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.

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.

Artificial intelligence-based classification of peripheral blood nucleated cells using label-free imaging flow cytometry.

Label-free image identification of circulating rare cells, such as circulating tumor cells within peripheral blood nucleated cells (PBNCs), the vast majority of which are white blood cells (WBCs), remains challenging. We previously described developing label-free image cytometry for classifying live cells using computer vision technology for pattern recognition, based on the subcellular structure of the quantitative phase microscopy images. We applied our image recognition methods to cells flowing in a flow cytometer microfluidic channel, and differentiated WBCs from cancer cell lines (area under receiver operating characteristic curve = 0.957). We then applied this method to healthy volunteers' and advanced cancer patients' blood samples and found that the non-WBC fraction rates (NWBC-FRs), defined as the percentage of cells classified as non-WBCs of the total PBNCs, were significantly higher in cancer patients than in healthy volunteers. Furthermore, we monitored NWBC-FRs over the therapeutic courses in cancer patients, which revealed the potential ability in monitoring the clinical status during therapy. Our image recognition system has the potential to provide a morphological diagnostic tool for circulating rare cells as non-WBC fractions.

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.

Coenzyme Q10 in the eye isomerizes by sunlight irradiation.

Photoisomerization of lipids has been well studied. As for the eyes, photoisomerization from 11-cis isomer to all-trans-retinal is well-known as the first step of the visual transduction in the photoreceptors. In addition to that, there would be other ocular lipids that undergo photoisomerization, which may be involved in ocular health and function. To explore any photoisomerizable lipids in the eyes, the nonirradiated and sunlight-irradiated eyeball extracts were subjected to liquid chromatography-mass spectrometry analysis, followed by the identification of the decreased lipid species in the irradiated extracts. Surprisingly, more than nine hundred lipid species were decreased in the irradiated extracts. Three lipid species, coenzyme Q10 (CoQ10), triglyceride(58:4), and coenzyme Q9, were decreased both significantly (p < 0.05) and by more than two-fold, where CoQ10 showed the most significant decrease. Later, photoisomerization was identified as the prominent cause underlying the decrease of CoQ10. Interestingly, CoQ10 in the sunlight-irradiated fresh eyeballs was also isomerized. Both the visible light and ultraviolet radiation were capable of producing CoQ10 isomer, while the latter showed rapid action. This study is believed to enhance our understanding of the biochemistry and photodamage of the eye and can potentially contribute to the advancement of opto-lipidomics.

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.

Molecular characteristics of the photosensitizer TONS504: Comparison of its singlet oxygen quantum yields and photodynamic antimicrobial effect with those of methylene blue.

TONS504 (C51H58O5I2) is a chlorin derivative that exhibits a photodynamic antimicrobial effect (PAE) on various infectious keratitis pathogens. However, the molecular characteristics of TONS504 are not well understood. This study aimed to investigate the molecular characteristics of TONS504 by comparing its singlet oxygen (1O2) quantum yields and PAE with those of methylene blue (MB). To measure the 1O2 quantum yields, TONS504 and MB were dissolved in phosphate-buffered saline and phosphate-buffered saline containing 1% Triton X-100. The solutions were then activated by a Nd:YAG laser with an average output power of 8 mW. Near-infrared 1O2 luminescence was detected as an indicator of the 1O2 quantum yields. To evaluate the PAE, TONS504 and MB were activated by a light-emitting diode with a total light energy of 30 J/cm2. We compared the minimum molar concentration of each photosensitizer to show apparent PAEs on Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. TONS504 exhibited higher 1O2 quantum yields than MB in PBS/Triton X-100 but not in PBS. S. aureus and C. albicans were reduced by TONS504 at lower concentrations than by MB, but this was not the case for P. aeruginosa. Our results provide insight on the molecular characteristics of TONS504 and suggest that TONS504 has excellent 1O2 quantum yields and PAE. Compared with MB, TONS504 in PBS has stronger efficacy toward some infectious keratitis pathogens but not others.

Correction for the Influence of Cataract on Macular Pigment Measurement by Autofluorescence Technique Using Deep Learning.

Purpose: Measurements of macular pigment optical density (MPOD) by the autofluorescence technique yield underestimations of actual values in eyes with cataract. We applied deep learning (DL) to correct this error. Subjects and Methods: MPOD was measured by SPECTRALIS (Heidelberg Engineering, Heidelberg, Germany) in 197 eyes before and after cataract surgery. The nominal MPOD values (= preoperative value) were corrected by three methods: the regression equation (RE) method, subjective classification (SC) method (described in our previous study), and DL method. The errors between the corrected and true values (= postoperative value) were calculated for local MPODs at 0.25°, 0.5°, 1°, and 2° eccentricities and macular pigment optical volume (MPOV) within 9° eccentricity. Results: The mean error for MPODs at four eccentricities was 32% without any correction, 15% with correction by RE, 16% with correction by SC, and 14% with correction by DL. The mean error for MPOV was 21% without correction and 14%, 10%, and 10%, respectively, with correction by the same methods. The errors with any correction were significantly lower than those without correction (P < 0.001, linear mixed model with Tukey's test). The errors with DL correction were significantly lower than those with RE correction in MPOD at 1° eccentricity and MPOV (P < 0.001) and were equivalent to those with SC correction. Conclusions: The objective method using DL was useful to correct MPOD values measured in aged people. Translational Relevance: MPOD can be obtained with small errors in eyes with cataract using DL.

Photooxidation Activity Control of Dimethylaminophenyl-tris-(N-methyl-4-pridinio)porphyrin by pH.

To control the activity of photodynamic agents by pH, an electron donor-connecting cationic porphyrin, meso-(N',N'-dimethyl-4-aminophenyl)-tris(N-methyl-p-pyridinio)porphyrin (DMATMPyP), was designed and synthesized. The photoexcited state (singlet excited state) of DMATMPyP was deactivated through intramolecular electron transfer under a neutral condition. The pK a of the protonated DMATMPyP was 4.5, and the fluorescence intensity and singlet oxygen-generating activity increased under an acidic condition. Furthermore, the protonation of DMATMPyP enhanced the biomolecule photooxidative activity through electron extraction. Photodamage of human serum albumin (HSA) was observed under a neutral condition because a hydrophobic HSA environment can reverse the deactivation of photoexcited DMATMPyP. However, an HSA-damaging mechanism of DMATMPyP under a neutral condition was explained by singlet oxygen production. Therefore, it is indicated that the protein photodamaging activity of DMATMPyP goes into an OFF state under a neutral hypoxic condition. Under an acidic condition, the HSA photodamaging quantum yield by DMATMPyP through electron extraction could be preserved in the presence of a singlet oxygen quencher. Photooxidation of nicotinamide adenine dinucleotide by DMATMPyP was also enhanced under an acidic condition. This study demonstrated the concept of using pH to control photosensitizer activity via inhibition of the intramolecular electron transfer deactivation and enhancement of the oxidative activity through the electron extraction mechanism. Specifically, biomolecule oxidation through electron extraction may play an important role in photodynamic therapy to treat tumors under a hypoxic condition.

Pathological application of carbocyanine dye-based multicolour imaging of vasculature and associated structures.

Simultaneous visualisation of vasculature and surrounding tissue structures is essential for a better understanding of vascular pathologies. In this work, we describe a histochemical strategy for three-dimensional, multicolour imaging of vasculature and associated structures, using a carbocyanine dye-based technique, vessel painting. We developed a series of applications to allow the combination of vessel painting with other histochemical methods, including immunostaining and tissue clearing for confocal and two-photon microscopies. We also introduced a two-photon microscopy setup that incorporates an aberration correction system to correct aberrations caused by the mismatch of refractive indices between samples and immersion mediums, for higher-quality images of intact tissue structures. Finally, we demonstrate the practical utility of our approach by visualising fine pathological alterations to the renal glomeruli of IgA nephropathy model mice in unprecedented detail. The technical advancements should enhance the versatility of vessel painting, offering rapid and cost-effective methods for vascular pathologies.

Label-free imaging flow cytometer for analyzing large cell populations by line-field quantitative phase microscopy with digital refocusing.

We propose a line-field quantitative phase-imaging flow cytometer for analyzing large populations of label-free cells. Hydrodynamical focusing brings cells into the focus plane of an optical system while diluting the cell suspension, resulting in decreased throughput rate. To overcome the trade-off between throughput rate and in-focus imaging, our cytometer involves digitally extending the depth-of-focus on loosely hydrodynamically focusing cell suspensions. The cells outside the depth-of-focus range in the 70-µm diameter of the core flow were automatically digitally refocused after image acquisition. We verified that refocusing was successful with our cytometer through statistical analysis of image quality before and after digital refocusing.

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.

A Twist-Assisted Biphenyl Photosensitizer Passable Through Glucose Channel.

While the development of low-molecular-weight drugs is saturating, agents for photodynamic therapies (PDTs) may become alternative seeds in pharmaceutical industry. Among them, orally administrative, cancer-selective, and side effect-free photosensitizers (PSs) that can be activated by tissue-penetrative near-infrared (NIR) lights are strongly demanded. We discovered such a PS from scratch by focusing on a twist-assisted spin-orbit charge transfer intersystem crossing (ISC) mechanism in a biphenyl derivative, which was demonstrated by thorough photophysical studies. The unique ISC mechanism enables the PS to be small and slim so as to pass through glucose transporters and exert a PDT effect selectively on a cancer cell line. The smallness will allow for oral administration and fast clearance, which have been agenda of approved PSs with larger molecular weights. We also demonstrated that our PS was able to be activated with an NIR pulse laser through two-photon excitation.

Label-free classification of cells based on supervised machine learning of subcellular structures.

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells.

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.

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.

Optical Detection of Macular Pigment Formation in Premature Infants.

PURPOSE: To demonstrate the presence of macular pigment in the retina of premature infants, and to examine its changes with age. METHODS: The participants included 40 premature infants. Infants who had received laser photocoagulation for retinopathy of prematurity were excluded. Macular pigment optical density (MPOD) was measured by fundus reflectometry using RetCam3, a digital fundus camera. The reflection imaging was performed for ROP screening. The imaging time points were from a post menstrual age (PMA) of 29 weeks 0 days to 46 weeks 5 days. RESULTS: The MPOD levels could be obtained from 39 premature infants. The levels at the first measurement ranged from 0 to 0.18 (mean 0.076, SD 0.044). The earliest time, when a nonvanishing MPOD level was obtained, was at a PMA of 33 weeks and 2 days, and that level was 0.05. The initial examination MPOD levels showed a moderate correlation with age (R2 = 0.32, P < 0.00017). The mean MPOD levels measured each week during the follow-up period showed a very strong correlation with age (R2 = 0.91, P < 0.0001). A regression line of MPOD = 0.0069 × age - 0.1783 was derived, where age is counted in PMA days. CONCLUSIONS: The MPOD levels of premature infants were for the first time measured in living eyes. Macular pigment increased linearly with age. TRANSLATIONAL RELEVANCE: Macular pigment increased with the development of macular morphology. This result suggested the importance of nutritional management of infants and mothers during perinatal period.

Aqueous-based tissue clearing in crustaceans.

BACKGROUND: Investigation of the internal tissues and organs of a macroscopic organism usually requires destructive processes, such as dissection or sectioning. These processes are inevitably associated with the loss of some spatial information. Recently, aqueous-based tissue clearing techniques, which allow whole-organ or even whole-body clearing of small rodents, have been developed and opened a new method of three-dimensional histology. It is expected that these techniques will be useful tools in the field of zoology, in which organisms with highly diverse morphology are investigated and compared. However, most of these new methods are optimized for soft, non-pigmented organs in small rodents, especially the brain, and their applicability to non-model organisms with hard exoskeletons and stronger pigmentation has not been tested. RESULTS: We explored the possible application of an aqueous-based tissue clearing technique, advanced CUBIC, on small crustaceans. The original CUBIC procedure did not clear the terrestrial isopod, Armadillidium vulgare. Therefore, to apply the whole-mount clearing method to isopods with strong pigmentation and calcified exoskeletons, we introduced several pretreatment steps, including decalcification and bleaching. Thereafter, the clearing capacity of the procedure was dramatically improved, and A. vulgare became transparent. The internal organs, such as the digestive tract and male reproductive organs, were visible through sclerites using an ordinary stereomicroscope. We also found that fluorescent nuclear staining using propidium iodide (PI) helped to visualize the internal organs of cleared specimens. Our procedure was also effective on the marine crab, Philyra sp. CONCLUSIONS: In this study, we developed a method to clear whole tissues of crustaceans. To the best of our knowledge, this is the first report of whole-mount clearing applied to crustaceans using an aqueous-based technique. This technique could facilitate morphological studies of crustaceans and other organisms with calcified exoskeletons and pigmentation.