Multicolor Imaging Research Articles

Peripheral Blood B-Lymphocytes Are Involved in Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus) via Cellular Receptor-Mediated Mechanism.

Previous studies imply that peripheral blood leukocytes (PBLs) may play an important role in systemic lymphocystis disease virus (LCDV) dissemination, but whether the PBLs are susceptible and permissive to LCDV infection and the dissemination mechanism need to be clarified. In this study, LCDV was firstly confirmed to infect the PBLs in flounder (Paralichthys olivaceus) in vivo, and to replicate in PBLs in vitro. Subsequently, the 27.8 kDa receptor protein (27.8R), a functional receptor mediating LCDV infection in flounder gill cells, was shown to locate on the cell membrane of PBLs and co-localize with LCDV in PBLs, while blocking of the 27.8R via pre-incubation of anti-27.8R MAb with the PBLs could obviously inhibit LCDV infection, revealing the 27.8R as a receptor for LCDV entry into PBLs. Multicolor fluorescence imaging studies verified that IgM+ and IgD+ B-lymphocyte were involved in LCDV infection. In the sorted IgM+ B-cells, 27.8R+ and LCDV+ signals were simultaneously observed, and LCDV copy numbers increased with time, indicating that IgM+ B-cells expressed the 27.8R and were permissive to LCDV infection. Furthermore, the dynamic changes of IgM+, 27.8R+, LCDV+ and LCDV+/IgM+ PBLs were monitored during the early phase of LCDV infection. It was found that the percentage of IgM+ B-cells in PBLs clearly declined first and then increased, suggesting LCDV infection facilitated damage to B-cells, whereas the amounts of 27.8R+ and LCDV+ PBLs, as well as LCDV-infected IgM+ B-cells, showed an opposite trend. These results proved that IgM+ B-lymphocytes could be infected by LCDV via a receptor-mediated mechanism and support viral replication, which provided novel insights for the first time into the role of B-lymphocytes in LCDV dissemination and pathogenesis in teleost fish.

MBeRFP: a versatile fluorescent tool to enhance multichannel live imaging and its applications.

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins offers interesting new perspectives to bypass this limitation. Here, we provide a detailed characterization of mBeRFP for in vivo live imaging and its applications in Drosophila. Briefly, we show that a single illumination source is sufficient to stimulate mBeRFP and GFP simultaneously. We demonstrate that mBeRFP can be easily combined with classical green and red fluorescent proteins without any crosstalk. We also show that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications, such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano-biologists in their current research.

Excitation-dependent perovskite/polymer films for ultraviolet visualization

Ultraviolet (UV) visualization has extensive applications in military and civil fields such as security monitoring, space communication, and wearable equipment for health monitoring in the internet of things (IoT). Due to their remarkable optoelectronic features, perovskite materials are regarded as promising candidates for UV light detecting and imaging. Herein, we report for the first time the excitation-dependent perovskite/polymer films with dynamically tunable fluorescence ranging from green to magenta by changing the UV excitation from 260 to 380 nm. And they still render dynamic multi-color UV light imaging with different polymer matrixes, halogen ratios, and cations of perovskite materials. The mechanism of its fluorescence change is related to the chloride vacancies in perovskite materials. A patterned multi-color ultraviolet visualization pad is also demonstrated for visible conversion of the UV region. This technique may provide a universal strategy for information securities, UV visualizations, and dynamic multi-color displays in the IoT.

Taking molecular pathology to the next level: Whole slide multicolor confocal imaging with the Pannoramic Confocal digital pathology scanner.

The emergence and fast advance of digital pathology allows the acquisition, digital storage, interactive recall and analysis of morphology at the tissue level. When applying immunohistochemistry, it also affords the correlation of morphology with the expression of one or two specific molecule of interest. The rise of fluorescence pathology scanners expands the number of detected molecules based on multiplex labeling. The Pannoramic Confocal (created by 3DHistech, Hungary) is a first-of-the-kind digital pathology scanner that affords not only multiplexed fluorescent detection on top of conventional transmission imaging, but also confocality. We have benchmarked this scanner in terms of stability, precision, light efficiency, linearity and sensitivity. X-Y stability and relocalisation precision were well below resolution limit (≤50 nm). Light throughput in confocal mode was 4-5 times higher than that of a point scanning confocal microscope, yielding similar calculated confocal intensities but with the potential for improving signal to noise ratio or scan speed. Response was linear with R2 ≥ 0.9996. Calibrated measurements showed that using indirect labeling ≥2000 molecules per cell could be well detected and imaged on the cell surface. Both standard-based and statistical post-acquisition flatfield corrections are implemented. We have also measured the point spread function (PSF) of the instrument. The dimensions of the PSF are somewhat larger and less symmetric than of the theoretical PSF of a conventional CLSM, however, the spatial homogeneity of these parameters allows for obtaining a specific system PSF for each optical path and using it for optional on-the-fly deconvolution. In conclusion, the Pannoramic Confocal provides sensitive, quantitative widefield and confocal detection of multiplexed fluorescence signals, with optical sectioning and 3D reconstruction, in addition to brightfield transmission imaging. High speed scanning of large samples, analysis of tissue heterogeneity, and detection of rare events open up new ways for quantitatively analyzing tissue sections, organoid cultures or large numbers of adherent cells.

Construction of Red Fluorescent Dual Targeting Mechanically Interlocked Molecules for Live Cancer Cell Specific Lysosomal Staining and Multicolor Cellular Imaging.

We have designed and synthesized red fluorescent mechanically interlocked molecules with dual targeting functionality for live cancer cell specific active targeting followed by selective internalization and imaging of malignant lysosomes along with real-time tracking, 3D, and multicolor cellular imaging applications.

Tunable emission color of [(2-mb)tpp]2Cd1-xMnxCl4 (x = 0–1) solid solutions synthesized via mechanochemical method for anti-counterfeit luminescent paper and WLEDs

Zero-dimensional (0D) hybrid metal halides (HMHs) have received widespread concern due to their unique structures and splendid luminescent properties. However, the syntheses of HMHs primarily rely on crystal growth methods in solution or ion-thermal conditions, which are time-consuming and require extensive use of organic solvents. On the other hand, the reported HMHs usually exhibiting only one emission color hinder their further applications. Herein, we report a series of 0D HMHs solid solutions of [(2-mb)tpp]2Cd1-xMnxCl4 [(2-mb)tpp = (2-methylbenzyl)triphenylphosphine and x = 0–1] synthesized through a facile mechanochemical method. The emission color of [(2-mb)tpp]2Cd1-xMnxCl4 can be tuned from purplish-blue, bluish-green, to green light by adjusting the Cd/Mn molar ratio or excitation wavelength. Thus different luminescent panda and quick response (QR) code patterns can be designed with [(2-mb)tpp]2Cd1-xMnxCl4 as luminescent dyes for potential applications in multicolor imaging and anti-counterfeiting encryption. Moreover, owing to the excellent thermal stability, the as-fabricated white light emitting diode (WLED) with [(2-mb)tpp]2MnCl4 as the green phosphor possesses an applicable correlated color temperature (CCT = 5305 K), an ultrahigh color rendering index (CRI, Ra = 95.4), and the CIE chromaticity coordinates of (0.3386, 0.3864) corresponding to pure white light. This work provides guidance for the rapid, facile, economical, and large-scale synthesis of HMHs with multiple luminescent centers, and exhibits the development of these materials in potential applications such as anti-counterfeiting luminescent papers and WLEDs.

Preprint Highlight: Multiplexed bioluminescence microscopy via phasor analysis

Luciferase‐based bioluminescence imaging requires no excitation light and thus can be advantageous over fluorescence imaging, but multicolor imaging has been a challenge due to the difficulties of spectral and substrate unmixing. To achieve effective multiplexing of bioluminescence imaging, this study applies phasor analysis that can cluster profiles, in the phasor space, of acquired light intensity according to specific bioluminescence probes. The authors demonstrate that bioluminescence phasor imaging can distinguish heterogeneous cell populations expressing up to six unique reporters. They also resolve two distinct reporters localized to the nucleus and plasma membrane within a single cell. The workflow and optical setup are simple and easily accessible to most cell and developmental biologists, which enables multicolor live‐cell imaging without concerns of photodamage.

Multi-color RNA imaging with CRISPR-Cas13b systems in living cells.

Visualizing RNA dynamics is important for understanding RNA function. Catalytically dead (d) CRISPR-Cas13 systems have been established to image and track RNAs in living cells, but efficient dCas13 for RNA imaging is still limited. Here, we analyzed metagenomic and bacterial genomic databases to comprehensively screen Cas13 homologies for their RNA labeling capabilities in living mammalian cells. Among eight previously unreported dCas13 proteins that can be used for RNA labeling, dHgm4Cas13b and dMisCas13b displayed comparable, if not higher, efficiencies to the best-known ones when targeting endogenous MUC4 and NEAT1_2 by single guide (g) RNAs. Further examination of the labeling robustness of different dCas13 systems using the GCN4 repeats revealed that a minimum of 12 GCN4 repeats was required for dHgm4Cas13b and dMisCas13b imaging at the single RNA molecule level, while >24 GCN4 repeats were required for reported dLwaCas13a, dRfxCas13d and dPguCas13b. Importantly, by silencing pre-crRNA processing activity of dMisCas13b (ddMisCas13b) and further incorporating RNA aptamers including PP7, MS2, Pepper or BoxB to individual gRNAs, a CRISPRpalette system was developed to successfully achieve multi-color RNA visualization in living cells.

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.

Interstitial Inflammation in the ISN/RPS 2018 Classification of Lupus Nephritis Predicts Renal Outcomes and is Associated With Bcl-2 Expression.

To investigate the histopathological characteristics of patients with lupus nephritis in the 2018 revised International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification and assess the prognostic factors. This study enrolled 92 patients with lupus nephritis, who had conventional treatment and renal biopsy. Each renal tissue was evaluated according to 2018 ISN/RPS classification, and quantified apoptotic regulator protein, the B-cell lymphoma-2 protein (Bcl-2), expressions in selected lymphocyte subsets were measured using novel computational approaches using multicolor confocal images. Histopathological characteristics and prognostic factors of end-stage renal disease (ESRD) and chronic kidney disease (CKD) were compared. Follow-up data were obtained, and survival analysis was conducted. During follow-up period (average 74.3 months), 16 and 18 patients progressed ESRD and CKD, respectively. Multivariable analysis of age, sex, disease activity and pathological features in ISN/RPS demonstrated the extent of interstitial inflammation (grade 0~3) was significantly associated with both ESRD and CKD. When interstitial inflammation was divided into mild (grade 0, 1) and severe (grade 2, 3), Cox regression analysis showed that patients with severe interstitial inflammation were significantly increased risk of both ESRD and CKD (hazard ratio 4.67 and 3.8, respectively). Bcl-2 expression in CD4+ and CD20 cells was significantly higher in the severe interstitial inflammation group compared to in mild interstitial inflammation patients (p=0.006 and 0.010, respectively). The extent of interstitial inflammation can predict clinical renal outcomes. Significantly elevated Bcl-2 expression in both CD4+ and CD20 cells was found in severe interstitial inflammation compared with mild interstitial inflammation.

Design and synthesis of biodegradable nonconjugated S[sbnd]S[sbnd]PAMAM dendrimers with unexpected Deep-red/NIR emission and cell membrane targeting ability for biological imaging

Fluorescent non-conjugated polymers without any π-aromatic building blocks are of great significance in biological applications due to low cytotoxicity and good biocompatibility. However, it is still a great challenge to achieve bright and deep-red/near-infrared (NIR) emissions. Herein, we have designed and synthesized a series of deep-red/NIR non-conjugated polymers with aggregation-induced emission based on disulfide-linked poly(amidoamine) (SSPAMAM) dendrimers, which exhibit excellent biodegradation for bioimaging. These SSPAMAM dendrimers (G0.5-G2.0) containing disulfide bonds in their backbone were synthesized in high yield by incorporating cystamine instead of common diacrylamide. Unexpectedly, the SSPAMAM dendrimers prepared exhibited bright and excitation dependent multicolor-tunable emission. More interestingly, G2.0 SSPAMAM showed a deep-red/NIR emission (λem = 625 nm with a shoulder at 800 nm) originated from an extraordinarily rigid and compact conformation via strong intramolecular hydrogen bonds. Meanwhile, these dendrimers displayed excellent biodegradability because of the large number of disulfide linkages in backbone. Due to the excellent specific cytomembrane location, SSPAMAM dendrimers can be used as a potential cell membrane imaging reagent for multi-color (blue, green, and deep-red) imaging. Furthermore, they could also be used in zebrafish for bioimaging. This investigation has expanded the applications of fluorescent non-conjugated polymers to the field of long-wavelength in vivo biological imaging.

Qudi-HiM: an open-source acquisition software package for highly multiplexed sequential and combinatorial optical imaging.

Multiplexed sequential and combinatorial imaging enables the simultaneous detection of multiple biological molecules, e.g. proteins, DNA, or RNA, enabling single-cell spatial multi-omics measurements at sub-cellular resolution. Recently, we designed a multiplexed imaging approach (Hi-M) to study the spatial organization of chromatin in single cells. In order to enable Hi-M sequential imaging on custom microscope setups, we developed Qudi-HiM, a modular software package written in Python 3. Qudi-HiM contains modules to automate the robust acquisition of thousands of three-dimensional multicolor microscopy images, the handling of microfluidics devices, and the remote monitoring of ongoing acquisitions and real-time analysis. In addition, Qudi-HiM can be used as a stand-alone tool for other imaging modalities.

Hyaluronic acid-coated ultrasmall BiOI nanoparticles as a potentially targeted contrast agent for X-ray computed tomography

Commercial X-ray computed tomography (CT) contrast agents (CAs) are not appropriate for multicolor CT imaging and are limited due to a lack of tumor targeting. In this contribution, to favor the combination of high Z elements like bismuth and iodine for a broad range of X-ray photon energy, BiOI nanoparticles (NPs) are synthesized with hyaluronic acid (HA) coating to target the tumor cells with CD44 overexpression. The crystal structure of BiOI NPs is determined by X-ray powder diffraction, and the size of NPs is determined by a transmission electron microscope at about 14 ± 3 nm. The dynamic diameter of the NPs (DLS) in PBS buffer was measured at about 100 nm. Fourier transform infrared spectroscopy and thermal gravimetric analysis confirm the coating of BiOI NPs with HA. The stability of the NPs was also investigated via UV–vis spectroscopy. The immunocytochemistry process is performed to investigate the targeting ability of synthesized NPs on the human colon cancer cells with CD44 antigen. Finally, the X-ray attenuation of prepared HA-coated BiOI NPs is higher than Iohexol as the commercially available iodinated contrasting agent at the same concentrations, which can be administrated at much lower doses to achieve similar contrast to Iohexol.

A photoactivatable theranostic probe for simultaneous oxidative stress-triggered multi-color cellular imaging and photodynamic therapy

Simultaneous in situ monitoring critical organelles upon oxidative stress and implementing therapeutics utilizing oxidative stress are of vital importance and remain challenging task. Herein, we rationally design and facilely synthesized a photoactivatable fluorescent probe bearing 1,4-dihydropyridine moiety with aggregation-induced emission (AIE) tendency, namely TPA-DHPy, which can rapidly transform into its pyridine counterpart TPA-Py via photo-oxidative dehydrogenation showing strong polarity sensitivity and largely red-shifted emission. TPA-DHPy- and TPA-Py-based type I/type II photosensitization is able to effectively generate reactive oxygen species to induce in situ oxidative stress under white light irradiation. TPA-DHPy can be taken up by cancer cells, and gradually light up lipid droplets (LDs) and endoplasmic reticulum (ER) during photoactivatable process, as well as in situ monitoring difference and alteration of their microenvironment upon oxidative stress by means of multi-color fluorescence imaging in lambda mode. Furthermore, the in situ generated TPA-Py is capable of further destroying the functions of LDs and ER with prolonging the irradiation time, and remarkably inhibiting tumor growth under white light irradiation by the way of photodynamic therapy. This study thus offers useful insights into designing a new generation of theranostic agents towards imaging-guided precise cancer therapy.

Green synthesis of multicolour fluorescence carbon quantum dots from sugarcane waste: Investigation of mercury (II) ion sensing, and bio-imaging applications

Initial identification is important in the predictive strategy of many life-threatening ailments, such as infectious diseases and cancer. These diseases' mortality rates could be significantly reduced if they were detected in the early stages of disease development. Natural carbon quantum dots (NCQDs) a new type of carbon-based nanomaterial have sparked the interest of researchers for many years in biomedical applications. The eco-friendly and cost-effective hydrothermal synthesis of fluorescent CQDs utilizing sugarcane baggage as a carbon source is discussed in this study. The RAMAN and XRD analysis confirmed the graphitic nature of CQDs. The FTIR, and XPS demonstrate the presence of functional groups such as CO, CC, and CN, hence it's regarded as CQDs. High-resolution transmission electron microscopy (HR-TEM) findings exhibiting that the synthesized CQDs are predominantly monodispersed spherical in shape, with particle sizes ranging from 2 to 8 nm. It is used to develop a more environmentally friendly and cost-effective fluorescent probe for detecting Hg2+ ions. MCF-7 cells were used to test cancer cell lines, which showed significant bio-labeling capacity and low cytotoxicity. The cell viabilities of the MCF-7 cells were assessed. As a result of their low cytotoxicity and good biocompatibility, we used the synthesized CQDs as an efficient fluorescent probe for multicolour live-cell imaging.

Preparation of lysosomal targeted fluorescent carbon dots and its applications in multi-color cell imaging and information encryption

In this study, carbon dots (CDs) were obtained by secondary hydrothermal method using ammonium citrate and folic acid as precursors. It had stable properties, small particle size and good water solubility, and its absolute quantum yield was up to 19.48%. Moreover, the emission centers of CDs shifted from green to yellow under different excitation and were successfully used for multi-color cell imaging and targeting of lysosomes. The CDs also showed different color in 254 nm and 365 nm UV light, which could be used to a fluorescent ink for information encryption. In addition, the fluorescence of CDs was quenched at pH 6–8 and showed a good linear relationship with a linear correlation coefficient of 0.9989, which had been implemented for the measurement of pH in tap water.

Ductal keratin 15+ luminal progenitors in normal breast exhibit a basal-like breast cancer transcriptomic signature

Normal breast luminal epithelial progenitors have been implicated as cell of origin in basal-like breast cancer, but their anatomical localization remains understudied. Here, we combine collection under the microscope of organoids from reduction mammoplasties and single-cell mRNA sequencing (scRNA-seq) of FACS-sorted luminal epithelial cells with multicolor imaging to profile ducts and terminal duct lobular units (TDLUs) and compare them with breast cancer subtypes. Unsupervised clustering reveals eleven distinct clusters and a differentiation trajectory starting with keratin 15+ (K15+) progenitors enriched in ducts. Spatial mapping of luminal progenitors is confirmed at the protein level by staining with critical duct markers. Comparison of the gene expression profiles of normal luminal cells with those of breast cancer subtypes suggests a strong correlation between normal breast ductal progenitors and basal-like breast cancer. We propose that K15+ basal-like breast cancers originate in ductal progenitors, which emphasizes the importance of not only lineages but also cellular position within the ductal-lobular tree.

Performance demonstration of a novel photon-counting CT for preclinical application

Photon-counting computed tomography (PC-CT) has attracted attention over the last few years as the next-generation CT technique that solves the problems encountered in clinical CT. In PC-CT, dark current and electronic noise can be reduced by setting the energy threshold to exceed the noise level, which leads to a low-dose scan. Furthermore, multiple energy thresholds realize multicolor CT imaging, which is not possible with clinical CT. Recently, we proposed a novel PC-CT system consisting of a multipixel photon counter (MPPC) coupled with a high-speed scintillator, performing simultaneous imaging of multiple contrast agents and estimate concentration. However, the PC-CT images obtained by our PC-CT system faces some limitations, such as degradation of image quality due to the lack of photon statistics and/or image resolution loss due to the pixel size of the detectors. In this study, the signal-to-noise ratio (SNR) of the PC-CT images was improved by applying machine-learning models, that is, U-Net and Noise2Noise, to the PC-CT images. In addition, a new imaging method was developed to acquire the high-resolution CT images required for clinical use. As a result, the resolution of the CT images improved from 1.04 mm to 0.77 mm. Finally, the visualization of contrast agents in plants was set as a challenge for the next step towards the clinical application of MPPC-based PC-CT. The results demonstrate that our PC-CT system can provide color imaging not only in phantom-based experiments, but also in plants close to an organism.

Selective rod outer segment disruption in commotio retinae - a finding identified on Multicolour® Imaging.

To report Multicolour® imaging (MCI) findings in commotio retinae (CR) involving macula and correlate topographically with outer retinal layers on optical coherence tomography (OCT). This retrospective study included participants with CR involving macula without any other type of traumatic maculopathy and imaged with OCT and MCI. The study included 16 eyes of 16 patients (14 males). Age of presentation ranged from 7 to 56years and presenting vision ranged from 6/6 to 6/24. On OCT, increased reflectivity and obliteration of hyporeflective ellipsoid zone (EZ) and interdigitation zone (IZ) at CR region were seen. Fovea and other retinal layers were spared. On MCI, white areas due to hyperreflectance corresponding to CR were noted on individual colour reflectance channels and on composite multicolour image. In all cases, foveal reflectance pattern was unaffected. The affection of the EZ and IZ at the CR on OCT was associated with increased reflectance on individual wavelength colour channels on MCI. Foveal sparing on MCI correlated with photoreceptor layer sparing at the fovea on OCT. In 6 (38%) cases with follow-up details, normal reflectivity of EZ and IZ was noted in the region of previous CR as early as 1-week post-presentation. White coloration on multicolour image showed resolution. Foveal sparing was common and rod-dominated areas were affected in CR. Corresponding changes on MCI showed hyperreflectance areas on individual wavelength colour channels. Studies combined with photoreceptor-specific electrophysiological tests, adaptive optics imaging and histological evidences would be required in future.

MULTICOLOR CONFOCAL SCANNING LASER OPHTHALMOSCOPE IMAGING IN POSTERIOR UVEITIS.

To study the utility of MultiColor confocal scanning laser ophthalmoscope imaging (MCI) in identifying the morphology of uveitic lesions compared with conventional color fundus photography (CFP) in patients with posterior uveitis. In this prospective observational study, subjects with posterior uveitis underwent MCI and CFP. The images obtained by the two modalities were analyzed by two independent reviewers for vitreoretinal surface abnormalities, retinal fluid and hemorrhages, and depth/location of lesions. These findings were compared with the clinical findings and other imaging techniques. Sixty-nine eyes of 43 patients (25 men) with mean age of 33.5 ± 13.9 years were studied. MultiColor imaging had better sensitivity and specificity in detecting vitreoretinal interface abnormalities, such as epiretinal membrane and inner retinal striae, compared with CFP. MultiColor imaging failed to detect retinochoroiditis lesions in 5 of 6 eyes (83%) and choroiditis in 9 46 eyes (20%), which were detected on CFP and clinical examination. Also, MCI showed a high false-positive rate of 34% in detecting intraretinal hemorrhages. Retinochoroidal lesions in posterior uveitis may be poorly identified on MCI compared with CFP and clinical examination. One must exercise caution in commenting on disease morphology based on MCI alone.