Development of a visible to 1600 nm hyperspectral imaging rigid-scope system using supercontinuum light and an acousto-optic tunable filter.

In this study, we developed a rigid-scope system that can perform hyperspectral imaging (HSI) between visible and 1600 nm wavelengths using a supercontinuum light source and an acousto-optic tunable filter to emit specific wavelengths. The system optical performance was verified, and the classification ability was investigated. Consequently, it was demonstrated that HSI (490-1600 nm) could be performed. In addition, seven different targets could be classified by the neural network with an accuracy of 99.6%, recall of 93.7%, and specificity of 99.1% when the wavelength range of over 1000 nm (OTN) was extracted from HSI data as train data.

Influence of the difference in refractive index on the interface of an object and the surroundings in near-infrared fluorescence tomography.

The refraction of fluorescence from the inside of a sample at the surface results in fluctuations in fluorescence computed tomography (CT). We evaluated the influence of the difference in refractive index (RI) between the sample body and the surroundings on fluorescence CT results. The brightest fluorescent point is away from the correct point on the tomograms owing to the refraction. The speculated position is determined as the exact point if the RI ratio ranges between 0.97 and 1.03 by immersing the body in an RI matching liquid. The results can help in experimental settings of fluorescence CT for acquiring three-dimensional positional information.

Size-controlled bimodal in vivo nanoprobes as near-infrared phosphors and positive contrast agents for magnetic resonance imaging.

Rare-earth-doped nanoparticles (NPs), such as NaGdF4 nanocrystals doped with light-emitting rare earth ions, are promising bimodal probes that allow the integration of over 1000 nm near-infrared (OTN-NIR; NIR-II/III) fluorescence imaging and magnetic resonance imaging (MRI) of live bodies. A precise control of the particle size is the key factor for achieving a high signal-to-noise ratio in both NIR fluorescence and MR images and for regulating their function in the body. In this study, size-controlled NaGdF4:Yb3+, Er3+ NPs prepared by stepwise crystal growth were used for in vivo bimodal imaging. Hexagonal NaGdF4:Yb3+,Er3+ NPs coated with poly(ethylene glycol)-poly(acrylic acid) block copolymer, with hydrodynamic diameters of 15 and 45 nm, were prepared and evaluated as bimodal NPs for OTN-NIR fluorescence imaging and MRI. Their longitudinal (T 1) and transverse (T 2) relaxation rates at the static magnetic field strength of 1.0 T, as well as their cytotoxicity towards NIH3T3 cell lines, were evaluated and compared to study the effect of size. Using these particles, blood vessel visualization was achieved by MRI, with the highest relaxometric ratio (r 1/r 2) of 0.79 reported to date for NaGdF4-based nanoprobes (r 1 = 19.78 mM-1 s-1), and by OTN-NIR fluorescence imaging. The results clearly demonstrate the potential of the size-controlled PEG-modified NaGdF4:Yb3+,Er3+ NPs as powerful 'positive' T 1-weight contrast MRI agents and OTN-NIR fluorophores.

Over 1000 nm Near-Infrared Multispectral Imaging System for Laparoscopic In Vivo Imaging.

In this study, a laparoscopic imaging device and a light source able to select wavelengths by bandpass filters were developed to perform multispectral imaging (MSI) using over 1000 nm near-infrared (OTN-NIR) on regions under a laparoscope. Subsequently, MSI (wavelengths: 1000-1400 nm) was performed using the built device on nine live mice before and after tumor implantation. The normal and tumor pixels captured within the mice were used as teaching data sets, and the tumor-implanted mice data were classified using a neural network applied following a leave-one-out cross-validation procedure. The system provided a specificity of 89.5%, a sensitivity of 53.5%, and an accuracy of 87.8% for subcutaneous tumor discrimination. Aggregated true-positive (TP) pixels were confirmed in all tumor-implanted mice, which indicated that the laparoscopic OTN-NIR MSI could potentially be applied in vivo for classifying target lesions such as cancer in deep tissues.

Delayed Increase in Near-Infrared Fluorescence in Cultured Murine Cancer Cells Labeled with Oxygen-Doped Single-Walled Carbon Nanotubes.

The labeling technique for cells with over-thousand-nanometer near-infrared (OTN-NIR) fluorescent probes has attracted much attention for in vivo deep imaging for cell tracking and cancer metastasis, because of low scattering and absorption of OTN-NIR light by biological tissues. However, the intracellular behavior following the uptake of the single-walled carbon nanotubes (SWCNTs), an OTN-NIR fluorophore, remains unknown. The aim of this study is to investigate the time-dependent change in OTN-NIR fluorescence images of cultured murine cancer cells (Colon-26) following treatment with a recently developed OTN-NIR fluorescent probe, epoxide-type oxygen-doped SWCNTs (o-SWCNTs). The o-SWCNTs were synthesized by oxygenation of SWCNTs by ozone under ultraviolet irradiation and were dispersed in an aqueous solution of N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine to prepare biocompatible o-SWCNTs (o-SWCNT-PEG). OTN-NIR fluorescent o-SWCNT-PEG showed an abnormal behavior following cellular uptake. OTN-NIR fluorescence was not observed in the cells after 24 h incubation with the o-SWCNT-PEG, but clearly increased with longer incubation time from three days after the treatment. This result was further confirmed by Raman microscopy, suggesting that OTN-NIR fluorescence intensity was associated with the cellular uptake of the o-SWCNT-PEG. These results suggest that the Colon-26 cells were successfully labeled by the o-SWCNT-PEG that emit OTN-NIR fluorescence. The o-SWCNT-PEG may aggregate in the cells over time, which could favor their internalization. This delayed concentration followed by a long retention of the o-SWCNT-PEG in cells will facilitate further biotechnological applications of the o-SWCNTs to in vivo deep OTN-NIR fluorescent imaging.

A new orthodontic force system for moment control utilizing the flexibility of common wires: Evaluation of the effect of contractile force and hook length.

BACKGROUND/PURPOSE: The application of an appropriate force system is indispensable for successful orthodontic treatments. Second-order moment control is especially important in many clinical situations, so we developed a new force system composed of a straight orthodontic wire and two crimpable hooks of different lengths to produce the second-order moment. The objective of this study was to evaluate this new force system and determine an optimum condition that could be used in clinics. METHODS: We built a premolar extraction model with two teeth according to the concept of a modified orthodontic simulator. This system was activated by applying contractile force from two hooks that generated second-order moment and force. The experimental device incorporated two sensors, and forces and moments were measured along six axes. We changed the contractile force and hook length to elucidate their effects. Three types of commercial wires were tested. RESULTS: The second-order moment was greater on the longer hook side of the model. Vertical force balanced the difference in moments between the two teeth. Greater contractile force generated a greater second-order moment, which reached a limit of 150 g. Excessive contractile force induced more undesired reactions in the other direction. Longer hooks induced greater moment generation, reaching their limit at 10 mm in length. CONCLUSION: The system acted similar to an off-center V-bend and can be applied in clinical practice as an unconventional loop design. We suggest that this force system has the potential for second-order moment control in clinical applications.

Extra-luminal detection of assumed colonic tumor site by near-infrared laparoscopy.

BACKGROUND: Localization of colorectal tumors during laparoscopic surgery is generally performed by tattooing into the submucosal layer of the colon. However, faint and diffuse tattoos may lead to difficulties in recognizing cancer sites, resulting in inappropriate resection of the colon. We previously demonstrated that yttrium oxide nanoparticles doped with the rare earth ions (ytterbium and erbium) (YNP) showed strong near-infrared (NIR) emission under NIR excitation (1550 nm emission with 980 nm excitation). NIR light can penetrate deep tissues. In this study, we developed an NIR laparoscopy imaging system and demonstrated its use for accurate resection of the colon in swine. METHODS: The NIR laparoscopy system consisted of an NIR laparoscope, NIR excitation laser diode, and an NIR camera. Endo-clips coated with YNP (NIR clip), silicon rubber including YNP (NIR silicon mass), and YNP solution (NIR ink) were prepared as test NIR markers. We used a swine model to detect an assumed colon cancer site using NIR laparoscopy, followed by laparoscopic resection. The NIR markers were fixed at an assumed cancer site within the colon by endoscopy. An NIR laparoscope was then introduced into the abdominal cavity through a laparoscopy port. RESULTS: NIR emission from the markers in the swine colon was successfully recognized using the NIR laparoscopy imaging system. The position of the markers in the colon could be identified. Accurate resection of the colon was performed successfully by laparoscopic surgery under NIR fluorescence guidance. The presence of the NIR markers within the extirpated colon was confirmed, indicating resection of the appropriate site. CONCLUSIONS: NIR laparoscopic surgery is useful for colorectal cancer site recognition and accurate resection using laparoscopic surgery.

Neuronal differentiation of human iPS cells induced by baicalin via regulation of bHLH gene expression.

Efficient differentiation is important for regenerative medicine based on pluripotent stem cells, including treatment of neurodegenerative disorders and trauma. Baicalin promotes neuronal differentiation of neural stem/progenitor cells of rats and mice. To evaluate the suitability of baicalin for neuronal differentiation of human iPS cells, we investigated whether it promotes neuronal differentiation in human iPS cells and monitored basic helix-loop-helix (bHLH) gene expression during neuronal differentiation. Baicalin promoted neuronal differentiation and inhibited glial differentiation, suggesting that baicalin can influence the neuronal fate decision in human iPS cells. Notch signaling, which is upstream of bHLH proteins, was not involved in baicalin-induced neuronal differentiation. Baicalin treatment did not down-regulate Hes1 gene expression, but it reduced Hes1 protein levels and up-regulated Ascl1 gene expression. Thus, baicalin promoted neuronal differentiation via modulation of bHLH transcriptional factors. Therefore, baicalin has potential to be used as a small-molecule drug for regenerative treatment of neurodegenerative disorders.

Pixel Screening in Lifetime-Based Temperature Mapping Using ß-NaYF4:Nd3+,Yb3+ by Time-Gated Near-Infrared Fluorescence Imaging on Deep Tissue in Live Mice.

Near-infrared fluorescence (NIRF) thermometry is an emerging method for the noncontact measurement of in vivo deep temperatures. Fluorescence-lifetime-based methods are effective because they are unaffected by optical loss due to excitation or detection paths. Moreover, the physiological changes in body temperature in deep tissues and their pharmacological effects are yet to be fully explored. In this study, we investigated the potential application of the NIRF lifetime-based method for temperature measurement of in vivo deep tissues in the abdomen using rare-earth-based particle materials. ß-NaYF4 particles codoped with Nd3+ and Yb3+ (excitation: 808 nm, emission: 980 nm) were used as NIRF thermometers, and their fluorescence decay curves were exponential. Slope linearity analysis (SLA), a screening method, was proposed to extract pixels with valid data. This method involves performing a linearity evaluation of the semilogarithmic plot of the decay curve collected at three delay times after cutting off the pulsed laser irradiation. After intragastric administration of the thermometer, the stomach temperature was monitored by using an NIRF time-gated imaging setup. Concurrently, a heater was attached to the lower abdomens of the mice under anesthesia. A decrease in the stomach temperature under anesthesia and its recovery via the heater indicated changes in the fluorescence lifetime of the thermometer placed inside the body. Thus, NaYF4:Nd3+/Yb3+ functions as a fluorescence thermometer that can measure in vivo temperature based on the temperature dependence of the fluorescence lifetime at 980 nm under 808 nm excitation. This study demonstrated the ability of a rare-earth-based NIRF thermometer to measure deep tissues in live mice, with the proposed SLA method for excluding the noisy deviations from the analysis for measuring temperature using the NIRF lifetime of a rare-earth-based thermometer.

Enhancing near-infrared fluorescence intensity and stability of PLGA-b-PEG micelles by introducing Gd-DOTA at the core boundary.

Micelles have been extensively used in biomedicine as potential carriers of hydrophobic fluorescent dyes. Their small diameters can potentially enable them to evade recognition by the reticuloendothelial system, resulting in prolonged circulation. Nevertheless, their lack of stability in physiological environments limits the imaging utility of micelles. In particular, when a dye sensitive to water, such as IR-1061, is encapsulated in the micelle core, the destabilized structure leads to interactions between water and dye, degrading the fluorescence. In this study, we investigated a method to improve micelle stability utilizing the electrical effect of gadolinium (Gd3+ ) and tetraazacyclododecane tetraacetic acid (DOTA), introduced into the micelles. Three micellar structures, one containing a poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) block copolymer, and two other structures, including PLGA-b-PEG with DOTA or Gd-DOTA introduced at the boundary of PLGA and PEG, were prepared with IR-1061 in the core. Structures that contained DOTA at the border of the PLGA core and PEG shell exhibited much higher fluorescence intensity than probes without DOTA. With Gd3+ ions at the DOTA center, fluorescence stability was enhanced remarkably in physiological environments. Most interesting is the finding that fluorescence is enhanced with increased Gd-DOTA concentrations. In conclusion, we found that overall fluorescence and stability are improved by introducing Gd-DOTA at the boundary of the PLGA core and PEG shell. Improving micelle stability is crucial for further biomedical applications of micellar probes such as bimodal fluorescence and magnetic resonance imaging.

Temperature imaging inside fluid devices using a ratiometric near infrared (NIR-II/III) fluorescent Y2O3: Nd3+, Yb3+, Er3+ nanothermometer.

Luminescence thermometry is a non-contact method that can measure surface temperatures and the temperature of the area where the fluorescent probe is located, allowing temperature distribution visualizations with a camera. Ratiometric fluorescence thermometry, which uses the intensity ratio of fluorescence peaks at two wavelengths with different fluorescence intensity dependencies, is an excellent method for visualizing temperature distributions independent of the fluorophore spatial concentration, excitation light intensity and absolute fluorescence intensity. Herein, Nd3+/Yb3+/Er3+-doped Y2O3 nanomaterials with a diameter of 200 nm were prepared as phosphors for temperature distribution measurement of fluids at different temperatures. The advantages of this designed fluorescent material include non-aggregation in water and the fact that its near-infrared (NIR) fluorescence excitation (808 nm) is not absorbed by water, thereby minimizing sample heating upon irradiation. Under optical excitation at 808 nm, the ratio of the fluorescence intensities of Yb3+ (IYb; 975 nm) and Er3+ (IEr; 1550 nm), which exhibited different temperature responses, indicated the temperature distribution inside the fluid device. Thus, this technique using Nd3+/Yb3+/Er3+-doped Y2O3 is expected to be applied for temperature distribution mapping analysis inside fluidic devices as a ratiometric NIR fluorescence thermometer, which is unaffected by laser-induced heating.

Micromolding in capillaries for calcination-free fabrication of flexible inorganic phosphor films consisting of rare-earth-ion-doped nanoparticles.

We discuss the micromolding in capillaries technique for the direct fabrication of calcination-free rare earth ion-doped (RE) phosphor films consisting of RE nanoparticles on plastic sheets. We synthesized two types of RE nanoparticles consisting of Y2O3 matrix doped with Er and Yb ions. Green upconversion luminescence, red upconversion luminescence, and near-infrared fluorescence appeared from the RE nanoparticles under excitation of near-infrared light. Adjusting the channel width and depth of polydimethylsiloxane molds led to control of the density of nanoparticles in the patterned RE nanoparticle films. Adjusting concentration of the RE nanoparticle dispersion and size of the RE nanoparticles allowed for the control of the density of nanoparticles in the patterned RE nanoparticle films. The density of nanoparticles in the patterned RE films on plastic sheets increased with an increase in the number of injection and drying of the RE nanoparticle dispersion. These results demonstrate that this technique enables us to directly fabricate the patterned RE phosphor films on plastic sheets, leading to the fabrication of inorganic flexible devices with small fabrication steps and material consumptions.

Controlling Molecular Dye Encapsulation in the Hydrophobic Core of Core-Shell Nanoparticles for In Vivo Imaging.

Polymeric nanoparticles with a hydrophobic core are valuable biomedical materials with potential applications in in vivo imaging and drug delivery. These materials are effective at protecting vulnerable molecules, enabling them to serve their functions in hydrophilic physiological environments; however, strategies that allow the chemical composition and molecular weight of polymers to be tuned, forming nanoparticles to control the functional molecules, are lacking. In this article, we review strategies for designing core-shell nanoparticles that enable the effective and stable encapsulation of functional molecules for biomedical applications. IR-1061, which changes its optical properties in response to the microenvironment are useful for in vitro screening of the in vivo stability of polymeric nanoparticles. An in vitro screening test can be performed by dispersing IR-1061-encapsulated polymer nanoparticles in water, saline, buffer solution, aqueous protein solution, etc., and measuring the absorption spectral changes. Through the screening, the effects of the polarity, molecular weight, and the chiral structure of polymers consisting of polymer nanoparticles on their stability have been revealed. Based on the findings presented here, more methodologies for the effective application of various biomolecules and macromolecules with complex high-dimensional structures are expected to be developed.

Visualization of hydrocarbon chain length and degree of saturation of fatty acids in mouse livers by combining near-infrared hyperspectral imaging and machine learning.

Fatty acids play various physiological roles owing to their diverse structural characteristics, such as hydrocarbon chain length (HCL) and degree of saturation (DS). Although the distribution of fatty acids in biological tissues is associated with lipid metabolism, in situ imaging tools are still lacking for HCL and DS. Here, we introduce a framework of near-infrared (1000-1400 nm) hyperspectral label-free imaging with machine learning analysis of the fatty acid HCL and DS distribution in the liver at each pixel, in addition to the previously reported total lipid content. The training data of 16 typical fatty acids were obtained by gas chromatography from liver samples of mice fed with various diets. A two-dimensional mapping of these two parameters was successfully performed. Furthermore, the HCL/DS plot exhibited characteristic clustering among the different diet groups. Visualization of fatty acid distribution would provide insights for revealing the pathophysiological conditions of liver diseases and metabolism.

Influence of Carboxyl Group Ratios on the Design of Breast Cancer Targeting Bimodal MR/NIR-II Imaging Probe from PLGA@Gd-DOTA@PEG Micelles Conjugating Herceptin.

We developed a small MRI/NIR-II probe to target HER2 (tetanucleotide) breast cancer cells. The probe is composed of PLGA-b-PEG micelles encapsulated NIR-II, and Gd-DOTA is conjugated at the border of PLGA/PEG. Herceptin was then conjugated to carboxyl residues of PLGA-b-PEG chains. We examined the influence of carboxyl group ratios on the probe property stability and Herceptin concentration and the binding affinity to HER2(+) cells corresponding to the -COOH ratios. The binding assays demonstrated that the optimal surface ratio of -COOH is 5%, which is less affected by fluorescence reduction and which exhibited the highest antigen-capturing activity.

Evaluating the identification of the extent of gastric cancer by over-1000 nm near-infrared hyperspectral imaging using surgical specimens.

Significance: Determining the extent of gastric cancer (GC) is necessary for evaluating the gastrectomy margin for GC. Additionally, determining the extent of the GC that is not exposed to the mucosal surface remains difficult. However, near-infrared (NIR) can penetrate mucosal tissues highly efficiently. Aim: We investigated the ability of near-infrared hyperspectral imaging (NIR-HSI) to identify GC areas, including exposed and unexposed using surgical specimens, and explored the identifiable characteristics of the GC. Approach: Our study examined 10 patients with diagnosed GC who underwent surgery between 2020 and 2021. Specimen images were captured using NIR-HSI. For the specimens, the exposed area was defined as an area wherein the cancer was exposed on the surface, the unexposed area as an area wherein the cancer was present although the surface was covered by normal tissue, and the normal area as an area wherein the cancer was absent. We estimated the GC (including the exposed and unexposed areas) and normal areas using a support vector machine, which is a machine-learning method for classification. The prediction accuracy of the GC region in every area and normal region was evaluated. Additionally, the tumor thicknesses of the GC were pathologically measured, and their differences in identifiable and unidentifiable areas were compared using NIR-HSI. Results: The average prediction accuracy of the GC regions combined with both areas was 77.2%; with exposed and unexposed areas was 79.7% and 68.5%, respectively; and with normal regions was 79.7%. Additionally, the areas identified as cancerous had a tumor thickness of >2 mm. Conclusions: NIR-HSI identified the GC regions with high rates. As a feature, the exposed and unexposed areas with tumor thicknesses of >2 mm were identified using NIR-HSI.

In vitro and in vivo investigations of upconversion and NIR emitting Gd2O3:Er³âº,Yb³âº nanostructures for biomedical applications.

The use of an "over 1000-nm near-infrared (NIR) in vivo fluorescence bioimaging" system based on lanthanide containing inorganic nanostructures emitting in the visible and NIR range under 980-nm excitation is proposed. It may overcome problems of currently used biomarkers including color fading, phototoxicity and scattering. Gd(2)O(3):Er(3+),Yb(3+) nanoparticles and nanorods showing upconversion and NIR emission are synthesized and their cytotoxic behavior is investigated by incubation with B-cell hybridomas and macrophages. Surface modification with PEG-b-PAAc provides the necessary chemical durability reducing the release of toxic Gd(3+) ions. NIR fluorescence microscopy is used to investigate the suitability of the nanostructures as NIR-NIR biomarkers. The in vitro uptake of bare and modified nanostructures by macrophages is investigated by confocal laser scanning microscopy. In vivo investigations revealed nanostructures in liver, lung, kidneys and spleen a few hours after injection into mice, while most of the nanostructures have been removed from the body after 24 h.

Heat Treatment Effects for Controlling Dye Molecular States in the Hydrophobic Core of Over-1000 nm Near-Infrared (NIR-II) Fluorescent Micellar Nanoparticles.

Organic molecules that emit near-infrared (NIR) fluorescence at wavelengths above 1000 nm, also known as the second NIR (NIR-II) biological window, are expected to be applied to optical in vivo imaging of deep tissues. The study of molecular states of NIR-II dye and its optical properties are important to yield well-controlled fluorescent probes; however, no such study has been conducted yet. Among the two major absorption peaks of the NIR-II dye, IR-1061, the ratio of the shorter wavelength (900 nm) to the longer one (1060 nm) increased with an increase in the dye concentration in tetrahydrofuran, suggesting that the 900 nm peak is due to the dimer formation of IR-1061. Both absorption peaks are also observed when IR-1061 is encapsulated in the hydrophobic (stearyl) core of micellar nanoparticles (MNPs) of a phospholipid-poly(ethylene glycol). The dimers in the MNP cores decreased via dimer dissociation by enhancing the mobility of the hydrophobic stearyl chains by heat treatment of the dye-encapsulating MNPs at 50-70 °C. The MNPs maintained the dissociated IR-1061 monomers in the core after recooling to 25 °C and showed a higher NIR-II fluorescence intensity than those before heat treatment. This concept will provide better protocols for the preparation of NIR-II fluorescent probes with well-controlled fluorescence properties.

Effect of the enantiomeric structure of hydrophobic polymers on the encapsulation properties of a second near infrared (NIR-II) fluorescent dye for in vivo deep imaging.

Over-thousand-nanometer (OTN) near-infrared (NIR) fluorophores are useful for biological deep imaging because of the reduced absorption and scattering of OTN-NIR light in biological tissues. IR-1061, an OTN-NIR fluorescent dye, has a hydrophobic and cationic backbone in its molecular structure, and a non-polar counter ion, BF4 -. Because of its hydrophobicity, IR-1061 needs to be encapsulated in a hydrophobic microenvironment, such as a hydrophobic core of polymer micelles, shielded with a hydrophilic shell for bioimaging applications. Previous studies have shown that the affinity of dyes with hydrophobic core polymers is dependent on the polarity of the core polymer, and that this characteristic is important for designing dye-encapsulated micelles to be used in bioimaging. In this study, the dye-polymer affinity was investigated using hydrophobic polymer films with different chiral structures of poly(lactic acid). IR-1061 showed higher affinity for l- and d-lactic acid copolymers (i.e., poly(dl-lactic acid) (PDLLA)) than to poly(l-lactic acid) (PLLA), as IR-1061 shows less dimerization in PDLLA than in PLLA. In contrast, the stability of IR-1061 in PDLLA was less than that in PLLA due to the influence of hydroxyl groups. Choosing hydrophobic core polymers for their robustness and dye affinity is an effective strategy to prepare OTN-NIR fluorescent probes for in vivo deep imaging.

The effect of Gd-DOTA locations within PLGA-b-PEG micelle encapsulated IR-1061 on bimodal over-1000 nm near-infrared fluorescence and magnetic resonance imaging.

Multimodal imaging is attractive in biomedical research because it can provide multidimensional information about objects that individual techniques cannot accomplish. In particular, combining over one-thousand-nanometer near-infrared (OTN-NIR) fluorescence and magnetic resonance (MR) imaging is promising for detecting lesions with high sensitivity and structural information. Herein, we describe the development of a bimodal OTN-NIR/MRI probe from gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) conjugated poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer (PLGA-b-PEG) micelle encapsulated IR-1061 at two different locations. One configuration contains Gd-DOTA at the end of the PEG of the hydrophilic shell and the other contains Gd-DOTA at the border of PLGA/PEG. The two structures show remarkable differences in fluorescence and R1 relaxation rates in biological environments; the structure with Gd-DOTA at the border of PLGA/PEG exhibits stable fluorescence and T1 signal distribution in live mice. The introduction ratio of Gd-DOTA to PEG is significant for controlling the properties of both structures; a higher Gd-DOTA ratio is preferable for the contrast enhancement effect. We found that Gd-DOTA ratios higher than 10% degraded the fluorescence intensity when Gd-DOTA was bound to the end of PEG. In contrast, the introduction of 70% Gd-DOTA at the border of PLGA/PEG did not exhibit a degraded signal, and the structural stability was enhanced with higher ratios of Gd-DOTA. In conclusion, we confirmed that the location of Gd-DOTA is a crucial factor in designing high-performance probes. The overall properties improve when Gd-DOTA is set on the border of PLGA/PEG. These improvements in the properties by controlling the probe structures are promising for future biomedical applications.