Protein Photodamaging Activity and Photocytotoxic Effect of an Axial-Connecting Phosphorus(V)porphyrin Trimer.

An axial-connecting trimer of the porphyrin phosphorus(V) complex was synthesized to evaluate the relaxation process of the photoexcited state and the photosensitizer activity. The photoexcitation energy was localized on the central unit of the phosphorus(V)porphyrin trimer. The photoexcited state of the central unit was relaxed through a process similar to that of the monomer phosphorus(V)porphyrin. The excited state of this axially connected type of phosphorus(V)porphyrin trimer was not deactivated through intramolecular electron transfer. The singlet oxygen generation quantum yield of the trimer was almost the same as that of the monomer. The phosphorus(V)porphyrin, trimer, and monomer bound to human serum albumin and oxidized the tryptophan residue via singlet oxygen generation and electron transfer during visible light irradiation. The photocytotoxicity of these phosphorus(V)porphyrins on two cell lines was examined. The monomer induced photocytotoxicity; however, the trimer did not show cytotoxicity with or without photoirradiation. In summary, the photoexcited state of the trimer was almost the same as that of the monomer, and these phosphorus(V)porphyrins demonstrated a similar protein-photodamaging activity. The difference in association between the photosensitizer molecules and cells is the key factor of phototoxicity by these phosphorus(V)porphyrins.

Synergistic effect of TONS504-mediated photodynamic antimicrobial chemotherapy and additives widely contained in ophthalmic solutions: benzalkonium chloride and ethylenediaminetetraacetic acid.

TONS504 (C51H58N8O5I2), a chlorine derivative, effectively generates singlet oxygen by light activation and exhibits photodynamic antimicrobial effects (PAEs) on various pathogens. However, this photosensitizer has some limitations: a high tendency to self-aggregate and a relatively weak PAE for Gram-negative bacteria compared with Gram-positive bacteria. To overcome these limitations, the present study investigated the synergistic effects of the PAE of TONS504 and two additives commonly contained in ophthalmic solutions: benzalkonium chloride (BAC) or ethylenediaminetetraacetic acid (EDTA). Staphylococcus aureus and Pseudomonas aeruginosa were exposed to TONS504 and/or each additive. Photodynamic antimicrobial chemotherapy was performed with light irradiation centered at a wavelength of 665 nm with a total light energy of 30 J/cm2. Following incubation, the number of colonies formed was counted. Additionally, we examined the inhibitory effects of the additives on TONS504 self-aggregation by observing its absorption spectrum. Consequently, the PAEs of TONS504 on S. aureus were enhanced by both additives, and BAC displayed stronger synergistic effects on the bacteria than EDTA. By contrast, only EDTA increased the PAE on P. aeruginosa. The peak of the TONS504 absorption spectrum shifted to a longer wave length and the absorbance increased in the presence of BAC, suggesting that BAC inhibited the self-aggregation of the photosensitizer. In conclusion, the combination of BAC or EDTA and TONS504-mediated photodynamic antimicrobial chemotherapy exhibits a synergistic antimicrobial effect on S. aureus and P. aeruginosa. The optimal additive to enhance the PAE may differ between bacterial strains.

Endosomal Escape of Peptide-Photosensitizer Conjugates Is Affected by Amino Acid Sequences near the Photosensitizer.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

Determination and analysis of singlet oxygen quantum yields of talaporfin sodium, protoporphyrin IX, and lipidated protoporphyrin IX using near-infrared luminescence spectroscopy.

In photodynamic therapy (PDT), singlet oxygen ([Formula: see text]) is the main species responsible for promoting tumor cell death. The determination of the quantum yield (ΦΔ) of a photosensitizer (PS) is important for dosimetry. The purpose of this paper is to quantify the [Formula: see text] generated by the PS by near-infrared spectroscopy (NIRS). The ΦΔ of different PS species were measured by the detection of near-infrared [Formula: see text] luminescence. From the measurement results, the ΦΔ of talaporfin sodium, protoporphyrin IX (PpIX), and lipidated PpIX (PpIX lipid) were measured as 0.53, 0.77, and 0.87, respectively. In addition, the ΦΔ values of PpIX in a hypoxic and oxic solution were evaluated, since tumors are associated with regions of hypoxia. The measured ΦΔ indicated a same value at high (DO: 20%) and low (DO: 1%) oxygen concentrations. Using the measured ΦΔ, the amount of [Formula: see text] generated by the PSs was estimated using [[Formula: see text]] = D*ΦΔ, where D* is the total excited PS concentration. The generated [Formula: see text] amounts were little different at the high and the low oxygen concentrations, and the generated [Formula: see text] amount for each PS was different depending on each ΦΔ. The NIRS measurement determined the ΦΔ of talaporfin sodium, PpIX, and PpIX lipid. The quantitative evaluation based on the measured ΦΔ will support the development of PDT treatment monitoring and design.

Controlled Photodynamic Action of Axial Fluorinated DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin through Self-Aggregation.

DiethoxyP(V)tetrakis(p-methoxyphenyl)porphyrin (EtP(V)TMPP) and its fluorinated derivative (FEtP(V)TMPP) were synthesized to examine their photodynamic action. These P(V)porphyrins were aggregated in an aqueous solution, resulting in the suppression of their photodynamic activity. In the presence of human serum albumin (HSA), a water-soluble protein, the aggregation states were resolved and formed a binding complex between P(V)porphyrin and HSA. These P(V)porphyrins photosensitized the oxidation of the tryptophan residue of HSA under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was explained by the electron transfer from tryptophan to the photoexcited state of P(V)porphyrins and singlet oxygen generation. The axial fluorination reduced the redox potential of the one-electron reduction of P(V)porphyrin and increased the electron transfer rate constant. However, this axial fluorination decreased the binding constant with HSA, and the quantum yield of photosensitized HSA damage through electron transfer was decreased. The photocytotoxicity of these P(V)porphyrins to HaCaT cells was also confirmed, and FEtP(V)TMPP demonstrated stronger phototoxicity than EtP(V)TMPP. In summary, a self-aggregation of porphyrin photosensitizers and resolving by targeting biomacromolecules may be used to target selective photodynamic action. The redox potential and an association with a targeting biomolecule are the important factors of the electron transfer-mediated mechanism, which is advantageous under hypoxic tumor conditions.

Elucidating the mode of action for thiophene-based organic D-π-A sensitizers for use in photodynamic therapy.

Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. The development of readily accessible templates that allow rapid structural modification for further improvement of PDT remains important. We previously reported thiophene-based organic D-π-A sensitizers consisted of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety as valuable templates for a photosensitizer that can be used in PDT. Our preliminary structure-activity relationship study revealed that the structure of the A moiety significantly influences its phototoxicity. In this study, we evaluated the photoabsorptive, cellular uptake, and photo-oxidizing abilities of D-π-A sensitizers that contained different A moieties. The level of phototoxicity of the D-π-A sensitizers was rationalized by considering those three abilities. In addition, we observed the ability of amphiphilic sensitizers containing either a carboxylic acid or an amide in an A moiety to form aggregates that penetrate cells mainly via endocytosis.

Reliability of a two-wavelength autofluorescence technique by Heidelberg Spectralis to measure macular pigment optical density in Asian subjects.

This study evaluates the accuracy of an objective two-wavelength fundus autofluorescence technique for the purpose of measuring the macular pigment optical density (MPOD) in Asian pigmented eyes. Potential differences between MPOD values obtained via autofluorescence technique and subjective heterochromatic photometry (HFP) were examined. Inter-examiner reproducibility between three examiners and test-retest reliability over five time points were also explored. Subjects were 27 healthy Japanese volunteers aged 24 to 58 (mean ±â€¯standard deviation, 40.2 ±â€¯9.0) years. An MPOD module of the Spectralis MultiColor instrument configuration (Spectralis-MP) was used for the autofluorescence technique, and a Macular Metrics Densitometer (MM) was used for HFP. The mean MPOD values at 0.25° and 0.5° eccentricities using the Spectralis-MP were 0.51 ±â€¯0.12 and 0.48 ±â€¯0.13, respectively. In comparison, the MM based values were 0.72 ±â€¯0.23 and 0.61 ±â€¯0.25, respectively. High correlations between the Spectralis-MP and MM instrument were found (Pearson's correlation coefficients of 0.73 and 0.87 at 0.25° and 0.5° eccentricities, respectively), but there was a systematic bias: the MPOD values by MM method were significantly higher than those by Spectralis-MP at 0.25° eccentricity. High inter-examiner reproducibility and test-retest reliability were found for MM measurements at 0.5° eccentricity, but not at 0.25°. The Spectralis-MP showed less inter-examiner and test-retest variability than the MM instrument at 0.25° and 0.5° eccentricities. We conclude that the Spectralis-MP, given its high agreement with the HFP method and due to its higher reproducibility and reliability, is well suited for clinical measurements of MPOD levels in Asian pigmented eyes.

Photosensitized Protein-Damaging Activity, Cytotoxicity, and Antitumor Effects of P(V)porphyrins Using Long-Wavelength Visible Light through Electron Transfer.

Photodynamic therapy (PDT) is a less-invasive treatment for cancer through the administration of less-toxic porphyrins and visible-light irradiation. Photosensitized damage of biomacromolecules through singlet oxygen (1O2) generation induces cancer cell death. However, a large quantity of porphyrin photosensitizer is required, and the treatment effect is restricted under a hypoxic cellular condition. Here we report the phototoxic activity of P(V)porphyrins: dichloroP(V)tetrakis(4-methoxyphenyl)porphyrin (CLP(V)TMPP), dimethoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (MEP(V)TMPP), and diethyleneglycoxyP(V)tetrakis(4-methoxyphenyl)porphyrin (EGP(V)TMPP). These P(V)porphyrins damaged the tryptophan residue of human serum albumin (HSA) under the irradiation of long-wavelength visible light (>630 nm). This protein photodamage was barely inhibited by sodium azide, a quencher of 1O2. Fluorescence lifetimes of P(V)porphyrins with or without HSA and their redox potentials supported the electron-transfer-mediated oxidation of protein. The photocytotoxicity of these P(V)porphyrins to HeLa cells was also demonstrated. CLP(V)TMPP did not exhibit photocytotoxicity to HaCaT, a cultured human skin cell, and MEP(V)TMPP and EGP(V)TMPP did; however, cellular DNA damage was barely observed. In addition, a significant PDT effect of these P(V) porphyrins on a mouse tumor model comparable with the traditional photosensitizer was also demonstrated. These findings suggest the cancer selectivity of these P(V)porphyrins and lower carcinogenic risk to normal cells. Electron-transfer-mediated oxidation of biomacromolecules by P(V)porphyrins using long-wavelength visible light should be advantageous for PDT of hypoxic tumor.

Water-Soluble Glucosyl Pyrene Photosensitizers: An Intramolecularly Synthesized 2- C-Glucoside and an O-Glucoside.

Prevalent photosensitizing agents for photodynamic therapy (PDT) suffer from their relatively large molecular weights causing photodermatosis. In this regard, low molecular weight pyrene could be an efficient photosensitizer except for its extreme hydrophobicity. To tackle the insolubility of pyrene, we synthesized 1-carboxypyren-2-yl C-glucoside 4 by a tethered C-glucosylation and 1-pyrenylmethyl O-glucoside 5 by a simple O-glucosylation. Compounds 4 and 5 showed modest water solubilities of 72 and 47 µg/mL, respectively. Whereas compound 4 partially underwent a cyclization reaction at pH 3 to give the corresponding δ-valerolactone 15b in 31% yield after 24 h, it is stable at pH 5-9 for at least a week. The 1O2-producing photosensitizabilities of 4 and 5 were sufficient to apply to PDT. Although compound 5 was uptaken by HeLa cells and showed a good PDT activity, compound 4 showed neither a sufficient cell uptake nor PDT effect. The binding modes of compounds 4 and 5 to concanavalin A were specific and unspecific, respectively. These results demonstrate that compounds 4 and 5 are within a pharmacologically acceptable range as oral drugs and could be a fluorescence imaging probe for α-glucose/mannose receptors and a photosensitizing agent for PDT, respectively.

Sequence selective photoinduced electron transfer of a pyrene-porphyrin dyad to DNA.

The binding modes of a pyrene-porphyrin dyad, (1-pyrenyl)-tris(N-methyl-p-pyridino)porphyrin (PyTMpyP), to various DNAs (calf thymus DNA (Ct-DNA), poly[d(G-C)2], and poly[d(A-T)2]) have been investigated using circular dichroism and linear dichroism measurements. Based on the polarization spectroscopic results, it can be shown that the pyrenyl and porphryin planes are skewed to a large extent for PyTMPyP in an aqueous environment and in the binding site of poly[d(G-C)2]. In this complex, a photoinduced electron transfer (PET) process between the pyrenyl and porphyrin moieties occurs. On the other hand, PET was not observed in the PyTMPyP-poly[d(A-T)2] complex, whereas the fluorescence intensity of TMPyP was enhanced. The molecular planes of the pyrene and porphyrin moieties are almost parallel in the poly[d(A-T)2] and Ct-DNA adducts. Moreover, the generation of 1O2 species occurs only for the PyTMPyP-Ct-DNA and PyTMPyP-poly[d(A-T)2] complexes. We discuss the photophysical properties of PyTMPyP which are attributed to the binding patterns and the sequence of DNA bases.

Correction of spherical aberration in multi-focal multiphoton microscopy with spatial light modulator.

We demonstrate that high-quality images of the deep regions of a thick sample can be obtained from its surface by multi-focal multiphoton microscopy (MMM). The MMM system incorporates a spatial light modulator to separate the excitation beam into a multi-focal excitation beam and modulate the pre-distortion wavefront to correct spherical aberration (SA) caused by a refractive index mismatch between the immersion medium and the biological sample. When fluorescent beads in transparent epoxy resin were observed using four SA-corrected focal beams, the fluorescence signal of the obtained images was ~52 times higher than that obtained without SA correction until a depth of ~1100 µm, similar to the result for single-focal multiphoton microscopy (SMM). The MMM scanning time was four times less than that for SMM, and MMM showed an improved fluorescence intensity and depth resolution for an image of blood vessels in the brain of a mouse stained with a fluorescent dye.

Free Radical Scavenging and Cellular Antioxidant Properties of Astaxanthin.

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health.

Determination of Singlet Oxygen and Electron Transfer Mediated Mechanisms of Photosensitized Protein Damage by Phosphorus(V)porphyrins.

The mechanism of photosensitized protein damage byphosphorus(V) tetraphenylporphyrin derivatives (P(V)TPPs) wasquantitatively clarified. P(V)TPPs bound to human serum albumin(HSA), a water-soluble protein, and damaged its tryptophan residueduring photoirradiation. P(V)TPPs photosensitized singlet oxygen ((1)O(2))generation, and the contribution of (1)O(2) to HSA damage was confirmedby the inhibitory effect of sodium azide, a (1)O(2) quencher. However,sodium azide could not completely inhibit HSA damage, suggesting thecontribution of an electron transfer mechanism to HSA damage. Thedecrement in the fluorescence lifetime of P(V)TPPs by HSA supportedthe electron transfer mechanism. The contribution of these processes could be determined by the kinetic analysis of the effect ofsodium azide on the photosensitized protein damage by P(V)TPPs.

An adaptive approach for uniform scanning in multifocal multiphoton microscopy with a spatial light modulator.

We propose high-quality generation of uniform multiple fluorescence spots (MFS) with a spatial light modulator (SLM) and demonstrate uniform laser scanning in multifocal multiphoton microscopy (MMM). The MFS excitation method iteratively updates a computer-generated hologram (CGH) using correction coefficients to improve the fluorescence intensity distribution in a dye solution whose consistency is uniform. This simple correction method can be applied for calibration of the MMM before observation of living tissue. We experimentally demonstrate an improvement of the uniformity of a 10 × 10 grid of MFS by using a dye solution. After the calibration, we performed laser scanning with two-photon excitation to observe fluorescent polystyrene beads, as well as the gastric gland of a guinea pig specimen.

Effect of age and other factors on macular pigment optical density measured with resonance Raman spectroscopy.

BACKGROUND: Macular pigment is a defense system against phototoxic damage of the retina by visible light. It is still under debate whether or not macular pigment optical density (MPOD) levels decline with age, because the age effect varied depending on the technique used to measure MPOD levels. Resonance Raman spectroscopy (RRS) is an objective method to measure MPOD, and studies using RRS showed a drastic age-related decline of MPOD levels; however, since RRS measurements are influenced by cataracts, it has been argued that the age-related decline of RRS measurements is an artifact from lens changes in aged subjects. In the present study, MPOD levels were measured with RRS in pseudophakic eyes, and the effects of age and other factors on MPOD levels were investigated. METHODS: The subjects included 144 patients with no fundus disorders who received cataract surgery with untinted intraocular lens implantation. MPOD levels were measured in 144 eyes using integral RRS 1 day post surgery. Factors potentially associated with MPOD levels such as age, gender, smoking habits, body mass index, diabetes, glaucoma, axial length, pupil diameter, spherical equivalent refractive error, and foveal thickness were examined by multiple regression analysis. RESULTS: The macular pigment RRS levels ranged from 776 to 11,815 Raman counts, with an average level of 4,375 ± 1,917 (standard deviation [SD]) Raman counts. Multiple regression analysis revealed that age and axial length were significantly correlated with low MPOD values (regression coefficient of -59 for age and -404 for axial length, respectively). No significant correlations were observed for other factors. CONCLUSIONS: After removing the potentially confounding effect of age-related lens yellowing on the RRS measurements, age remained a significant patient parameter for lowered MPOD levels. MPOD levels were found to decline by more than 10 % each decade. Axial length was also a negative predictor of MPOD levels. Since the present study included only patients aged 50 years and older, the effects of age and other factors on MPOD levels for younger subjects remain unknown.

Deep learning-based correction of cataract-induced influence on macular pigment optical density measurement by autofluorescence spectroscopy.

PURPOSE: Measurements of macular pigment optical density (MPOD) using the autofluorescence spectroscopy yield underestimations of actual values in eyes with cataracts. Previously, we proposed a correction method for this error using deep learning (DL); however, the correction performance was validated through internal cross-validation. This cross-sectional study aimed to validate this approach using an external validation dataset. METHODS: MPODs at 0.25°, 0.5°, 1°, and 2° eccentricities and macular pigment optical volume (MPOV) within 9° eccentricity were measured using SPECTRALIS (Heidelberg Engineering, Heidelberg, Germany) in 197 (training dataset inherited from our previous study) and 157 eyes (validating dataset) before and after cataract surgery. A DL model was trained to predict the corrected value from the pre-operative value using the training dataset, and we measured the discrepancy between the corrected value and the actual postoperative value. Subsequently, the prediction performance was validated using a validation dataset. RESULTS: Using the validation dataset, the mean absolute values of errors for MPOD and MPOV corrected using DL ranged from 8.2 to 12.4%, which were lower than values with no correction (P < 0.001, linear mixed model with Tukey's test). The error depended on the autofluorescence image quality used to calculate MPOD. The mean errors in high and moderate quality images ranged from 6.0 to 11.4%, which were lower than those of poor quality images. CONCLUSION: The usefulness of the DL correction method was validated. Deep learning reduced the error for a relatively good autofluorescence image quality. Poor-quality images were not corrected.

Luminescence of singlet oxygen in photosystem II complexes isolated from cyanobacterium Synechocystis sp. PCC6803 containing monovinyl or divinyl chlorophyll a.

The luminescence spectrum of singlet oxygen produced upon excitation at 674nm in the photochemically active photosystem II (PS II) complexes isolated from cyanobacterium Synechocystis sp. PCC 6803 containing different types of chlorophyll, i.e., monovinyl (wild-type) or divinyl (genetically modified) chlorophyll a. The yield of singlet oxygen, estimated using methylene blue as the standard, from the divinyl-chlorophyll PS II complex was more than five times greater than that from the monovinyl-chlorophyll PS II complex. These results are consistent with the observed difference in the sensitivity towards high intensity of light between the two cyanobacterial strains. The yield of singlet oxygen appeared to increase with the level of triplet chlorophyll, in the divinyl-chlorophyll PS II complex. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin.

For the purpose of the basic study of photodynamic therapy, the activity of the water-soluble P(V)porphyrin, dimethoxyP(V)tetraphenylporphyrin chloride (DMP(V)TPP), on photosensitized protein damage was examined. The quantum yield of singlet oxygen generation by DMP(V)TPP (0.64) was comparable with that of typical porphyrin photosensitizers. Absorption spectrum measurement demonstrated the binding interaction between DMP(V)TPP and human serum albumin, a water-soluble protein. Photo-irradiated DMP(V)TPP damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of DMP(V)TPP by human serum albumin supported the electron transfer mechanism. The estimated contribution of the electron transfer mechanism is 0.64. These results suggest that the activity of DMP(V)TPP can be preserved under lower oxygen concentration condition such as tumor.

Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe.

We have rationally designed and synthesized a novel near-infrared (NIR) photoactivating probe, designated by iDOPE, in which an indocyanine green (ICG) fluorophore is covalently conjugated with a phospholipid moiety, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), to incorporate into liposome bilayers. NIR irradiation showed that iDOPE retained the optical and fluorescence properties of ICG and demonstrated photoactivator characteristics: fluorescence emission at around 820 nm in a solvent, singlet oxygen production, and concentration-dependent heat generation. Additionally, iDOPE was incorporated into liposome bilayers and maintained stable liposomally formulated iDOPE (LP-iDOPE) over 1week under physiological conditions. We also observed the tumor-specific biodistribution of LP-iDOPE of in vivo xenografts. These findings suggest that LP-iDOPE might be a promising tool for NIR optical imaging, photodynamic therapy, and photothermal therapy.

Complex-Amplitude-Modulation Vectorial Excitation Beam for High-Resolution Observation of Deep Regions in Two-Photon Microscopy.

In two-photon microscopy, aberration correction is an essential technique for realizing high resolution in deep regions. A spatial light modulator (SLM) incorporated into an optical system for two-photon microscopy performs pre-compensation on the wavefront of the excitation beam, restoring the resolution close to the diffraction limit even in the deep region of a biological sample. If a spatial resolution smaller than the diffraction limit can be achieved along with aberration correction, the importance of two-photon microscopy for deep region observation will increase further. In this study, we realize higher resolution observations in the deep region by combining two resolution-enhancement methods and an aberration correction method. Therefore, a z-polarizer is added to the aberration-correction optical system, and the SLM modulates the amplitude and phase of the excitation beam; in other words, complex-amplitude modulation is performed. The lateral resolution is found to be approximately 20% higher than the diffraction limit obtained using a circularly polarized beam. Verification was conducted by simulation and experimentation using model samples and ex vivo biological samples. The proposed method has the potential to be effective for live imaging and photostimulation of the deep region of the sample, although it requires only minor changes to the conventional optical system that performs aberration correction.