Fluorescence Imaging of DMDG-ICG Across NIR-I and NIR-II Windows Using a Single-Camera System

Abstract

Near-infrared (NIR) imaging, including NIR-I (800–1000 nm) and NIR-II (1000–1700 nm), has been primarily evaluated using separate cameras with different detectors, limiting comparison. We investigated whether using a single-camera system capable of both NIR-I and NIR-II acquisition, NIR-II improves spatial resolution and contrast-to-noise ratio (CNR) for nanoparticle-based imaging. Dual-mode, dual-Gd ICG (DMDG-ICG) nanoparticles were characterized for absorption and fluorescence. A custom NIR imaging system using a single InGaAs camera enabled visualizing both NIR-I and -II windows. In vitro, capillary tubes containing nanoparticles in water, in tissue-mimicking Intralipid, or covered with mouse skin were imaged, and full-width-half maximum (FWHM) and CNR were measured. In vivo, the mouse femoral artery was imaged after IV nanoparticle delivery. DMDG-ICG showed strong fluorescence at both NIR-I and NIR-II. Scatter greater at NIR-I than NIR-II increased with depth and tissue layers. FWHM was lower and CNR higher at NIR-II versus NIR-I for up to 10 mm depth (p < 0.05, n = 3) in Intralipid. In vivo, femoral artery CNR was also higher at NIR-II (p < 0.05, n = 3). Using a single-camera system allowing direct comparison, NIR-II imaging provided greater penetration, spatial resolution, and CNR compared to NIR-I. The findings support the utility of NIR-II for vascular and molecular imaging applications.

1. Introduction

Optical imaging in the near-infrared (NIR) window offers advantages over visible light imaging in biological tissues due to reduced absorption, scattering, and autofluorescence. The NIR window can be further divided into NIR-I and NIR-II regions. At NIR-I, absorption and scattering from blood significantly decreases [1]. At NIR-II, absorption and scattering from water also decreases, particularly at 1000–1300 nm [2,3,4,5], which should lead to greater depth of penetration with enhanced contrast. At wavelengths above 1300 nm, strong water absorption bands dominate, limiting imaging depth [4]. Cameras for imaging fluorophores have primarily used silicon-based detectors, which are highly efficient, but their efficiency drops significantly beyond 800 nm [3]. For NIR-II imaging, indium gallium arsenide (InGaAs) cameras are commonly used, but their efficiency is typically low at wavelengths below 950 nm [3]. Direct comparisons of NIR-I and NIR-II fluorescence imaging have been limited because most prior studies have been primarily evaluated using separate camera systems, which are typically a silicon detector camera for NIR-I and an InGaAs camera for NIR-II that could influence the results [6,7,8]. As detector performance, noise characteristics, and optical responses differ substantially between camera types, it remains challenging to isolate intrinsic differences in NIR-I and NIR-II imaging performance using two different cameras, particularly with two different detector types. Recent advances in InGaAs camera technology have enabled broadband detection with extended spectral range and improved quantum efficiency (See Supplementary Figure S1), covering both NIR-I and NIR-II wavelengths using a single detector. Acquisition using a single InGaAs camera system is needed for quantitative comparison of NIR-I and NIR-II imaging performance. For such a comparison, a fluorophore capable of emission at both wavelengths is desirable. For in vivo imaging, encapsulation in nanoparticles can result in greater fluorescence and aid localization for applications such as vascular imaging and tumor imaging.

Indocyanine green (ICG) is a near-infrared (NIR) fluorescent dye that is widely used for NIR imaging in medical diagnostics [9,10]. In 1959, it became the only NIR dye approved by the US Food and Drug Administration for clinical applications such as angiography and oncologic image-guided surgery [11,12,13]. As with all fluorophores, ICG exhibits a distinct emission peak along with a broader emission spectrum. Recently, it was reported that ICG has a long-wavelength emission tail that extends into NIR-II, also referred to as the short-wave infrared (SWIR) region [8,14]. Also, it was recently found that ICG exists as a monomer in not only a planar form, but also in a TWIST form with shift in peak emission from around 820 nm in the planar form to a twisted intramolecular charge transfer (TICT) peak emission near 900 nm, closer to the NIR-II window [15,16,17]. Optical imaging using the NIR-II region theoretically should improve spatial resolution and imaging contrast due to lower background noise and greater tissue penetration of longer wavelengths, compared to optical imaging in the NIR-I or visible region. ICG fluorescence can be enhanced by encapsulation and appropriate nanoparticle formulations can increase circulation time and stability [18,19,20].

The dual-mode-dual-gadolinium (DMDG-ICG) liposome is an advanced nanoparticle imaging agent designed to enable both fluorescence and magnetic resonance imaging (MRI) modalities [18,21]. These liposomes encapsulate ICG, a near-infrared fluorescent dye, as well as encapsulate and surface display gadolinium-based contrast agents, enabling dual-mode imaging modalities that integrate percutaneous MRI with the real-time, near-infrared optical visualization provided by ICG [18,21,22]. These nanoparticles have shown utility to image tumors, for example, in ovarian cancer models [18,21]. PEGylation of these liposomes was performed to impart stealth properties to extend their circulation time for in vivo imaging [18,21,23]. These nanoparticles fluoresce at NIR-I and NIR-II upon a single wavelength excitation.

Using a single camera capable of both NIR-I and NIR-II imaging, and a single nanoparticle capable of NIR-I and NIR-II emission from a single wavelength excitation, we evaluated whether the spatial resolution and contrast-to-noise ratio (CNR) during nanoparticle imaging are superior in NIR-II compared to NIR-I.

2. Results

2.1. In Vitro NIR Fluorescence Imaging and In Vivo-Enabled NIR-I and NIR-II Imaging Set up

Figure 1a presents the schematic diagram of the key components in our custom-built NIR fluorescence setup designed for both phantoms and in vivo imaging in the NIR-I and NIR-II regions using a single NIR detector. The setup includes the 785 nm laser, the holder for either the phantom or mouse, and the InGaAs camera (spectral range: 600–1700 nm) capable of capturing images in both NIR-I and NIR-II windows. Appropriate optical filters were used for the detection range: 800–1000 nm for NIR-I and 1000–1700 nm for NIR-II.

In vitro imaging of capillary phantoms was conducted using a single detector to minimize errors that may be caused by dual-camera systems (silicon-based CCD camera for visible and NIR-I and InGaAs focal plane arrays for NIR-II). To achieve uniform and wide-area illumination, a collimator and an engineered diffuser, which provide a non-Gaussian intensity distribution, were utilized following the laser fiber output. Fluorescence signals were captured using the InGaAs camera equipped with interchangeable, high transmittance NIR-I and NIR-II filters, which were placed in front of the camera lens. The sample was positioned on a stage, and specifically, a capillary phantom was placed on a cylindrical reservoir filled with varying volumes of 1% Intralipid solution to assess the penetration depth, ranging from 0 mm to 10 mm.

Figure 1b,c present the absorption and fluorescence spectra of DMDG-ICG nanoparticle, respectively. The raw absorption spectrum (black solid line) of DMDG-ICG showed the characteristic absorption features of incorporated ICG, along with scattering effects from the ~100 nm nanoparticles (gray dotted line), which scale inversely with the fourth power of the wavelength. After subtracting the scattering effects, the scattering-subtracted spectrum of DMDG-ICG is displayed as the blue line, revealing the ICG absorption with a peak around 820 nm. In the fluorescence spectrum of DMDG-ICG, the emission peaks observed around 820 nm and 950 nm correspond to planar and TWIST emission forms of ICG, respectively [15,16]. Notably, upon the same excitation, fluorescence emission was observed both below and above 1000 nm, indicating that the fluorescence of DMDG-ICG emits in NIR-I and NIR-II regions. The InGaAs sensor employed in our system provided high quantum efficiency across the NIR-I and NIR-II ranges. (See Supplementary Figure S1). This single np capable of NIR-I and NIR-II fluorescence upon single excitation was used for subsequent experiments. The DMDG–ICG liposomes exhibited a hydrodynamic diameter of 149 nm with a polydispersity index (PDI) of 0.083 and a zeta potential of −17 mV.

2.2. Transparent Phantom Imaging

NIR-I and NIR-II images were acquired from a capillary tube containing 0.5 µM DMDG-ICG in histidine buffer, a concentration selected based on its confirmed linear relationship between fluorescence intensity and concentration (see Supplementary Figure S2). The capillary tube was immersed in DI water, which served as a transparent medium, at depths of 1, 3, 5, and 7 mm from the surface (Figure 2). The fluorescence images were normalized by the maximum intensity of the images to evaluate relative signal distribution and penetration depth. The absorbance of DI water in the NIR region is negligible, except for the combination vibration bands around 1500 nm. This minimal absorbance allows for the 785 nm excitation and NIR fluorescence of ICG without significant interference, making DI water an ideal transparent medium for phantom imaging. The fluorescence emitted from the capillary tubes was clearly visible in both the NIR-I and NIR-II windows at all four penetration depths, with minimal background signals from scattering and autofluorescence. Cross-sectional fluorescence intensities are shown for NIR-I (Figure 2b) and NIR-II (Figure 2c). NIR-I and NIR-II fluorescence remained largely unaffected by scattering or re-absorption in the DI water medium. As the penetration depth increased in DI water, fluorescence intensity decreased slightly, while changes in the FWHM remained largely unchanged.

2.3. Intralipid Phantom Imaging

To mimic the optical properties of biological tissues, 1% Intralipid was used as a scattering medium, as its optical characteristics approximate those of many soft tissues in the visible and NIR spectral ranges [24]. NIR-I and NIR-II images were obtained of a capillary tube filled with 0.5 µM DMDG-ICG in histidine buffer and immersed in 1% Intralipid (Figure 3a), with fluorescence images normalized as described above for the DI water experiments. Four representative images were taken at depths of 1, 3, 5, and 7 mm from the surface, respectively. The phantoms at 1 mm penetration depth in the Intralipid medium are clearly visible with minimal scattering. As the penetration depth increased in Intralipid, the NIR fluorescence images became progressively blurrier due to the scattering properties of the medium. However, images acquired in the NIR-II window exhibited reduced scattering and lower background compared to those in the NIR-I window, particularly at greater depths. Cross-sectional fluorescence intensity profiles of the capillary tube were obtained at NIR-I and NIR-II (Figure 3c). As penetration depth increased, the FL intensity decreased, and the apparent width of the capillary broadened. To further assess spatial resolution, fluorescence attenuation, and overall image quality of the system, additional analyses were performed, including measurements of FWHM for spatial resolution (image sharpness), FL intensity normalized to the intensity at 1 mm depth, and contrast-to-noise ratio (CNR) (Figure 4).

Figure 4a shows the FWHM as a function of penetration depth in both DI water and Intralipid phantoms for NIR-I and NIR-II imaging. Across all depths in the Intralipid phantom, the FWHM in the NIR-II region was approximately 40% narrower than in the NIR-I region (p < 0.05, n = 3), indicating superior spatial resolution and image sharpness for NIR-II imaging at all depths. In contrast, no significant difference in FWHM was observed between NIR-I and NIR-II under DI water conditions (p > 0.05, n = 3). Figure 4b shows the attenuation of fluorescence intensity as a function of depth. A greater loss of fluorescence intensity at 2 mm penetration depth and beyond was observed in the Intralipid phantom, a scattering medium, compared to transparent DI water, in both the NIR-I (p < 0.05, n = 3) and NIR-II (p < 0.05, n = 3) regions. In Intralipid, fluorescence attenuation followed an exponential decay with depth at NIR-I and NIR-II. These results suggest that fluorescence intensity loss is more pronounced in the tissue-mimicking Intralipid medium than in water.

Image quality was evaluated by CNR. Overall, CNR values in the NIR-II regions were significantly higher than those at NIR-I with either water or Intralipid phantom imaging (Figure 4c). In the Intralipid phantom, CNR was significantly greater at NIR-II compared to NIR-I (p < 0.05, n = 3) up to the penetration depth of approximately 6 mm. These findings suggest that NIR-II imaging provides improved CNR over NIR-I when using tissue-mimicking Intralipid phantoms incorporating DMDG-ICG nanoparticles.

Images were captured with exposure times of 20 ms for NIR-I (Figure 5a) and 200 ms for NIR-II (Figure 5b). As the number of skin layers increased, the NIR fluorescence images became progressively blurrier due to increased scattering; however, the phantom appeared sharper at NIR-II compared to NIR-I, with less background noise.

The cross-sectional fluorescence profiles of the capillary tubes in the NIR-I and NIR-II regions, shown in Figure 5c,d, span three conditions: uncovered, single-layer, and double-layer mouse skin coverage. As the number of mouse skin layers increases, fluorescence intensity is attenuated and exhibits broader profiles, consistent with trends observed with Intralipid phantom imaging. Notably, NIR-II imaging demonstrated reduced background noise and improved signal clarity, despite requiring an exposure time ten times longer than that of NIR-I imaging.

To evaluate spatial resolution and imaging quality, FWHM (Figure 5e) and CNR values (Figure 5f) were assessed. In air (without skin coverage), the FWHM values were equivalent for NIR-I and NIR-II. However, FWHM values increased to a greater extent for NIR-I compared to NIR-II with one (p < 0.05, n = 3) or two nude mouse skin layers (p < 0.05, n = 3). Consistent with less background and scatter at NIR II, the CNR plot (Figure 5f, Supplementary Table S1) indicates that NIR-II imaging provided CNR improvements of 7-fold with air (p < 0.05, n = 3), 10-fold with a single skin layer (p < 0.05, n = 3), and approximately 18-fold with a double skin layer (p < 0.05, n = 3) compared to NIR-I imaging.

2.4. In Vivo Imaging in NIR-I and NIR-II Windows

Next, the NIR fluorescence imaging system was tested in vivo using a nude mouse vascular imaging model. Mice were injected with 200 μL of DMDG-ICG via tail vein injection, and 5 min post-injection, they were imaged with exposure times of 50 ms for NIR-I and 500 ms for NIR-II, respectively, using appropriate filters. Representative images of the hind limb in the NIR-I (Figure 6a) and NIR-II (Figure 6b) windows demonstrated the femoral blood vessel at both NIR-I and -II imaging. However, the background signal in the mouse was lower at NIR-II than -I. Quantitative analysis revealed that the CNR was approximately two-fold higher in NIR-II than in NIR-I imaging (p < 0.05, n = 3), indicating improved imaging contrast in vivo with NIR-II.

3. Discussion

Using the same camera for image acquisition, we found, in both phantoms and in living subjects, improved imaging performance, such as CNR, at NIR-II over NIR-I when employing a nanoparticle capable of fluorescing in both windows. The second near-infrared (NIR-II) window has recently emerged as a promising alternative to the conventionally utilized NIR-I window [8,14,25]. However, previous studies have used different types of camera systems for NIR-I and NIR-II comparisons, introducing potential systematic errors [7,8,26,27]. Most conventional NIR imaging has relied on silicon-based scientific complementary metal-oxide semiconductor (sCMOS) cameras, which tend to lose efficiency beyond ~800 nm. Cameras designed for NIR-II imaging typically utilize compound semiconductor detector elements, such as InGaAs, which have tended to be sensitive in the 950–1700 nm range, where silicon-based detectors experience a loss of efficiency. In comparing NIR-I and-II imaging, the two different cameras may introduce bias and limit direct comparisons. In addition, most comparative studies between NIR-I and NIR-II have employed longpass filters, which do not allow for detection strictly within the NIR-I window [8,25,26]. In this study, we employed a single InGaAs camera with broader wavelength efficiency, along with appropriate filters, to enable accurate imaging in both NIR-I and -II windows. Notably, the InGaAs camera used here demonstrated higher quantum efficiency than a conventional CCD camera across both NIR-I and -II regions (see Supplementary Figure S1). To further improve consistency and reduce variability, enhancing robustness to the study design, we also employed a single nanoparticle (DMDG-ICG) capable of fluorescing at both NIR-I and -II upon a single excitation wavelength. Using our custom-built NIR fluorescence imaging system, employing a single camera and a single type of nanoparticle for both NIR-I and -II, we found that NIR-II imaging can provide improved spatial resolution and CNR compared to NIR-I

Compared to visible optical imaging, reduced scattering and lower tissue autofluorescence are noted at NIR-I imaging due to decreased fluorescence from hemoglobin [1,3]. In the NIR-II region, and in particular <1200 nm [3], there is also less fluorescence from water, which should further improve imaging of biological tissue [3]. NIR-I imaging is enabled by widely available CMOS cameras and has been successfully employed for many biological and clinical fluorescence imaging applications but suffers from loss of signal as penetration depth in tissues increases. NIR-II cameras and fluorophores are being developed [8,14,16,25] and should improve NIR imaging performance at greater tissue depth. Beyond current NIR applications, these advancements may enable deeper vascular imaging in animal models, and deeper tumor imaging, including beneath the few mm deep epithelial surfaces.

For this study, we used a nanoparticle formulation containing clinically approved ICG. We recently discovered that ICG exists in a second predominant emissive form. Classically, ICG fluorescence has been described using CMOS detectors that have a rapid drop off in sensitivity after ~800 nm, and using such detectors, the ICG fluorescence peak has been suggested to be at ~820 nm. Using a spectrophotometer with an InGaAs detector, we noted a second predominant peak fluorescing at 880 nm corresponding to the TWIST form, rather than the classically described planar form with 820 nm peak. The TWIST form pushes the fluorescence towards the NIR-II window. In the nanoparticle formulation incorporating ICG used for this study, we found a broader peak that started at ~840 nm and extended beyond 950 nm, and had a fluorescence tail beyond 1000 nm. The nanoparticle served as a single imaging agent for NIR-I and -II fluorescence with a single excitation laser. In addition to phantom studies, it had long-term vascular residence, as we have seen previously with the Dual-Gd platform [8,18,21], and enabled vascular imaging for in vivo studies [8]. Furthermore, the dual-window functionality enables versatile imaging applications [28], allowing the use of NIR-I for high fluorescence intensity in superficial tissues and NIR-II for improved clarity in deeper tissues, all without the need to switch probes.

A custom-built near-infrared (NIR) fluorescence imaging system was developed to explore both NIR-I and NIR-II regions using a single NIR detector, effectively minimizing artifacts that can be associated with dual-camera setups. In conventional dual-camera systems, a CCD is typically used for NIR-I and an InGaAs camera for NIR-II, resulting in differences in pixel size, field of view, and readout noise, including due to distinct cooling systems, which make precise image registration and direct comparison of NIR-II and NIR-I signals challenging. The current system incorporated a collimator and an engineered diffuser to ensure uniform, wide-area illumination (~100 cm²), which is essential for consistent excitation in NIR fluorescence imaging. In vitro imaging of a capillary phantom in DI water demonstrated clear fluorescence signals in both NIR-I and NIR-II windows, with minimal interference from scattering and autofluorescence across varying penetration depths. In contrast, imaging within an Intralipid phantom, used to mimic the light scattering of biological tissues, revealed that fluorescence images became increasingly blurry with greater depth. However, NIR-II imaging, with its reduced autofluorescence noise and photon scattering at longer wavelengths, demonstrated superior spatial resolution and higher CNR values compared to NIR-I in highly scattering media. This was despite the lower fluorescence intensity at NIR-II than NIR-I by the nanoparticle and the longer acquisition time by the camera. Similar findings were seen with biological tissue of one or two layers of nude mouse skin, resulting in multiple-fold increases in CNR at NIR-II versus NIR-I. Previous studies have demonstrated enhanced visualization of hindlimb and cranial vasculature using NIR-II fluorescence imaging in dual-camera systems [8,18,29,30]. In contrast, the present study demonstrates that comparable advantages can be achieved using a single ICG-based nanoparticle, along with a single excitation light source, and a single camera detection system for both NIR-I and NIR-II imaging as systematically evaluated through capillary phantoms and biological tissues. These findings were further validated in vivo, where blood vessel imaging exhibited approximately a two-fold increase in CNR values in the NIR-II window compared to NIR-I.

The implications of NIR-II imaging using clinically approved ICG in a liposomal formulation are significant. Our findings demonstrate that NIR-II imaging under strong scattering conditions provides superior CNR and enhanced spatial resolution compared to the clinically utilized NIR-I window, indicating improved overall image quality at greater tissue penetration depths. This performance was achieved using a clinically approved excitation wavelength of 785 nm at safe power levels (~10 mW/cm²), well below the established safety threshold of 329 mW/cm² [29]. Under these conditions, DMDG nanoparticles incorporating ICG enabled effective visualization of both capillary phantoms and in vivo vasculature in a mouse model. These results suggest that NIR-II imaging systems—without the need for major changes to clinical protocols—could potentially be translated into clinical applications such as vascular and tumor imaging [7,25], as well as image-guided surgery [12,31], offering significant improvements in NIR image quality. Using agents with NIR-I and NIR-II capabilities and a camera with NIR-I and NIR-II capabilities would simplify protocols and enable high signal superficial NIR-I imaging and high CNR deeper tissue NIR-II imaging without the need for multiple fluorophores or multiple camera systems.

While NIR-II imaging demonstrated superior spatial resolution and contrast-to-noise ratio compared to NIR-I, several limitations remain. Although new NIR-II fluorophores are being developed, different from the nanoparticles used here, they still suffer from low quantum yields and limited availability, and additional time will be required for clinical approval of these agents [32,33]. In comparison, ICG is already FDA-approved. In addition, NIR-II imaging requires specialized InGaAs cameras, which are significantly more expensive, offer lower spatial resolution than silicon-based cameras used in visible or NIR-I imaging, and exhibit higher dark noise, necessitating active cooling [3,27,34]. These hurdles are being met, and reasonably priced instruments with wider spectral range; acceptable image quality, including reduced readout noise and increased dynamic range; and more compact design are becoming increasingly available beyond traditional NIR technologies. Furthermore, optimized optical components such as filters, lenses, and imaging systems for NIR-II wavelengths remain less mature, more expensive and more difficult to source than those for visible light, and can present challenges in system development. Although NIR-II fluorescence intensity was lower than that of NIR-I in our study, NIR-II imaging achieved superior CNR and spatial resolution due to reduced photon scattering and reduced autofluorescence at longer wavelengths. Further development of brighter NIR-II fluorophores is expected to further enhance image contrast and extend the advantages of NIR-II imaging for biomedical applications [32,35].

4. Materials and Methods

4.1. Materials

Indocyanine green (ICG, Sigma-Aldrich, St. Louis, MO, USA) was used as a United States Pharmacopeia (USP) reference standard. Deionized (DI) water with a resistivity of 18 MΩ·cm was obtained from a Milli-Q ultrapure water system (MilliporeSigma, Burlington, MA, USA). All chemicals and reagents were used as received without further purification. A stock solution of ICG (1 mg/mL, ~1.3 mM) was prepared in DI water. To minimize ICG J-aggregation, absorption and fluorescence measurements were performed using freshly prepared solutions on the same day as the experiments. 1,2-Dipalmitoyl-sn-Glycero-3-Phosphatidylcholine (DPPC), N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-Distearoyl-sn-Glycero-3-Phosphatidylethanolamine (mPEG-2000-DSPE), and diethylenetriaminepentaacetic acid-bis(stearylamide) gadolinium salt (DTPA-BSA-Gd) were obtained from Avanti Polar Lipids (Alabaster, AL, USA), and cholesterol (Chol) was obtained from Corden Pharma (Eichenweg 1, Liestal, Switzerland).

4.2. Synthesis of DMDG-ICG Liposomes

The synthesis of DMDG-ICG liposomes followed previously reported procedures [18,21]. Briefly, Liposomes were formulated at a molar ratio of DPPC:Chol:DSPE-mPEG-2000:Gd-DTPA-BSA = 30:40:5:25. A 125 µM ICG solution was prepared by diluting the 1.3 mM stock solution in gadobenate dimeglumine (505 mg/mL, Bracco Diagnostics Inc., Princeton, NJ, USA). The liposomes were extruded sequentially through 400 nm and 100 nm polycarbonate membranes using a mini-extruder (Avanti Polar Lipids, Alabaster, AL, USA, Cat # 610000) to produce unilamellar liposomes. The resulting liposomes were suspended in 10 mM histidine with 140 mM saline (pH ≈ 7.4) and purified via diafiltration to remove unencapsulated ICG and Gd complex.

4.3. Hydrodynamic Size Measurement

Liposome particle size, polydispersity, and zeta potential were determined by dynamic light scattering (DLS) using a Zetasizer ZS (Malvern Panalytical Inc., Westborough, MA, USA) and analyzed with the cumulant method. Measurements were conducted on three independent samples (n = 3).

4.4. Absorption and Fluorescence Spectra

Measurements were conducted using 0.5 µM DMDG-ICG dissolved in histidine buffer. Absorbance spectra were obtained from 500 to 1700 nm using a UV-VIS-NIR spectrophotometer (UV-3600, Shimadzu, Japan). Fluorescence spectra of DMDG-ICG were acquired using a spectrofluorometer (Nanolog, Horiba, Piscataway, NJ, USA) equipped with an InGaAs detector, following previously described procedures [15]. The fluorescence spectra were collected and averaged over five measurements, with an acquisition time of 1 s per measurement. An excitation wavelength of 680 nm was used to minimize nanoparticle scattering and account for Stokes shift in order to acquire a clear and complete fluorescence spectrum. The reported fluorescence spectra were corrected by subtracting the background spectrum obtained from dark counts in the liquid nitrogen-cooled InGaAs detector.

4.5. NIR Fluorescence Imaging Setup

Figure 1 shows the experimental schema for the NIR imaging system, including an InGaAs camera with filter sets for NIR-I and NIR-II imaging, a 785 nm laser, and a base for holding the phantoms or mouse. The 785 nm continuous-wave diode laser (Laserglow Technologies, LRD-0785, North York, ON, Canada) at approximately absorption maximum to maximize fluorescence was coupled to a 600 µm core multimode fiber, via featuring epi-illumination geometry and fiber-based configurations, with a collimating lens (F810SMA-780, Thorlabs, Newton, MA, USA) and engineered diffuser (ED1-C50-MD, Thorlabs) providing uniform illumination in the irradiation area (5–10 mW/cm²). For NIR fluorescence detection, an ultrabroad bandpass filter (800 nm–1000 nm, Semrock, Rochester, NY, USA) and a bandpass filter (1000 nm–1700 nm, Semrock, Rochester, NY, USA) were employed in front of an InGaAs camera (Ninox 640 II, Raptor Photonics, Larne, Northern Ireland; 640 × 512 pixels with pixel size of 15 × 15 μm, response 600–1700 nm, binning 1, 14 bits digital output,) with efficiency at NIR-I and NIR-II (see Supplementary Figure S1) equipped with a shortwave infrared C-mount lens (NMV-35M1-VIS-SWIR, Navitar, Rochester, NY, USA) set to F-number = 4. The InGaAs camera was operated at a working (thermoelectric cooling) temperature of −15 °C with the setting of high gain (on) and gain value (1). The images were acquired with µManager (a free, open-source software package) and analyzed with Excel (ver. 2408, Microsoft Office, Redmond, WA, USA) and MATLAB (R2024a, MathWorks, Natick, MA, USA). The overall image acquisition time for NIR-II (500 ms) was ten times longer than that of NIR-I (50 ms) to compensate for the lower emission intensity of the nanoparticle in the NIR-II window. Analyses included CNR (see Data Analysis below), which is a normalizing metric that accounts for both fluorescence intensity and background noise, enabling comparison between the two imaging windows despite differences in acquisition time.

4.6. Capillary Phantom in DI Water or Intralipid

In vitro imaging using either DI water or Intralipid phantom was performed following methods similar to those described previously [8,25], but with the added advantage of a single detector to minimize artifacts typically caused by dual-camera systems (silicon-based CCD camera for visible and NIR-I and InGaAs focal plane arrays for NIR-II). DI water was obtained from a Milli-Q ultrapure water system. A 1% Intralipid solution was prepared by diluting 20% Intralipid (Sigma-Aldrich, St. Louis, MO, USA) with DI water. Intralipid is capitalized here as it refers to the brand name of a commercially available fat emulsion. A cylindrical reservoir containing either DI water or 1% Intralipid solution was placed on a plate. Capped glass capillary tubes (OD = 1.5 mm/ID = 1.1 mm) filled with 0.5 µM DMDG-ICG in histidine buffer were immersed in the reservoir. The capillary tube was imaged at depths from 1 to 10 mm from the top surface. All the images were collected with the custom-built NIR fluorescence imaging system and normalized to their maximum signal intensity to enable quantitative comparison of image quality across different penetration depths.

$${Intensity}_{normalized}(x,y)={\displaystyle \frac{Intensity(x,y)}{{Intensity}_{\left(max\right)}}}$$

4.7. Capillary Phantom in Mouse Skin

Using the same method as in the capillary phantom imaging in water or 1% Intralipid, the capillaries containing DMDG-ICG nanoparticles were overlaid with either a single or double layer of nude mouse skin. Subsequently imaging was conducted in the NIR-I and NIR-II spectral regions.

4.8. Animal Experiments

Female athymic nude mice were purchased from Envigo (Indianapolis, IN, USA) and housed under specific pathogen-free conditions. All experiments were conducted in accordance with the animal care guidelines at the University of Maryland School of Medicine approved by the Institutional Animal Care and Use Committee (IACUC, approval number AUP-00000407). For in vivo imaging, 200 μL of DMDG-ICG was administered via intravenous injection. Imaging was performed under anesthesia using the custom-built in vivo NIR fluorescence imaging setup. NIR imaging was initiated within five minutes post-injection.

4.9. Data Analysis

Image processing and analysis were performed with Microsoft Excel and MATLAB. Quantitative enhancement was evaluated based on the average signal intensities (SI) in the manually selected regions of interest (ROIs). Signal-to-noise ratios (SNR) were calculated as mean signal intensity in ROIs divided by noise, where noise was defined as the standard deviation of the signal intensity in air. Contrast-to-noise ratio (CNR) for the tissue phantoms were calculated as a difference between SNR of a capillary tube and adjacent regions:

$${CNR}_{in tube}={SNR}_{capillary tube}-{SNR}_{adjacent region}$$

For the in vivo vascular imaging, the CNR was calculated similarly by estimating the SNR in the vessel and SNR in the adjacent region:

$${CNR}_{in vessel}={SNR}_{vessel}-{SNR}_{adjacent region}$$

For the CNR calculations, signal ROIs were precisely positioned along the tube or vessel, aligning with the central axis of the capillary in vitro with dimensions of 1 × 5 mm. Corresponding ROIs of identical dimensions were placed adjacent to the capillary tube or vessel, maintaining a 10 mm separation. To assess background noise, larger ROIs (four times the size of the signal ROI) were positioned in air regions free from artifacts. These ROI configurations were consistently applied for in vitro analyses of phantom imaging in water, Intralipid, or overlying skin, and for in vivo vessel imaging. SNR and CNR were calculated for three repeated images, and the mean values were reported for each group.

4.10. Statistical Analysis

Student’s one-tailed t-tests were conducted using Microsoft Excel for group comparisons. Differences were considered statistically significant at p < 0.05.

5. Conclusions

This study presents a custom-built NIR imaging system employing a single InGaAs camera, eliminating the need for a dual-camera setup, to capture both NIR-I and NIR-II windows. It also used a single-nanoparticle construct that fluoresces at NIR-I and NIR-II upon a single excitation. Comparative in vitro and in vivo imaging analyses demonstrated that NIR-II imaging outperforms NIR-I by achieving enhanced spatial resolution and improved CNR values at greater penetration depth. These findings underscore the potential of NIR-II imaging technology for bench and bedside deeper-tissue and high-resolution fluorescence imaging for multiple applications, such as lymphatic, vascular, and tumor imaging. Furthermore, the combined use of dual window fluorophores and a single dual-band camera would simplify protocols and enable high-signal superficial NIR-I imaging and high-CNR deeper-tissue NIR-II imaging without the need for multiple fluorophores or multiple camera systems.