Two-photon Fluorescence Imaging Research Articles

Two-photon imaging of rabbit mammary tissue with 3D reconstruction

A broadband two-photon fluorescence (TPF) imaging system, based on a femtosecond oscillator, has been constructed. This system enables TPF imaging of rabbit mammary gland tissue samples by detecting TPF signals generated from the tissue upon excitation by broadband femtosecond pulses. Specifically, TPF imaging utilizes a detection window of 565–615 nm to capture TPF signals from the fluorescent adenine dinucleotide (FAD) present in lactating cells within breast tissue. Moreover, the 3D reconstruction algorithm has been optimized to visualize the 3D structure of the rabbit mammary gland acinar cavity.The optimized three-dimensional reconstruction algorithm can reduce ambiguous signal regions, making the reconstructed three-dimensional structure more reliable and accurate. This is of significant importance for studying the microscopic structure and cell distribution of breast tissue, contributing to a deeper understanding of the tissue's physiological status.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE

BackgroundCysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. ResultsHere, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL). SignificanceThe outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Instantaneous thermometry imaging using two-photon laser-induced fluorescence.

Measuring temperature in complex two-phase flows is crucial for understanding the dynamics of heat and mass transfer. In this Letter, we introduce a novel, to the best of our knowledge, optical approach based on the combination of two-photon laser-induced fluorescence (2p-LIF) imaging and two-color laser-induced fluorescence (2CLIF) for instantaneous temperature mapping of complex liquid media. Using Kiton Red (KR) and Rhodamine 560 (R560), a temperature sensitivity of 1.54%/∘C has been achieved over a range of 17-60°C. The monitoring of two-dimensional transient temperature dynamics in the heating and degassing of water shows the efficiency of the 2p-2CLIF. This new approach contributes to the toolkit of optical temperature measurement techniques, providing a robust solution for studying transient scattering media and high-speed two-phase flows.

Two spirobifluene-based turn-on fluorescent probes for highly selective detection of Cysteine and the applications in cells two-photon fluorescence imaging

Two spirobifluene-based fluorescent probes SPF1 and SPF2, were designed and synthesized. The probes displayed “turn-on” fluorescence response for Cysteine. One of the challenges in developing a Cysteine probe is to secure high selectivity. SPF1/SPF2 can discriminate Cysteine from GSH as well as Hcy, and showed high substrate selectivity. The detection limit of SPF1 is 36 nM, which is excellent comparing with other optical sensors for Cysteine. The sensing mechanism of SPF1/SPF2 was verified by experimental data and theoretical calculations. There was a good linear relationship between the fluorescence intensity of SPF1/SPF2 and the concentration of Cysteine. The MTT tests indicated that SPF1/SPF2 had low cytotoxicity and good biocompatibility. Theoretical calculations demonstrated that SPF1, SPF2, and their related reaction products with Cysteine exhibited good two-photon absorption properties. Finally, SPF1/SPF2 had been successfully applied to the imaging of Cysteine in living cells under two-photon excitation.

Intelligent Biogenic Missile for Two-Photon Fluorescence Imaging-Guided Combined Photodynamic Therapy and Chemotherapy in Tumors.

Photodynamic therapy (PDT) is a significant noninvasive therapeutic modality, but it is often limited in its application due to the restricted tissue penetration depth caused by the wavelength limitations of the light source. Two-photon (TP) fluorescence techniques are capable of having an excitation wavelength in the NIR region by absorbing two NIR photons simultaneously, which offers the potential to achieve higher spatial resolution for deep tissue imaging. Thus, the adoption of TP fluorescence techniques affords several discernible benefits for photodynamic therapy. Organic TP dyes possess a high fluorescence quantum yield. However, the biocompatibility of organic TP dyes is poor, and the method of coating organic TP dyes with silica can effectively overcome the limitations. Herein, based on the TP silica nanoparticles, a functionalized intelligent biogenic missile TP-SiNPs-G4(TMPyP4)-dsDNA(DOX)-Aptamer (TGTDDA) was developed for effective TP bioimaging and synergistic targeted photodynamic therapy and chemotherapy in tumors. First, the Sgc8 aptamer was used to target the PTK7 receptor on the surface of tumor cells. Under two-photon light irradiation, the intelligent biogenic missile can be activated for TP fluorescence imaging to identify tumor cells and the photosensitizer assembled on the nanoparticle surface can be activated for photodynamic therapy. Additionally, this intelligent biogenic missile enables the controlled release of doxorubicin (DOX). The innovative strategy substantially enhances the targeted therapeutic effectiveness of cancer cells. The intelligent biogenic missile provides an effective method for the early detection and treatment of tumors, which has a good application prospect in the real-time high-sensitivity diagnosis and treatment of tumors.

Highly crystallization-induced emissive luminophores with mechanoluminescent features for two-photon harvesting fluorescence imaging and latent fingerprint identification

Highly crystallization-induced emissive luminophores with mechanoluminescent features for two-photon harvesting fluorescence imaging and latent fingerprint identification

Idebenone ameliorates statin-induced myotoxicity in atherosclerotic ApoE−/− mice by reducing oxidative stress and improving mitochondrial function

Statins are the first line of choice for the treatment for atherosclerosis, but their use can cause myotoxicity, a common side effect that may require dosage reduction or discontinuation. The exact mechanism of statin-induced myotoxicity is unknown. Previous research has demonstrated that the combination of idebenone and statin yielded superior anti-atherosclerotic outcomes. Here, we investigated the mechanism of statin-induced myotoxicity in atherosclerotic ApoE−/− mice and whether idebenone could counteract it. After administering simvastatin to ApoE−/− mice, we observed a reduction in plaque formation as well as a decrease in their exercise capacity. We observed elevated levels of lactic acid and creatine kinase, along with a reduction in the cross-sectional area of muscle fibers, an increased presence of ragged red fibers, heightened mitochondrial crista lysis, impaired mitochondrial complex activity, and decreased levels of CoQ9 and CoQ10. Two-photon fluorescence imaging revealed elevated H2O2 levels in the quadriceps, indicating increased oxidative stress. Proteomic analysis indicated that simvastatin inhibited the tricarboxylic acid cycle. Idebenone treatment not only further reduced plaque formation but also ameliorated the impaired exercise capacity caused by simvastatin. Our study represents the inaugural comprehensive investigation into the mechanisms underlying statin-induced myotoxicity. We have demonstrated that statins inhibit CoQ synthesis, impair mitochondrial complex functionality, and elevate oxidative stress, ultimately resulting in myotoxic effects. Furthermore, our research marks the pioneering identification of idebenone's capability to mitigate statin-induced myotoxicity by attenuating oxidative stress, thereby safeguarding mitochondrial complex functionality. The synergistic use of idebenone and statin not only enhances the effectiveness against atherosclerosis but also mitigates statin-induced myotoxicity.

The use of NADH anisotropy to investigate mitochondrial cristae alignment

Life may be expressed as the flow of electrons, protons, and other ions, resulting in large potential difference. It is also highly photo-sensitive, as a large proportion of the redox capable molecules it relies on are chromophoric. It is thus suggestive that a key organelle in eukaryotes, the mitochondrion, constantly adapt their morphology as part of the homeostatic process. Studying unstained in vivo nano-scale structure in live cells is technically very challenging. One option is to study a central electron carrier in metabolism, reduced nicotinamide adenine dinucleotide (NADH), which is fluorescent and mostly located within mitochondria. Using one and two-photon absorption (340–360 nm and 730 nm, respectively), fluorescence lifetime imaging and anisotropy spectroscopy of NADH in solution and in live cells, we show that mitochondria do indeed appear to be aligned and exhibit high anisotropy (asymmetric directionality). Aqueous solution of NADH showed an anisotropy of ~ 0.20 compared to fluorescein or coumarin of < 0.1 and 0.04 in water respectively and as expected for small organic molecules. The anisotropy of NADH also increased further to 0.30 in the presence of proteins and 0.42 in glycerol (restricted environment) following two-photon excitation, suggesting more ordered structures. Two-photon NADH fluorescence imaging of Michigan Cancer Foundation-7 (MCF7) also showed strong anisotropy of 0.25 to 0.45. NADH has a quantum yield of fluorescence of 2% compared to more than 40% for photoionisation (electron generation), when exposed to light at 360 nm and below. The consequence of such highly ordered and directional NADH patterns with respect to electron ejection upon ultra-violet (UV) excitation could be very informative—especially in relation to ascertaining the extent of quantum effects in biology, including electron and photonic cascade, communication and modulation of effects such as spin and tunnelling.

Tumor Microenvironment-Regulating Two-Photon Probe Based on Bimetallic Post-Coordinated MOF Facilitating the Dual-Modal and Deep Imaging-Guided Synergistic Therapies.

The intricate tumor microenvironment (TME) always brings about unsatisfactory therapeutic effects for treatments, although nanomedicines have been demonstrated to be highly beneficial for synergistic therapies to avoid the side effects caused by the complexity and heterogeneity of cancer. Developing nanotheranostics with the functionalities of both synergistic therapies and TME regulation is a good strategy but is still in its infancy. Herein, an "all-in-one" nanoplatform for integrated diagnosis and treatment, namely, Carrier@ICG@DOX@FA (CIDF), is constructed. Benefiting from the bimetallic coordination of Eu3+-HTHA (4,4,4-trifluoro-1-(9-hexylcarbazol-3-yl)-1,3-butanedione) and Fe3+ with the ligands in UiO-67, CIDF can simultaneously achieve two-photon fluorescence imaging, fluorescent lifetime imaging in deep tumors, and regulation of TME. Owing to its porosity, CIDF can encapsulate indocyanine green as photosensitizers and doxorubicin as chemotherapeutic agent, further realizing light-controlled drug release. Moreover, CIDF exhibited good biocompatibility and tumor targeting by coating with folic-acid-modified polymers. Both in vitro and in vivo experiments demonstrate the excellent therapeutic efficacy of CIDF through dual-modal-imaging-guided synergistic photothermal-, photodynamic-, and chemotherapy. CIDF provides a new paradigm for the construction of TME-regulated synergistic nanotheranostics and realizes the complete elimination of tumors without recurrence.

Label-free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy.

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two-photon excitation fluorescence and second-harmonic generation imaging to label-free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM.

Monitoring the response of a model protocell to dye and surfactant molecules through second harmonic generation and fluorescence imaging.

Probing the interaction between molecules and protocells is crucial for understanding the passive transport of functional molecules in and out of artificial and real cells. Second-harmonic generation (SHG) has been proven to be a powerful method for analyzing the adsorption and cross-membrane transport of molecules on lipid bilayers. In this study, we used SHG and two-photon fluorescence (TPF) imaging to study the interaction of charged dye molecules (D289) with a lipid vesicle. Unexpectedly, it was observed that the transport of D289 at a relatively high concentration is not as efficient as that at a lower dye concentration. Periodic shrinking of the model protocell and discharging of D289 out from the vesicle were revealed by combined analyses of SHG and TPF images. The response of the vesicle to a surfactant was also analyzed with D289 as a probe. This work demonstrates that the combined SHG and TPF imaging method is a unique approach that can provide detailed information on the interaction of molecules and lipids (both morphology and molecular kinetics). Determining these subtle interfacial kinetics in molecules is important for understanding the mechanism of many biophysical processes occurring on lipids.

Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging.

In this paper, three fluorescent protein chromophore analogs PFPAr (PFPP, PFPC, and PFPT) were synthesized and proved to be useful for photodynamic therapy and two-photon fluorescence imaging. By adding five- or six-membered aromatic heterocycles to the photosensitizer PFP, we obtained three fluorescent protein photosensitizers PFPAr with better performances. As a demonstration, compared with the reported photosensitizer PFP, photosensitizer PFPP exhibits larger emission wavelengths (701 nm) and achieves a slight enhancement in the efficiency of singlet oxygen (ΦΔ = 23%). Notably, PFPP can perform good two-photon fluorescence imaging with an 800 nm femtosecond laser in zebrafish. In in vitro cytotoxicity assays, PFPP shows good phototoxicity (IC50 = 4.12 μM) and acceptable dark toxicity (cell viability assay >90%). The reactive oxygen imaging experiments and AO/EB double staining assay indicate that PFPP can generate singlet oxygen to eliminate A-549 tumor cells effectively with photoexcitation of 460 nm blue light (20 mW cm-2). Furthermore, PFPP can label the lysosomes of tumor cells with high specificity for lysosomes (Pearson's correlation coefficient of 0.91). Thus, our study demonstrated that the rational introduction of aromatic heterocycles into fluorescent protein photosensitizers can effectively enhance the key parameters of photosensitivity and pave the way for further two-photon photodynamic therapy.

Characterization of degradation effects on wood ultrastructure by non-linear imaging

The characterization of deterioration processes in wooden artifacts is crucial for assessing their state of conservation and ensuring their preservation. Advanced imaging techniques are currently being explored to study the effect of chemical changes on the structural and mechanical properties of wood. Combined second harmonic generation and two-photon excited fluorescence (SHG/TPEF) imaging is a recently introduced non-destructive method for the analysis of cellulose-based samples. The study of age-related wood degradation based on nonlinear signal variation is a promising avenue. This work involves nonlinear multimodal analysis of naturally aged and dendrochronologically dated spruce samples. SHG/TPEF imaging and fluorescence lifetime imaging microscopy (FLIM) were used to demonstrate the influence of molecular deterioration and rearrangement of biopolymers on the fluorescence emitted by lignin and the second harmonic signal generated by cellulose. Imaging based on spectral filter detection and time-resolved analysis of the nonlinear fluorescence signal was used to delineate and potentially quantify ageing-induced morpho-chemical changes in the ultrastructure of wood cells. The analysis of cell structures by optical sectioning revealed variations between wood samples of different ages and different cell structures.

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy.

In this study, two water-soluble red fluorescent protein (RFP) dimers, FP2R' and FP2R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP2R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient (β) was 2.1 × 10-11 cm W-1. Further, the RFP dimer FP2R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1O2 and O2˙- simultaneously. Under 460 nm illumination, the photosensitizer FP2R'' showed high phototoxicity with an IC50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP2R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP2R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP2R'' effectively produced 1O2 and O2˙- in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP2R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP2R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.

Near-infrared BODIPY photosensitizers for two-photon excited singlet oxygen generation and tumor cell photodynamic therapy.

In this paper, two near-infrared BODIPY photosensitizers, Id-BDPI and Cz-BDPI, were obtained by modifying the indole and carbazole aromatic heterocycles in the core of BODIPY. The maximum absorption wavelengths of Id-BDPI and Cz-BDPI were 694 nm and 722 nm, and their singlet oxygen yields were 48% and 48.4%, respectively. In the simulated tumor cell photodynamic therapy, Id-BDPI and Cz-BDPI could effectively inhibit the growth of A549 tumor cells under near-infrared light. Meanwhile, the lysosomal co-localization coefficients of Id-BDPI and Cz-BDPI with A549 tumor cells were 0.94 and 0.89, respectively, showing high lysosomal targeting ability and biocompatibility. The two-photon absorption cross sections measured at 1050 nm by the Z-scanning method were 661.8 GM and 715.6 GM, respectively, and Cz-BDPI was further successfully applied to two-photon fluorescence imaging and two-photon excited singlet oxygen generation in zebrafish. The above results indicate that the introduction of aromatic heterocycles can effectively enhance the photodynamic efficacy of BODIPY photosensitizers, and the larger two-photon absorption cross section also brings potential for two-photon photodynamic therapy applications.

Heavy-atom-free BODIPY dendrimer: utilizing the spin-vibronic coupling mechanism for two-photon photodynamic therapy in zebrafish.

In this study, the heavy-atom-free BODIPY dendrimer TM4-BDP was synthesized for near-infrared photodynamic therapy, and was composed of a triphenylamine-BODIPY dimer and four 1-(2-morpholinoethyl)-1H-indole-3-ethenyl groups. The TM4-BDP could achieve near-infrared photodynamic therapy through two different photosensitive pathways, which include one-photon excitation at 660 nm and two-photon excitation at 1000 nm. In the one-photon excitation pathway, the TM4-BDP could generate singlet oxygen and superoxide radicals under 660 nm illumination. In addition, the one-photon PDT experiment in human nasopharyngeal carcinoma (CNE-2) cells also indicated that the TM4-BDP could specifically accumulate in lysosomes and show great cell phototoxicity with an IC50 of 22.1 μM. In the two-photon excitation pathway, the two-photon absorption cross-section at 1030 nm of TM4-BDP was determined to be 383 GM, which means that it could generate reactive oxygen species (ROS) under 1000 nm femtosecond laser excitation. Moreover, the two-photon PDT experiment in zebrafish also indicated the TM4-BDP could be used for two-photon fluorescence imaging and two-photon induced ROS generation in biological environments. Furthermore, in terms of the ROS generation mechanism, the TM4-BDP employed a novel spin-vibronic coupling intersystem crossing (SV-ISC) process for the mechanism of ROS generation and the femtosecond transient absorption spectra indicated that this novel SV-ISC mechanism was closely related to its charge transfer state lifetime. These above experiments of TM4-BDP demonstrate that the dendrimer design is an effective strategy for constructing heavy-atom-free BODIPY photosensitizers in the near-infrared region and lay the foundation for two-photon photodynamic therapy in future clinical trials.

Biodegradable fluorescent protein chromophore nanoparticles for hypoxic two-photon photodynamic therapy.

In this paper, biodegradable red fluorescent protein (RFP) chromophore analogue DPFP-SS-FA nanoparticles were synthesized for hypoxic two-photon photodynamic therapy. The maximum emission wavelength of DPFP-SS-FA is in the red-to-near-infrared region at 674 nm. Interestingly, these DPFP-SS-FA nanoparticles remain stable under physiological conditions, but deplete glutathione and disintegrate into the RFP chromophore analogue monomer in the tumor microenvironment. Meanwhile, electron paramagnetic resonance data have shown that DPFP-SS-FA produced enhanced 1O2/O2˙- signals after glutathione depletion causing an enhanced PDT effect. DPFP-SS-FA has negligible cell dark toxicity and high phototoxicity in hypoxic environments, indicating the outstanding hypoxia-overcoming ability of DPFP-SS-FA. In addition, due to its folic acid receptor and lysosome dual-targeting ability, DPFP-SS-FA is highly enriched in A-549 tumor cells. In particular, the hypoxic two-photon photodynamic therapy mediated by DPFP-SS-FA nanoparticles was validated in a zebrafish tumor model. Under 800 nm two-photon excitation, DPFP-SS-FA enabled bright two-photon fluorescence imaging and significantly inhibited the growth of tumor cells in zebrafish. The biodegradable DPFP-SS-FA nanoparticles reasonably constructed in this study can serve as excellent candidates for efficient hypoxic two-photon photosensitizers to treat deep tumor tissues.

PLANAR TWO-PHOTON FLUORESCENCE IMAGING OF DENSE SPRAY TO ESTIMATE SPRAY CHARACTERISTICS: APPLICATION IN PRESSURE-SWIRL ATOMIZERS

The dense spray produced at the primary stage of atomization in a pressure-swirl atomizer is characterized in this work. The optically dense regime, from continuous liquid stream to first-step breakup into liquid structures, is acquired using a two-photon planar laser-induced fluorescence (2p-PLIF) technique. A notable advantage of 2p-PLIF over conventional PLIF is the attenuation of multiple scattering by simultaneous absorption of two photons in an ultra-short pulse duration. This approach is able to capture the complex interface morphology of spray structures. A curvature-based analysis of the near field is carried out to predict far-field spray characteristics. This methodology was recently introduced by Palanti et al. (2022) to investigate numerical simulation of atomizing liquid flows. The present work extends its application to experimental images. The atomization process is described through the curvature distribution in different regimes. The spray characteristics are predicted from the early stage of atomization and are reasonably comparable with those of direct measurement by phase Doppler anemometry (PDA) in the later stage of atomization. The present analysis shows how it is possible to obtain information about the dispersed phase of the spray in advance based on the dense spray curvature distribution.

Development of hydrogel-based standards and phantoms for non-linear imaging at depth.

Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques. We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth. Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers. These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and , respectively. Once fabricated, the phantoms were found to be stable for many months. The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities.

Restoration of metabolic functional metrics from label-free, two-photon human tissue images using multiscale deep-learning-based denoising algorithms.

Label-free, two-photon excited fluorescence (TPEF) imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, noise and other artifacts present in these images severely complicate the extraction of biologically useful information. We aim to employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-signal-to-noise ratio (SNR), TPEF images. TPEF images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used to assess the impact of various denoising models, preprocessing methods, and data on metrics of image quality and the recovery of six metrics of metabolic function from the images relative to ground truth images. Optimized recovery of the redox ratio and mitochondrial organization is achieved using a novel algorithm based on deep denoising in the wavelet transform domain. This algorithm also leads to significant improvements in peak-SNR (PSNR) and structural similarity index measure (SSIM) for all images. Interestingly, other models yield even higher PSNR and SSIM improvements, but they are not optimal for recovery of metabolic function metrics. Denoising algorithms can recover diagnostically useful information from low SNR label-free TPEF images and will be useful for the clinical translation of such imaging.