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Abstract
The application of molecular and cellular imaging in ophthalmology has numerous benefits. It can enable the early detection and diagnosis of ocular diseases, facilitating timely intervention and improved patient outcomes. Molecular imaging techniques can help identify disease biomarkers, monitor disease progression, and evaluate treatment responses. Furthermore, these techniques allow researchers to gain insights into the pathogenesis of ocular diseases and develop novel therapeutic strategies. Molecular and cellular imaging can also allow basic research to elucidate the normal physiological processes occurring within the eye, such as cell signaling, tissue remodeling, and immune responses. By providing detailed visualization at the molecular and cellular level, these imaging techniques contribute to a comprehensive understanding of ocular biology. Current clinically available imaging often relies on confocal microscopy, multi-photon microscopy, PET (positron emission tomography) or SPECT (single-photon emission computed tomography) techniques, optical coherence tomography (OCT), and fluorescence imaging. Preclinical research focuses on the identification of novel molecular targets for various diseases. The aim is to discover specific biomarkers or molecular pathways associated with diseases, allowing for targeted imaging and precise disease characterization. In parallel, efforts are being made to develop sophisticated and multifunctional contrast agents that can selectively bind to these identified molecular targets. These contrast agents can enhance the imaging signal and improve the sensitivity and specificity of molecular imaging by carrying various imaging labels, including radionuclides for PET or SPECT, fluorescent dyes for optical imaging, or nanoparticles for multimodal imaging. Furthermore, advancements in technology and instrumentation are being pursued to enable multimodality molecular imaging. Integrating different imaging modalities, such as PET/MRI (magnetic resonance imaging) or PET/CT (computed tomography), allows for the complementary strengths of each modality to be combined, providing comprehensive molecular and anatomical information in a single examination. Recently, photoacoustic microscopy (PAM) has been explored as a novel imaging technology for visualization of different retinal diseases. PAM is a non-invasive, non-ionizing radiation, and hybrid imaging modality that combines the optical excitation of contrast agents with ultrasound detection. It offers a unique approach to imaging by providing both anatomical and functional information. Its ability to utilize molecularly targeted contrast agents holds great promise for molecular imaging applications in ophthalmology. In this review, we will summarize the application of multimodality molecular imaging for tracking chorioretinal angiogenesis along with the migration of stem cells after subretinal transplantation in vivo.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Retinal diseases, including age-related macular degeneration and diabetic retinopathy, pose significant challenges to vision health worldwide [1–9]. Uncontrolled proliferation of blood vessels in the eye, including choroidal neovascularization (CNV) and retinal neovascularization (RNV), are major causes of vision loss and blindness [10,11]. CNV can occur in age-related macular degeneration (AMD), which affected 196 million people in 2020 [12,13]. RNV can occur with ischemic retinopathies that cause severe damage to retinal vessels, including diabetic retinopathy and retinal vein occlusions, which can ultimately cause vision loss and eventual blindness [10,14]. The standard therapy for alleviating neovascularization targets vascular endothelial growth factors (VEGF) using anti-VEGF drugs such as ranibizumab, aflibercept, and bevacizumab [15,16]. Other types of ocular neovascularization, including corneal neovascularization, are also a common cause of blindness in the world [11].
In recent years, molecular and cellular imaging techniques have emerged as powerful tools for diagnosing and monitoring animal models of these complex ocular conditions [17–23]. Furthermore, with the advent of stem cell transplantation as a potential therapy for retinal diseases, there is an increasing need for non-invasive imaging methods to assess the efficacy and safety of these regenerative approaches. Molecular and cellular imaging techniques enable researchers and clinicians to visualize and track specific molecules, cells, and biological processes in living organisms, providing valuable insights into disease progression and therapeutic interventions [17,24,25]. By leveraging the principles of molecular biology, optics, and imaging technologies, these techniques have revolutionized our understanding of retinal diseases and the transplantation of stem cells to restore visual function. One of the primary goals of molecular and cellular imaging in the context of retinal diseases is to non-invasively visualize and quantify specific molecular targets associated with disease pathology. This includes the identification of biomarkers, such as inflammatory molecules, angiogenic factors, and cellular components, that play crucial roles in disease development and progression. Currently, several imaging modalities, such as scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), OCT angiography (OCTA), fluorescein angiography (FA), indocyanine green angiography (ICGA), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) have been used in the clinic to screen and diagnose retinal diseases. These techniques have shown remarkable capabilities to aid in early diagnosis, treatment planning, and monitoring therapeutic response.
Moreover, the emerging field of stem cell transplantation for retinal diseases holds great promise for restoring visual function by replacing damaged or degenerated retinal cells [26–29]. However, monitoring the survival, integration, and functional outcomes of transplanted cells in vivo presents unique challenges. Cellular imaging techniques, such as magnetic resonance imaging (MRI) [30–32], bioluminescence imaging [33–36], and fluorescence imaging [37], allow for the non-invasive tracking and visualization of transplanted stem cells, enabling researchers to assess their viability, migration, integration into host tissue, and potential therapeutic effects.
This review delves into recent advancements in molecular and cellular imaging techniques used in the study of retinal diseases and stem cell transplantation. We will explore the principles, methodologies, and applications of various imaging modalities employed in preclinical and clinical studies. We will present the application of numerous contrast agents for specific targeting of retinal diseases such as CNV and RNV as well as the tracking and labeling of stem cells. Additionally, we will discuss the potential challenges, limitations, and future directions in the field of molecular and cellular imaging, aiming to shed light on the exciting possibilities of these technologies for improving the diagnosis and treatment of retinal diseases and enhancing the success of stem cell-based therapies.
2. Molecular imaging of the eye
2.1 Principle, applications, and limitations of molecular imaging of the eye
Multimodality ocular molecular imaging, a set of advanced imaging techniques, holds great promise in impacting medicine due to its ability to visualize specific molecules or cellular processes within ocular tissues. A number of imaging modalities are primarily utilized for medical imaging, including PET [38–41], SPECT [42,43], MRI [44–47], magnetic resonance spectroscopy (MRS) [48–51], ultrasound (US) [19,52–55], and computed tomography (CT) [56,57]. Clinical applications of these techniques include oncology [58], cardiology [59,60], and neurology [61]. One challenge of these imaging systems is that the resolution is not high enough to visualize different structures in the back of the eye. In addition, ionizing radiation is another barrier of some of these imaging systems for application in ophthalmology.
Current real-time in vivo optical imaging techniques, including OCT [62–65] and OCTA [66,67] as well as SLO, FA, and ICGA are widely used in ophthalmology [68,69]. While SLO is useful in diagnosing many retinal disorders, its resolution is limited by the anatomical shape of the eye and its ability to diffract light [70–72]. FA and ICGA are two commonly used imaging techniques in ophthalmology for evaluating vascular changes and pathological processes in the eye [73–75]. FA provides excellent visualization of the retinal and choroidal vasculature, allowing for the identification of abnormalities such as neovascularization, microaneurysms, and vascular leakage [76–78]. FA and ICGA also provide dynamic real-time imaging, enabling the assessment of blood flow patterns and the evaluation of disease progression. While both techniques have their advantages, a major limitation is the limited depth penetration, making it less effective for imaging deeper structures such as the choroid or optic nerve head. In addition, FA and ICGA rely on the nonspecific binding of fluorescein dye to albumin, limiting its ability to specifically target and image molecular markers or specific cell types [79]. Due to invasive intravenous administration of fluorescent compounds, adverse reactions may potentially occur, such as allergic reactions or nausea and vomiting [74,80].
Unlike FA and ICGA, OCT and OCTA are noninvasive and allow the visualization of high-resolution cross sections of the retina. They are helpful in diagnosing structural changes to the eye without the need to remove and process specimens, as in conventional excisional biopsy [81,82] or histopathology [83]. OCT and OCTA have been used to diagnose different retinal diseases such as AMD [84,85], retinitis pigmentosa [86–88], diabetic retinopathy [89–91], myopia [92–94], glaucoma [95–98], and corneal neovascularization [99,100]. The benefits of OCT are that it can provide high resolution, is noninvasive, and has rapid acquisition time within seconds, allowing for detailed visualization of retinal layers, the optic nerve head, and other ocular structures. It enables the identification and measurement of subtle changes associated with various retinal diseases by offering real-time imaging capabilities, allowing for the assessment of dynamic changes in retinal morphology and fluid dynamics. This is particularly useful in monitoring disease progression and response to treatment [101]. OCTA is also non-invasive and can create 3D images of the retina as well as the neovascularization. However, OCTA is unable to detect vessel leakage and choroidal microvasculature.
Recently, photoacoustic (PA) imaging has risen in popularity as a method of 3D imaging that is non-ionizing and non-invasive with micron-scale spatial resolution and millimeter-scale depth penetration (Fig. 1) [102,103]. In photoacoustic imaging, a short-pulsed laser is used to illuminate the tissue of interest. When this laser light is absorbed by the tissue, it generates a rapid increase in temperature, causing local thermal expansion and resulting in the emission of ultrasound waves. These ultrasound waves are then detected by an ultrasound transducer, which converts them into electrical signals that can be processed to create an image [104,105]. PA molecular imaging offers deep tissue penetration, high spatial resolution, molecular specificity, functional imaging capabilities, and real-time imaging. These advantages make it a powerful imaging modality with significant potential for advancing our understanding of disease processes and improving diagnostics and therapeutics.
Fig. 1. Photoacoustic (PA) imaging systems and applications in the medical field: (a) Linear-array photoacoustic tomography (PAT) of methylene blue concentration in a rat sentinel lymph node (SLN), (b) circular-array PAT of cerebral hemodynamic changes in a rat, and (c) photoacoustic endoscopy (PAE) of a rabbit esophagus. UST: ultrasound transducer. (d) optical-resolution photoacoustic microscopy (OR-PAM) used for quantification of oxygen saturation in a mouse ear. (e) acoustic-resolution photoacoustic microscopy (AR-PAM) of normalized total hemoglobin concentration in a human palm [106]. Adapted with permission from Ref. [12].
Due to the modularity of PA imaging, it can be merged with other imaging modalities as well as various contrast agents and specific cell targeting techniques to improve the diagnosis of retinal disease [108,109]. PA imaging can be used in conjunction with OCT imaging to visualize retinal vessels, choroidal vessels, and retinal pigment epithelial (RPE) cells as shown in Fig. 2. The resolution of PA imaging and its ability to recognize abnormal vessels can further be improved upon using various contrast agents. The use of optically excitable contrast agents is a key feature of photoacoustic imaging. These contrast agents can be designed to selectively absorb the laser light at specific wavelengths, converting the absorbed energy into heat and facilitating the generation of acoustic waves.
2.2 Molecular Imaging with organic contrast agents
Molecularly targeted contrast agents can be engineered to bind to specific molecular targets, such as biomarkers or disease-related molecules, promoting the specificity and sensitivity that can enhance the clarity of ocular structures and pathologies in various imaging techniques. Contrast agents are essential for successful molecular imaging of the eye as they improve the contrast between different structures of the eye which aids in identifying ocular abnormalities and diseases. Contrast agents fall into two main groups: organic contrast agents (i.e, ICG, IR780, dendrimer, Methylene Blue) [110], and carbon nanoparticles [111] and inorganic contrast agents such as semiconductors [112] and gold nanoparticles (e.g., sphere, stars, tripods, plates, rods, and prisms) [80,113–118]. Organic contrast agents are composed of organic carbon compounds while inorganic contrast agents are composed of inorganic compounds. The type of contrast agent used is dependent on the type of imaging modality used and the specific visualization needed for the ocular structures.
Indocyanine green (ICG) dye is a low-risk and nontoxic organic contrast agent that has been used for molecular imaging [119]. ICG has many benefits for molecular imaging. One of its advantages is that it absorbs and emits in the near-infrared range and exhibits low background interference which allows for clear visualization of structures within the eye [120]. Furthermore, ICG is highly protein bound, which prevents rapid evacuation from capillaries as is seen with other substances like fluorescein dye. It is bound by albumin in the intravascular space until cleared through hepatic metabolism [121,122]. The advantages and feasibility of ICG dye make it useful for imaging of ocular conditions like choroidal neovascularization (CNV) through ICG angiography (ICGA). ICGA is valuable in the diagnosis of choroidal conditions and has helped with understanding the different patterns of hyperfluorescence in eyes with both neovascular and dry age-related macular degeneration (AMD) [123]. ICGA can currently be performed using scanning laser ophthalmoscopy which produces high-definition images. It is divided into three phases: early, middle, and late. The early phase clearly visualizes the choroidal vessels that are beneath the retinal vasculature. In the middle phase, abnormal lesions are best detected as the choroidal veins become less distinct. In the late phase, retinal and choroidal details are lost and can demonstrate late leakage [124]. Overall, ICG dye offers several advantages for imaging in ocular conditions, particularly choroidal neovascularization (CNV). ICGA provides clear visualization of ocular structures in high-definition images, divided into early, middle, and late phases. Its near-infrared properties make it a valuable tool for precise diagnosis and understanding of ocular diseases.
Another advantage of ICG is that it has been approved by the United States Food and Drug Administration (FDA) for clinical use, and it has been explored as a great contrast agent to improve PA imaging. By conjugation with arginylglycylaspartic acid (RGD) peptides, ICG can target CNV created by subretinal injection of Matrigel and VEGF in a rabbit model [125]. This method allows tracking of CNV progression for up to 14 days post injection (Fig. 3). In addition, targeted ICG helped to improve PAM signal by 15.7-fold. By using the excitation wavelength in near-infrared window at 700 nm, CNV was clearly differentiated from the surrounding blood vessels without using post image processing like segmentation or machine learning. This helps to precise diagnosis of CNV and planning for the therapeutic treatment. However, its limited photostability and functionalization make it a suboptimal contrast agent.
Fig. 3. Longitudinal visualization of choroidal neovascularization in rabbit choroidal neovascularization (CNV) model: (a) Merged 3D visualization photoacoustic microscopy (PAM) image of CNV acquired at two different excitation wavelengths of 578 (pseudo-red) and 700 (pseudo-green) nm (post 24 h). (b) Horizontal (x–y) PAM image (post 24 h). (c) Vertical (y–z) PAM image (post 24 h). Green color shows the position of ICG-RGD bound at CNV. (d-f) Longitudinal PAM images visualizing CNV obtained at the excitation wavelengths of 578 nm, (d), and 700 nm, (e), pre-injection of ICG-RGD, and after-injection at 2 h, 4 h, 24 h, 48 h, 72 h, day 5, 7, 9, 11 and 14 post-injection of ICG-RGD (0.4 mL, 2.5 mg/mL). White arrows show the location of CNV. CNV was not readily detectable in the PAM image before the injection of ICG-RGD. (f) Overlay 3D PAM images. (g) Graph of the measured PAM signal in the CNV. PA signal reached a peak PA amplitude at 24 h post-injection and gradually decreases over time. (h-j) in vivo overlay 3D PAM images of CNV acquired at 578 and 700 nm pre- and post-administration of 0.4 mL ICG without conjugation with RGD at concentration of 2.5 mg/mL at 15 min, and 1 h. Pseudo-green color shows the location of CNV. Adapted with permission from Ref. [125].
2.3 Molecular Imaging with inorganic contrast agents
There are many inorganic contrast agents that are significantly utilized in the field of molecular imaging. One of these agents that has gained a lot of attention is gold nanoparticles. Gold nanoparticles are promising contrast agents due to their unique properties of high light scattering on their surface plasmon resonance, absorption, and strong electromagnetic fields on the particle surface [126]. Gold nanoparticles show low cytotoxicity, high functionalization ability, and dual absorbance for visualization of anatomy and vasculature [113–115,116,127]. These properties of gold nanoparticles allow for high transmissivity through biological tissues and therefore enhance detection using various imaging modalities, especially OCT and PA imaging [128]. Figure 4 shows the principal application of gold nanoparticles as the multifunctional contrast agents for PAM and OCT. The surface of GNPs can be modified and conjugated with a diverse range of targeting moieties, including targeting peptides, antibodies, nucleic acid sequences, drugs, and fluorescence dyes. This strategic modification enabled the GNPs to specifically bind to integrin αvβ3, which is overexpressed on newly developed angiogenic retinal blood vessels. Furthermore, the functionalized GNPs exhibited the ability to penetrate retinal cell nuclei, facilitating efficient drug release at the desired target sites.
Fig. 4. Schematic illustration of gold nanoparticles (GNPs) as multimodality photoacoustic microscopy (PAM) and optical coherence tomography (OCT) image contrast agents for molecular imaging of the eye. Non-targeting (NT-GNPs) and targeting GNPs (T-GNPs) can be administered via intravitreal injection (IVT) or intravenous injection (IV) routes. GNPs’ strong plasmonic properties enable them to generate robust back-scattered light or acoustic signals when irradiated with an appropriate laser wavelength. These signals are captured by an OCT photodiode to form OCT images or by ultrasound detection to reconstruct photoacoustic (PA) images. Utilizing multiple optic wavelengths within the near-infrared (NIR) window facilitates the detection of GNPs’ extravasation at targeted vessels, enabling differentiation of neovascularization.
Changing the scattering light wavelength and absorption properties of the nanoparticles allows for changes in its morphology and its optical properties, resulting in different types of gold nanoparticles like nanorods and nanostars. One such way is by reducing the number of metal ions on the surfaces of gold sheets to allow for the creation of gold nanorods. Similarly, optical properties of gold nanostars (GNS) can easily be adjusted by changing the seeding solution, resulting in formation of GNS with different size, branching, and absorption and scattering spectrum as shown in Fig. 5–6. Nguyen et. al demonstrated a study in which PAM signal amplitudes were enhanced by up to 27.2-fold and OCT signal intensities were enhanced by 171.4% due to gold nanorods conjugated with RGD, allowing for clear visualization and differentiation of CNV pathogenesis as shown in Fig. 7. The functionalized gold nanorods proved to be an excellent contrast agent for multimodal PAM and OCT imaging systems [113]. Gold nanostars have a strong optical response at near-infrared (NIR) wavelengths and excellent biocompatibility as well [129]. Nguyen et. al developed functionalized gold nanostars with an RGD peptide to use as contrast agents for PAM and OCT imaging modalities to visualize CNV. It was found that gold nanostars increased PA contrast up to 17-fold and OCT intensities by 167%, respectively [115].
Fig. 5. Morphology and size different GNPs as contrast agents for biomedicine: (a) Small nanospheres, (b) large nanospheres, (c) nanorods, (d) sharpened nanorods, (e) nanoshells, (f) nanocages/frames, (g) hollow nanospheres, (h) tetrahedra/octahedra/cubes/icosahedra, (i) rhombic dodecahedra, (j) octahedra, (k) concave nanocubes, (l) tetrahexahedra, (m) rhombic dodecahedra, (n) obtuse triangular bipyramids, (o) trisoctahedra, and (p) nanoprisms. Reproduced from Ref. [130].
Fig. 6. Morphology and optical properties of various GNPs. (a) Gold nanoshells (GNS). (b) Gold nanocages (GNC). (c) Gold nanorods (GNR). (d) Gold nanostars (GNST) (e) Gold nanochain-like clusters (CGNP). (f–j) Corresponding UV-Vis absorption spectra of GNS (f), GNC (g), GNR (h), GNST (i), and CGNP (j) of different aspect ratios, respectively. Adapted with permission from ref. [115,131–133].
Fig. 7. In vitro PAM of targeted GNR in a rabbit model of laser-induced choroidal neovascularization (CNV): (a) color fundus photograph of the rabbit retina. The color fundus shows the retinal vessels (RVs) and optic nerve. (b–d) Fluorescein angiography (FA) images acquired at different time points after IV injection of fluorescein sodium: (b) early phase, (c) middle phase, and (d) late phase. The FA shows the morphology of retinal vessels and capillaries and the location of CNV (white arrows). The red rectangle shows the selected scanning area. (e–k) Corresponding PAM images along the selected area outlined in panel (d) obtained at two different excitation wavelengths of 578 and 700 nm. (e) 3D volumetric PAM visualization image (post 1 h). (f–h) Horizontal (x–y) maximum intensity projection (MIP) PAM images (post 1 h). (i–k) Vertical (y–z) PAM images (post 1 h). The pseudo-yellow color represents the accumulation of GNRs in CNV. Note that GNRs were laid under retinal vessels and above the choroidal vessels (k). (l) Sequential longitudinal in vivo PAM images (overlaid 3D images acquired at 578 and 700 nm) acquired on the same rabbit at various time points post-administration of targeted GNR-RGD (0.4 mL, 2.5 mg/mL) and followed sequentially for 28 days. Adapted with permission from Ref. [113].
Another novel technique using gold nanoparticles to enhance imaging modalities is the fabrication of chain-like gold nanoparticles (CGNPs). Gold nanoparticles with an average diameter of 20 nm are fabricated by femtosecond pulsed laser ablation of gold target in deionized (DI) water, then assembled into CGNP clusters by surface modification with the peptide Cys–Ala–Leu–Asn–Asn–OH (CALNN) and cysteamine ligands. The CGNP clusters are then conjugated with polyethylene glycol (PEG) and RGD ligands [114]. In a longitudinal study, Nguyen et. al reported that CGNPs targeted at CNV stayed up to 28 days as shown in Fig. 8. Quantification data demonstrated that CGNPs enhanced photoacoustic signals by 25.3 fold and OCT signals by 150% in visualizing CNV [127]. This proves the effectiveness of CGNPs as contrast agents in multimodal molecular imaging. In conclusion, gold nanoparticles have emerged as promising inorganic contrast agents in the field of molecular imaging. Their unique properties, such as high light scattering, absorption, and strong electromagnetic fields, enable enhanced detection using various imaging modalities. The ability to modify their morphology and optical properties has led to the development of specialized gold nanoparticles, such as nanorods and nanostars, which exhibit improved imaging capabilities. Additionally, the fabrication of chain-like gold nanoparticles (CGNPs) has shown promising results as contrast agents in multimodal molecular imaging. These advancements in gold nanoparticle-based contrast agents offer great potential for precise visualization and characterization of ocular conditions, such as choroidal neovascularization (CNV). Further research and development in this area will advance improvement of diagnostics and understanding of ocular disease
Fig. 8. In vivo PAM visualization of CGNP clusters accumulated at CNV. (a-b) PAM images of CNV before and after the injection of 0.5 mL CGNP clusters-RGD at a concentration of 2.5 mg/mL acquired along the selected area outlined in fundus image (d) under nanosecond pulsed laser illumination at wavelength of 578 and 650 nm, respectively. (c) Overlay 3D images showed the distribution of CGNP clusters-RGD accumulated at CNV location in rabbit retina (pseudo-green color). (e) Rabbit injected with CGNP clusters-RGD exhibited significantly higher PA signal than pre-injection. Note that the peak PA signal occurred at 24 h post injection. Then, the PA signals gradually decreased over time. (f) In vivo photostability of CGNP clusters-RGD. The error bars in e and f represent standard error of the average PA signal measured from three different animals (N = 3), p < 0.05. Adapted with permission from Ref. [114].
3. Cellular Imaging of the eye
Age-related macular degeneration (AMD) is the leading causes of blindness in Americans aged 64 and older [1]. It is estimated 288 million people will be affected by AMD by 2040 [131]. While treatments like anti-vascular endothelial growth factor (anti-VEGF) exist, they are ineffective against dry AMD. Stem cell therapy (SCT) has the potential to improve vision lost and blinding from several retinal degenerative diseases and has progressed significantly in the past century (Fig. 9). Treatment using human-induced pluripotent stem cells (hiPSC) differentiated into retinal pigment epithelial (RPE) cells is a promising idea that has been heavily researched, because the transplanted stem cell can replace cells lost in disease. This has the potential to restore vision by replacing dead or degenerated RPE cells in AMD and retinitis pigmentosa (RP) and dying retinal ganglion cells in glaucoma. Many fish and amphibians exhibit exceptional ability to regenerate the RPE after damage [132,133], while in elderly humans and some other mammals, regeneration of the RPE is limited and localized to smaller-sized lesions [134]. Proliferative response of the RPE is rare and typically leads to the formation of a myofibroblast phenotype resulting from epithelial-mesenchymal transition (EMT) as opposed to a functional RPE monolayer [135].
Fig. 9. Timeline of major discoveries and advances in basic research and clinical applications of stem cell-based therapy. Adapted with permission from Ref. [136].
In 1998, human embryonic stem cells (hESC) and their derivatives were first applied as a new therapeutic approach for several diseases and became a new era of modern medicine [137]. Later, hESC-derived RPE cells were investigated for clinical application in retinal degenerative diseases including AMD, and the first trial was tested in human in 2011 and completed in 2017 at multiple world-leading eye centers in the United States, United Kingdom, and South Korea (Clinicaltrial.gov identifiers: UK-SMD: NCT01469832; US-SMD: NCT01345006; US-AMD: NCT01344993). There is no evidence indicating that the transplanted RPE cells cause severe acute or chronic immune inflammation. The immunological rejection can be well-managed by injection of immunosuppressants and steroids before and after transplantation. The survival and migration of the transplanted cells have been monitored by OCT imaging. There are several routes of delivery of stem cells to the retina, including intravitreal injection, subretinal delivery, and suprachoroidal transplantation across the sclera and choroid [138]. However, in order to quantify treatment outcome, long-term tracking of the migration of stem cells in the eye has posed a difficult challenge.
Multiple past studies demonstrate the ability of human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESC), and human umbilical cord mesenchymal stem cells (HUCMSCs) to be guided to differentiating into viable RPE cells, providing important biotechnological opportunities for vision-restorative stem cell therapies [139–146]. Previously, Petrus-Reurer et al. have tested the transplantation of RPE cells derived from hESC in a geographic atrophy (GA) rabbit model to determine the extent of stem cell transplant integration at injury sites [24]. In untreated naive eyes, follow-up imaging showed the hESC-RPE transplanted cells were successfully integrated between the photoreceptors and Bruch’s membrane. Yet, the expression of RPE65, a gene responsible for encoding all-trans retinyl ester isomerase needed for the regeneration of pigment in the visual cycle, only became detectable over time, and the hESC-RPE cells were absent from areas of natural RPE degeneration. After subretinal injection of PBS and NaIO3 to induce a GA-like phenotype, the study findings revealed a lack of formation of a robust RPE monolayer at the damaged sites as well as evidence of immune rejection, showing a lack of successful hESC-RPE integration after transplantation, likely due to their chemically-induced RPE damage protocol. In a more recent 2022 study, Petrus-Reurer et al. identified progenitor cells positive for the cell-surface marker NCAM1 as holding the greatest potential for differentiating into functional RPE cells [147]. The researchers subsequently investigated the integration efficacy in vivo of such hESC-RPE cells that were cultured for 60 days and transplanted subretinally into two albino rabbits (Fig. 10). Follow-up imaging at four weeks with infrared and spectral domain optical coherence tomography (SD-OCT) revealed a hyper-reflective layer of RPE cells, and further analysis with histology, immunofluorescence, and sc-RNA sequencing confirmed successful integration of the hESC-RPE cells, with no alternative progenitor cell lineages observed after transplantation.
Fig. 10. Transplantation of hESC-RPE in naive and PBS- or NaIO3-pretreated eyes. Subretinal transplantation of hESC-RPE in suspension (dotted circle) into non-pretreated naive albino rabbits shows patchy areas of pigmentation in eyes with injection-induced native RPE loss (a1). Large RPE denuded hypo-BAF areas are present 3 months after transplantation. On the corresponding multicolor cSLO image, pigmented areas are seen between bright atrophic areas. On SD-OCT, the neuroretina overlying the area with integrated hESC-RPE is well-preserved, in contrast to the adjacent area denuded of native RPE that shows outer retinal layer loss extending to the inner plexiform layer. The transition between the native RPE-denuded and hESC-RPE integrated area is marked (arrowhead), and the corresponding box magnified below. The SD-OCT scan plane is marked (green arrow). Hematoxylin and eosin (a2) and immunostaining (a3) for RPE65 demonstrates loss of native RPE adjacent to the integrated and weakly RPE65-positive hESC-RPE. Also note the striking difference in outer neuroretinal preservation between these areas. Suspension transplantation of hESC-RPE 1 week after pretreatment of the same area with PBS (dotted circle) showed presence of pigmented subretinal dots (open arrowhead) on multimodal imaging after 3 months (b1). Hematoxylin and eosin (b2) and immunostaining for RPE65 [b3], corresponding bright field image [b4]) demonstrated occasional pigmented RPE-like cells exhibiting an abnormal morphology in the subretinal space. When eyes were pretreated with 1 mM NaIO3 for 1 week (c1) followed by hESC-RPE transplantation, no pigmented areas were detected after 3 months (c2). In addition, the neuroretina overlying the transplanted area showed extensive atrophy. The margins of the bleb (closed arrowhead) and the SD-OCT scan planes are marked (green arrow). A hypo-BAF area is also outlined (dashed line in [c1]) where intravitreal triamcinolone particles (asterisks) partially block the BAF and IR signals. IR, infrared scanning laser ophthalmoscopy; MC, multicolor scanning laser ophthalmoscopy. Scale bars: (a1, b1, c1, c2) 200 µm; (a2, a3) 50 µm; (b2, b3, b4) 20 µm. Adapted with permission from Ref. [114].
Many such previous studies have made advancements in facilitating successful stem cell integration in the retina but are technologically limited in detailed tracking of the fate of these stem cells over time to determine long-term effects. In addition to OCT, transplanted stem cells in the retinal can be tracked with fluorescent imaging. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas-9 gene editing technology allows for the knock-in of the enhanced green fluorescent protein (eGFP) tag sequence in hiPSCs, resulting in green fluorescence of eGFP-tagged proteins [148,149]. Using light to excite eGFP at 488 nm, stem cells which have been labeled with the protein can easily be visualized in vivo for real-time location tracking in the retina. While fluorescent imaging using gene edited biomarkers provides real-time tracking, it is limited in depth of penetration, imaging resolution, and potential photobleaching after prolonged and successive eGFP excitation [18,150,151]. Additionally, inducing fluorescence of stem cells is limited by the nature of gene editing, where in some cell lines, the target gene transcript for knock-in of eGFP may be sparse in number and may result in some stem cells not producing enough eGFP to be sufficiently visualized with fluorescent imaging. It is also possible for eGFP to localize to small regions of the cells at cell junctions, leading to imprecise stem cell visualization [148]. Although there have been recent improvements to technology and technique for fluorescent microscopy, many require highly advanced equipment and training.
Nguyen et al. have developed a non-invasive and rapid dual-modality imaging system consisting of spectral-domain OCT (SD-OCT) and photoacoustic microscopy (PAM) [79,152,153]. Using low-coherence interferometry, OCT is able to detect backscattered photons, while PAM provides high-resolution and high-contrast imaging coupled with a far depth of penetration, allowing for effective real-time, longitudinal tracking of stem cells. Exogenous contrast agents are often used with PAM to significantly boost photoacoustic signals and produce higher resolution images not afforded by endogenous chromophores [116]. Indocyanine green (ICG) and gold nanoparticles (GNPs) are two highly effective contrast agents that enhance imaging with PAM and OCT.
ICG is a Food and Drug Administration (FDA)-approved biodegradable organic dye that has high optical absorption and contrast sensitivity. ICG has been applied in different biomedical applications such as enhanced visualization of lymph nodes [154], tumors [155–158], or tracking human stem cells for spinal cord injury in mice [159]. Previously, Nguyen et al. have utilized ICG to label adult retinal pigment epithelial cell line 19 (ARPE-19) cells, which exhibit the ability to form structurally and functionally viable RPE cells in vivo [160,161]. The study found that treatment of ARPE-19 with ICG did not significantly reduce cell viability for most ICG concentrations and incubation durations, demonstrating the biocompatibility of ICG for ARPE-19 stem cell tracking. Follow-up imaging for 28 days of the ICG-labeled ARPE-19 stem cells after subretinal injection into the rabbit model showed a 19-fold increase of fluorescence intensity as compared to images obtained pre-injection. Furthermore, PAM signal intensity was increased 20-fold by day 5 with the introduction of ICG-labeled ARPE-19 stem cells as compared to pre-injection. The highest image contrast with fluorescent imaging was present during days 0, 1, 3, and 5 post-treatments, with contrast gradually decreasing over time and disappearing completely by day 28. PAM imaging revealed the highest image contrast from ICG at day 5 and minimal signal by day 28. This signal attenuation is due to the relatively high rate of biodegradation of the dye with an estimated half-life of 72 hours after transplantation, meaning that decline of fluorescent signal intensity restricts the length of time available for follow-up and is a substantial limitation of ICG for long-term stem cell tracking [160,162]. ICG ultimately serves as a highly effective and biocompatible, nontoxic contrast agent for tracking the migration and integration of stem/progenitor cells such as ARPE-19 in the subretinal space that can help improve understanding of in vivo ocular stem cell therapies.
Gold nanoparticles are another contrast agent that can provide significant increases in imaging depth, resolution, and contrast. GNPs offer flexibility in engineering a wide range of different material properties and have markedly slower rates of biodegradation, enabling GNPs to remain present over a longer period of time [116,163]. Past research has shown that GNPs can be biocompatible and effective contrast agents for stem cell imaging. Donnelly et al. and Dhada et al. successfully labeled mesenchymal stem cells (MSCs) with gold nanospheres and gold nanorods, respectively, and successfully tracked MSC migration using an ultrasound- and photoacoustic-paired imaging system, finding no evidence of toxicity [153,164]. Another study successfully labeled human Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) with gold nanoparticles and tracked stem cell movement with micro-computed tomography following transplantation and subsequent localization in the subretinal layer of an Royal College of Surgeons (RCS) retinal degeneration rat model for over 30 days [165].
More recently, Nguyen et al. have carried out longitudinal tracking of ARPE-19 stem cells labeled with chain-like gold nanoparticles (CGNPs) using a custom non-invasive PAM and OCT multimodal imaging system [17]. Additional tests were performed in vitro and in vivo to determine the potential for photobleaching, level of biocompatibility, and level of cytotoxicity of CGNPs in ARPE-19 stem cells and found no significant photobleaching or impact on stem cell viability. Optimal cell density threshold testing revealed a 7-fold increase in PAM and OCT contrast when imaging CGNP-labeled ARPE-19 stem cells, even at the lowest cell concentration. These results are from labeling a mere 10,000 stem cells, almost 1000 times lower than the clinically injected dose of stem cells, demonstrating that their contrast agent is sensitive enough to track stem cell migration in clinical settings [17,164]. Following real-time OCT image-guided subretinal injection of the CGNP-labeled ARPE-19 stem cells into two living rabbit models. PAM images obtained post-injection displayed high-contrast and showed the ARPE-19 cell distribution to a depth of ∼500 µm. PAM images obtained at a wavelength of 578 nm showed both the CGNP-labeled ARPE-19 cells and the adjacent retinal and choroidal blood vessels, while images obtained at 650 nm displayed only the CGNP-labeled ARPE-19 stem cells, demonstrating the strength of imaging selectivity offered by photoacoustic imaging with gold nanoparticles (Fig. 11(a)–(g)). As a result, 90-day longitudinal tracking of the stem cells post-injection with PAM enabled comparison between the two rabbit models. In the rabbit model with laser-induced photocoagulation lesions, the CGNP-labeled ARPE-19 cells were observed to migrate towards the induced laser lesions two days post-injection, while in the model without induced photocoagulation lesions, the labeled ARPE-19 cells were found at the site of injection and distributed randomly in the subretinal space over time (Fig. 11(a)–(d)). Overall, this and other previous studies demonstrate the high biocompatibility of GNPs with stem cells, specifically ARPE-19 and MSCs, to significantly enhance signal detection, contrast, and imaging depth for PAM and other imaging modalities important for advancing stem cell tracking in the development of new clinical treatment methods.
Fig. 11. In vivo PAM images of labeled ARPE-19 cells post-transplantation into rabbit retina: (a–d) In vivo PAM images of CGNP clusters-labeled ARPE-19 cells post-transplantation into rabbit eyes with laser-induced RPE injury. (a) Baseline fundus photograph and PAM images acquired at 578 nm and 650 nm before the transplantation of the ARPE-19 cells. White arrows indicate the locations of retinal vessels (RVs) and photocoagulation lesions. (b–d) 2D and 3D volumetric PAM images of the ARPE-19 cells acquired at different time points post-transplantation, illustrating cell viability and migration toward photocoagulation lesions. The dotted line in (b) indicates the distribution of ARPE-19 cells post-transplantation, while the dotted circles in (a–c) show the lesion areas. The pseudo-green color in PAM images acquired at 650 nm shows the distribution of the transplanted ARPE-19 cells. See Supplementary Visualization 3 for 3D volumetric presentation. (e–h) In vivo PAM images of CGNP clusters-labeled ARPE-19 cells post-transplantation into rabbit eyes without laser-induced RPE injury. (e) Fundus photograph and PAM images acquired at 578 nm and 650 nm before the transplantation of ARPE-19 cells. (f–h) 2D and 3D volumetric PAM images acquired at different time points post-transplantation, illustrating cell viability and migration in a random fashion. The dotted line in (f) indicates the distribution of cells immediately post-injection, while the dotted line in (g) illustrates cell migration over time. Adapted with permission from Ref. [17].
4. Pre-clinical
PA imaging serves a crucial role as a non-invasive platform for anatomical, functional, and molecular imaging of retinal diseases in various animals, ranging from small (mice and rats) to larger species (rabbits), displaying promising potential for clinical applications. This technique could prove valuable in researching human retinal diseases like retinal neovascularization, choroidal neovascularization, and retinal vein occlusion, offering high resolution and deep tissue penetration.
The diverse applications of all three PA imaging modes (photoacoustic tomography (PAT), AR-PAM, and OR-PAM) in biomedical imaging have been extensively explored, covering multiscale structural imaging to functional imaging with both endogenous biomolecules and exogenous contrast agents. Notable examples include PAT being used for imaging the retinal vasculature in rabbits [166], AR-PAM studying variations in retinal blood oxygenation [18], and OR-PAM capturing images of retinal, corneal, and choroidal neovascularization, as well as melanin in mice [167–170]. Additionally, OR-PAM has been employed to map blood oxygen concentrations and blood flow at the capillary level of the eye [104,156]. Dynamic monitoring of normal and abnormal retinal blood vessels in larger animals, such as rabbits, and the acquisition of maps detailing damaged choroidal vessels are among the varied applications [156].
A comprehensive summary of PAM applications for evaluating the eye is provided in Table 1, emphasizing its potential as a tool for both fundamental research and clinical practice.
Table 1. Prospects of Ocular PAM
However, before integrating PAM imaging into clinical settings for human use, it is crucial to ensure that research validates the capability of capturing high-contrast and high-resolution ocular images without adversely affecting the sensitive neural tissue of the eye. Currently, there is no FDA-approved PAM imaging system for ocular use, leading most studies to conduct safety evaluations based on the ANSI safety limit for eyes, allowing a maximum single laser pulse energy of 160 nJ. The laser energy used in these studies typically ranges from 40 to 80 nJ/pulse for posterior imaging of the eyes, which is 50-75% lower than the ANSI safety limit.
While laser energy below the ANSI safety limit is generally considered safe, it is essential to recognize that when the laser irradiates the eye, a significant portion of the energy is transmitted to the retina, posing a risk of retinal laser injury upon overexposure. Considering the focal magnification (optical gain) of the eye, approximately 100,000 times [171], an irradiance of 80 nJ entering the eye effectively increases to 8,000 nJ when it reaches the retina. While some safety studies have been conducted in rabbits [172], further safety evaluations are necessary before applying this imaging technique to humans. Long-term safety studies, encompassing both structural and functional assessments of vision, are imperative to identify any evidence of cell injury, inflammation, or death following imaging procedures.
5. Clinical challenges and the future development of ocular molecular and cellular imaging
Ocular molecular and cellular imaging can play a crucial role in the diagnosis and monitoring of treatment of various ocular diseases. However, like any medical imaging technique, it faces several challenges and opportunities for future development. Here are some of the key challenges and potential advancements in ocular molecular and cellular imaging:
Spatial and Temporal Resolution: One of the challenges in ocular imaging is achieving high spatial and temporal resolution at the cellular and molecular levels. Currently, the axial and lateral resolution of PAM and OCT ranges from 3-37 µm for PAM and 2-4 µm for OCT [14,104,169,178,179]. Although the resolutions have been improved, future developments may focus on improving imaging technologies such as adaptive optics, super-resolution microscopy, and advanced imaging probes to enhance the resolution and visualization of cellular and molecular structures within the eye.
Noninvasive Imaging: Noninvasive imaging techniques are desirable to reduce patient discomfort and minimize the risk of complications. While many current ocular imaging methods are noninvasive, PAM imaging still uses an ultrasound transducer in contact with conjunctiva to record the acoustic signals, which could impede clinical translation and adoption. Further advancements can be made to develop novel imaging modalities that are even less invasive, such as using new contrast agents, non-contact ultrasound transducer [180,181], utilizing light-based technologies like OCTA [182,183], or photoacoustic remote sensing microscopy (PRSM) [184].
Real-time Imaging: Real-time imaging is important for dynamic processes and surgical interventions. Developing imaging techniques that provide real-time visualization of cellular and molecular events in the eye can aid in understanding disease progression and guide therapeutic interventions. This may involve combining imaging technologies with advanced computational algorithms for rapid image processing and analysis. Carrasco-Zevallos et al. have demonstrated a 4D OCT system that can visualize a needle inserted into the human vitreous [185–187]. In a previous study, our group has also described real-time OCT monitoring subretinal delivery of Matrigel and VEGF solution into the retina [17,188]. Further investigation will be performed to improve visualization the angiogenesis process.
Quantitative Imaging: Quantitative imaging techniques enable the measurement and analysis of molecular and cellular features, allowing for objective assessment and comparison of different ocular conditions. Further developments in image analysis algorithms and image quantification tools will enhance the ability to extract quantitative information from ocular molecular and cellular imaging data.
Multimodal Imaging: Combining multiple imaging modalities can provide complementary information and a more comprehensive assessment of ocular diseases. Future developments may involve integrating molecular and cellular imaging techniques with other imaging modalities, such as OCT, fluorescence imaging, or positron emission tomography (PET), to enable multimodal imaging approaches that capture different aspects of ocular pathologies.
Molecular Probes and Contrast Agents: The development of novel molecular probes and contrast agents is crucial for specific targeting and labeling of molecular and cellular structures within the eye. Advancements in molecular biology, nanotechnology, and chemistry can lead to the design and synthesis of more selective and sensitive probes for ocular imaging, enabling better detection and characterization of ocular diseases.
Translation to Clinical Practice: To realize the full potential of ocular molecular and cellular imaging, there is a need for translating these technologies from research settings to routine clinical practice. This requires addressing regulatory considerations, standardization of imaging protocols, validation of imaging biomarkers, and integration of imaging systems into clinical workflows. Addressing these challenges and advancing ocular molecular and cellular imaging techniques will likely significantly improve our understanding, diagnosis, and management of ocular diseases, leading to better patient outcomes and personalized treatments in the future.
Another challenge of PA imaging is the ultrasound coupling between transducer and targeted tissue to maximize the detection of PA signal. Water-based liquid and ultrasound gel have been widely used for acoustic coupling, which can help to minimize the acoustic impedance mismatch and improve the detected acoustic signal amplitude [20,104,167,189,190]. In addition to ultrasound gel, balanced salt solution (BBS) can be used as an alternative coupling media for PA imaging. BSS is similar to water but has a physiological pH and isotonic salt concentration to minimize irritation to the ocular surface, which is exquisitely sensitive. In addition, non-contact remote optical detection of the sound waves could be employed using optical interferometry to detect the photoacoustic signal [191–195].
Various imaging technologies and contrast agents exhibit promising potential for clinical translation. For instance, fluorescence imaging, with its high sensitivity and specificity, enables the real-time visualization of biological processes at the cellular and molecular levels. However, it is worth noting that in most situations fluorescence imaging is limited to 2D visualization of biological structures. In contrast, photoacoustic microscopy (PAM) imaging stands out by offering 3D imaging along with functional information, enhancing the capabilities for diagnosis and treatment in patient care. The present state of imaging technology enables the acquisition of real-time PAM images characterized by high resolution and contrast. When coupled with advanced contrast agents, this technology allows for precise observation of the location of diseases. Notably, ongoing improvements in contrast agents aim to enhance targeting capabilities and facilitate improved renal excretion, thereby mitigating long-term toxicity concerns.
The future of stem cell transplantation holds significant promise, but it also faces several challenges. These include engraftment monitoring to ensure successful engraftment of transplanted stem cells, graft-versus-host disease (GvHD) monitoring, cell fate and function monitoring, and long-term safety and tumorigenicity evaluation. Overcoming these challenges for tracking successful integration into the host tissue is critical for therapeutic efficacy. The challenges associated with stem cell transplantation can be effectively addressed by the integration of molecular and cellular imaging technologies. These non-invasive techniques offer valuable insights into the fate, function, and safety of transplanted cells, ultimately enhancing the success and safety of stem cell therapies in clinical practice. Continued advancements in imaging technology will likely contribute to the ongoing evolution of stem cell transplantation protocols and their application in personalized medicine.
6. Conclusion
In conclusion, this review provides a comprehensive overview of the current state-of-the-art ophthalmic molecular and cellular imaging techniques, ultimately driving the development of innovative strategies to combat eye diseases and improve visual outcomes for patients worldwide. By harnessing the power of molecular and cellular imaging, researchers and clinicians are poised to gain deeper insights into the intricacies of eye diseases and stem cell transplantation, offering new avenues for personalized medicine and regenerative approaches.
Funding
Unrestricted departmental support from Research to Prevent Blindness; Fight for Sight (FFSGIA16002); Alcon Research Institute Young Investigator Grant; Department of Defense Congressionally Directed Medical Research Programs (HT9425-23-10179); National Eye Institute (1K08EY027458, 1R01EY033000).
Acknowledgments
The research wishes to thank the generous support of the Helmut F. Stern Career Development Professorship in Ophthalmology and Visual Sciences (YMP) and the University of Michigan Department of Ophthalmology and Visual Sciences.
This research was supported by grants from the National Eye Institute 1R01EY034325 and 1R01EY033000 (YMP), Department of Defense CDMRP HT9425-23-10179 (YMP), Fight for Sight- International Retinal Research Foundation FFSGIA16002 (YMP), Alcon Research Institute Young Investigator Grant (YMP), and unrestricted departmental support from Research to Prevent Blindness.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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