excitations, revealing a two- to five-fold intensity increase. The activity of the used DNDs was tested using the agar diffusion method and observed against the bacteria of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli and the fungi of Aspergillus niger, Aspergillus fumigatus, and the yeast of Candida albicans, showing DND activity against all the test strains of fungi. Full article

The stratum corneum (SC) forms a strong barrier against topical drug delivery. Therefore, understanding the penetration depth and pathways into the SC is important for the efficiency of drug delivery and cosmetic safety. In this study, TPT-FLIM (two-photon tomography combined with fluorescence lifetime imaging) was applied as a non-invasive optical method for the visualization of skin structure and components to study penetration depths of exemplary substances, like hydrophilic propylene glycol (PG), sodium fluorescein (NaFl) and lipophilic Nile red (NR) into porcine ear skin ex vivo. Non-fluorescent PG was detected indirectly based on the pH-dependent increase in the fluorescence lifetime of SC components. The pH similarity between PG and viable epidermis limited the detection of PG. NaFl reached the viable epidermis, which was also proved by laser scanning microscopy. Tape stripping and confocal Raman micro-spectroscopy were performed additionally to study NaFl, which revealed penetration depths of ≈5 and ≈8 μm, respectively. Lastly, NR did not permeate the SC. We concluded that the amplitude-weighted mean fluorescence lifetime is the most appropriate FLIM parameter to build up penetration profiles. This work is anticipated to provide a non-invasive TPT-FLIM method for studying the penetration of topically applied drugs and cosmetics into the skin. Full article

This study was aimed to investigate the applicability of the exosome fluorescence-lifetime imaging microscopy (FLIM) for colorectal cancer (CRC) diagnosis. Differential ultra-centrifugation was used to extract exosomes from the blood plasma of 11 patients with colon polyps (CPs) and 13 patients with CRC at the T2-4, N0-3, and M0-1 stages. Analysis was performed using a two-photon FLIM device. In total, 165 and 195 FLIM images were recorded for the CP and CCR patient groups, respectively. Two classes of exosomes differentiated by autofluorescence average lifetime ${\mathrm{t}}_{\mathrm{m}}$were discovered in the samples. The first class of exosomes with ${\mathrm{t}}_{\mathrm{m}}$ = (0.21 ± 0.06) ns was mostly found in samples from CRC patients. The second class with ${\mathrm{t}}_{\mathrm{m}}$ = (0.43 ± 0.19) ns was mostly found in samples from CP patients. The relative number of “CRC-associated” exosomes $N_{ch}$in the FLIM dataset was shown to be very small for the CP patient group and large for the CRC patient group. This difference was statistically significant. Therefore, the suggested CRS diagnostics criterion can be as follows. If $N_{ch}$ > 0.5, the probability of CRC is high. If $N_{ch}$ < 0.3, the probability of CRC is low. Full article