The nervous system includes the central nervous system (CNS) and the peripheral nervous system (PNS), which take information from the surrounding environment and dominate the organism’s response to stimulus signals or stress. It controls muscle activity and glandular secretion and coordinates tissues and organs by transmitting signals and receiving feedback between body parts (Arafa et al., 2007; Carlstedt, 2008). The structural composition of the nerve varies from person to person, and the nerve is usually covered with a protective layer of tissue. Surgeons rely primarily on their anatomical knowledge, surgical expertise, and extensive clinical experience to identify important nerves during surgery. CNS or PNS damage produced by surgery may have devastating effects on a patient’s sensory, motor, and other bodily functions, negatively affecting the patient’s quality of life (Chaudhary et al., 2007; Shi et al., 2013; Musat et al., 2016). Hence, surgical planning frequently relies on imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and high-resolution ultrasonography (United States) to minimize the risk of nervous system injury during the procedure (Senden et al., 2006; Walsh et al., 2019). Intraoperative CT and MRI have the potential to provide valuable image guidance for surgeons. However, the operation of these systems can disrupt the normal surgical process, leading to prolonged surgery or anesthesia times. In addition, their expensive price and large floor space requirements can also impact their intraoperative use (Carl et al., 2018; Fuchs-Buder et al., 2018). In reagent applications, MRI can only identify the brain and spinal cord, but not peripheral nerve tissue due to the similar signal intensity between the PNS and surrounding tissues (Maravilla and Bowen, 1998). United States is a viable method for examining PNS, however, it is limited to superficial nerves due to its reliance on a network comprised of hypoechoic bands and hyperechoic lines (Misgeld and Kerschensteiner, 2006; Carvalho et al., 2019). The two most commonly utilized methods for testing nerve function are physical examination and electromyography (EMG). EMG is the prevailing approach for monitoring peripheral nerve function, achieved by stimulating target nerves to create action potentials (Kim et al., 2013; Barth and Gibbs, 2017). Based on current understanding, recognizing motor nerves is possible, but nonmotor sensory fibers are not recognized by the system (Walz et al., 2010). Although various techniques are available for nerve tissue treatment planning, they lack sensitivity and specificity, and some of them may pose radiological risks. Moreover, these techniques fail to achieve precise positioning of nerve tissue and anatomical details required for effective treatment planning.

In the report presented at the World Molecular Imaging Conference in 2009, Prof. Roger Yonchien Tsien first proposed the concept of optical molecular imaging-guided surgery. In 2011, the Leiden University Medical Center in the Netherlands and the Helmholtz Center Munich in Germany conducted a phase I clinical trial to evaluate the efficacy of their self-developed fluorescence imaging guidance system for the integrated treatment of peritoneal metastases in ovarian cancer (van Dam et al., 2011). In this field, the United States Food and Drug Administration (FDA) and the American Association for Image-Guided Surgery collaborated with 26 universities to establish standards. Additionally, the Canadian company Novadaq developed the first commercial real-time intraoperative imaging system called “Spy Fluorescence Imager,” which utilizes a CCD camera with filtering performance to achieve dynamic image acquisition (Liu et al., 2011). Subsequently, Roger Yonchien Tsien et al. successfully excised mouse tumor tissue using this technology proposed by their groups (Nguyen and Tsien, 2013). Xiaorong Xu’s team at the University of Science and Technology of China developed a miniaturized optical molecular image navigation eyepiece system by observing intraoperative fluorescence image information through a helmet display (Xu et al., 2014). In 2020, Jie Tian’s team at the Chinese Academy of Sciences, together with Sanjiv Sam Gambhir and Zhen Cheng’s team at Stanford University developed a near-infrared (NIR) I/II region imaging navigation system. This system achieved real-time multi-mode image detection on the micron level for the first time during the surgical resection of primary and metastatic liver tumors (Hu et al., 2020). In 2022, our team developed a solution for multi-mode three-dimensional optical molecular imaging intraoperative navigation imaging technology. This solution achieved a precise fusion of targeted optical molecular imaging and visible light images, accurately depicting the relationship between the three-dimensional structure of the target lesion and the surrounding tissue’s three-dimensional anatomy (Yang et al., 2022; Hao et al., 2023).

The advancement of optical molecular imaging technology has led to the emergence of new concepts such as optical molecular imaging surgery (Nguyen and Tsien, 2013; Wang et al., 2018), targeted surgery (Gandaglia et al., 2020), and molecular-guided surgery (Zhong et al., 2021; Mieog et al., 2022). These concepts involve the utilization of fluorescent dyes to label the target area, enabling real-time color contrast during surgical procedures. This technology utilizes fluorescent agents to mark target tissues and obtain real-time intraoperative images by irradiating them with light sources of varying wavelengths. It is capable of achieving high sensitivity at the skin level and high spatial resolution at the micron level, with the ability to capture hundreds of images per second. Fluorescence images provide a more intuitive representation of the tissue being imaged (Chi et al., 2014). The use of fluorescent agents to label nerve tissue has shown great potential in various surgeries. This technique enables surgeons to visualize nerves by observing optical signals hidden under the muscle or fat tissue during surgery, especially in situations where the normal anatomical position is displaced or destroyed due to external violence, which significantly reduces the difficulty of anatomy, maximizes the accuracy of the operation, shortens the duration of the operation, and minimizes the damage caused to the patient during the operation (Ietto et al., 2016).

Near-infrared light, an electromagnetic wave with a wavelength ranging from 700 nm to 2500 nm, is extensively employed in biological analysis research and biomedical diagnosis (Figure 1 (Beć et al., 2020)). Traditional optical imaging technology is mainly carried out in the spectral region of visible light (400–700 nm) and NIR I (700–900 nm) (Dip et al., 2014; Bennett et al., 2016; Lee et al., 2017; van Willigen et al., 2017; He et al., 2018; Du et al., 2021). However, when imaged in the visible region, the spatial resolution, imaging sensitivity, and contrast of these suffer from severe tissue scattering, absorption, and autofluorescence, leading to a decrease (Ren et al., 2021). NIR-I wavelengths are ideal for optical imaging as they can penetrate biological tissues such as skin and blood more deeply and effectively because they are not easily absorbed and scattered by these tissues (Prajapati et al., 2010; Vahrmeijer et al., 2013; Burggraaf et al., 2015). The autofluorescence of biological tissues in this wavelength range is minimal, resulting in a relatively high signal-to-background ratio (SBR) (Sakudo, 2016; Donnelly and Fine-Goulden, 2020). Currently, NIR-I wavelengths are the most suitable for deep tissue imaging of animals. Compared to NIR-I region optical imaging, the utilization of NIR-II (1,000–1700 nm) can result in lower levels of tissue autofluorescence, photon scattering, and absorption (Li et al., 2018; Li and Wang, 2018; He et al., 2019; Zhu et al., 2019). Additionally, it offers a higher SBR and can penetrate deeper into biological tissue (Li et al., 2020; Li et al., 2022b; Li et al., 2022a; Zhao et al., 2020; Zhao et al., 2021). Therefore, it is also regarded as the next-generation of optical imaging technology for assisting in biomedical diagnosis.

Since the beginning of the 21st century, optical molecular imaging has demonstrated both safety and effectiveness in numerous applications for surgical guidance (Tipirneni et al., 2017; Li et al., 2022). Medical devices utilizing this technology, particularly in the NIR region, are increasingly being approved for clinical use and hold a significant market share. These devices are currently capable of serving in endoscopic, laparoscopic, and open surgery procedures (Pogue and Rosenthal, 2021). With the advancement of imaging technology, the optical molecular imaging system has evolved to include independent imaging in visible light (Alfano et al., 1998) and NIR regions (Ntziachristos et al., 2003), bimodal imaging (Jathoul et al., 2014), multimodal imaging (Wang et al., 2020; Liu et al., 2022), 3D fluorescence imaging (Choi et al., 2020), color fusion imaging (Hao et al., 2023), and other modes. These improvements have significantly enhanced the doctor’s ability to visualize during operations.

In the realm of optical molecular imaging technology, the efficacy of surgical procedures is heavily reliant on the use of fluorescent agents. Unlike the medical device market, clinical approval has only been granted for a select few fluorescent agents, namely, fluorescein (FS), methylene blue (MB), 5-aminolevulinic acid (5-ALA), and indocyanine green (ICG). These agents have been clinically approved for uses such as tumor labeling (Waqas et al., 2017), angiography (Chotard et al., 2019), ureterography (Kaplan-Marans et al., 2019), sentinel lymph node identification (Chandrappa et al., 2020; Zhang et al., 2022) and cholangiography (Pardo Aranda et al., 2023). Compared to tubular structures such as blood vessels, ureters, and bile ducts, the bundled structure of nerve fibers makes it challenging for fluorescent agents to color, thus in vivo, imaging is difficult to achieve (Figures 2A,B). This problem has led researchers to develop neural fluorescent agents, and the number of studies in this field has increased in recent years. Although preliminary results of these probes are promising, there is insufficient research on their drug metabolism, dosage, pre-injection time, and toxicity. The clinical use of fluorescent agents in nerve bioimaging is limited, and there are few relevant clinical reports.

This paper provides a systematic summary of clinical and preclinical studies on various fluorescent agents used in optical molecular imaging over the past two decades. The functions and characteristics of these agents in neural visualization under the guidance of optical molecular imaging technology are also described. The discussion includes a prospectus on the development prospects of optical molecular imaging technology-guided neural visualization, aimed at assisting researchers in related fields.

Neuro-nonspecific agents are primarily utilized to enhance the visibility of nerves and their surrounding structures. This can be achieved through local injection or systemic injection, leveraging the physiological anatomy of the peripheral nerve (Samarasena et al., 2016). Furthermore, fluorescent agents can accumulate in peripheral nerve tissue through free diffusion and active reuptake (Schmued, 2016). By observing the timing and metabolic characteristics of fluorescent dyes in various tissues in vivo, we can effectively visualize nerve tissue intraoperatively (de Melo et al., 2016; Debie and Hernot, 2019; Walsh et al., 2019).

The discovery of multiple nerve marker binding sites has enabled the use of fluorescence imaging in vivo to quickly locate nerves in targeted areas, thereby significantly reducing the risk of nerve damage during surgery. Different fluorescent agents are utilized for various therapeutic applications, owing to their unique modes of action, binding locations, and methods of administration. (Davila et al., 2008). Neuro-specific agents are classified into distinct categories based on the specific target binding sites they bind to.

In the past decades, surgeons have adopted various diagnosis and treatment techniques and auxiliary methods. Among these, CT, MRI, United States, and nuclear medicine imaging have become common tools for preoperative diagnosis. These non-invasive imaging techniques help surgeons in planning surgery and improving treatment. However, some questions regarding surgical guidance and decision-making remain unanswered. Although United States, CT, and MRI are widely used, they have limited sensitivity and accuracy when it comes to detecting subtle lesions, and they are not suitable for real-time intraoperative feedback. United States is a valuable tool for diagnosing tumor diseases that have well-defined contours and clear boundaries in pathology, especially for PNS tumors (Misgeld and Kerschensteiner, 2006). However, it is important to note that high-resolution ultrasound relies on a network of hypoechoic bands and hyperechoic lines to identify nerves, which limits its ability to visualize deep tissue and hyperechoic structures of nerves, the United States can only detect approximately 30% of the total number of nerve bundles currently (Ohana et al., 2014; Carvalho et al., 2019). Optical molecular imaging technology utilizes the properties of fluorescent substances or externally introduced fluorescent compounds to aid physicians in making medical decisions. This is achieved by capturing optical signals using specific optical imaging equipment (Nishio et al., 2019; Liu et al., 2021). Compared to the United States, optical molecular imaging technology offers notable advantages in terms of imaging depth and clarity. In addition, optical molecular imaging technology can potentially visualize the parts that the fluorescent agent can reach during the operation. Recently, researchers have proposed a multimodal tumor diagnosis strategy by combining MRI and fluorescence imaging systems (McCann et al., 2009; Reynders et al., 2021; Song et al., 2021). This approach has demonstrated unique advantages in the multiple imaging of target tissues in vivo. The strategy leverages MRI to obtain accurate tumor volumes and utilizes fluorescent probes to measure tumor function and protease activity responses. This enables the quantification and tracking of tumor histological changes in a non-invasive manner. In molecular imaging of nuclear medicine, the imaging effect is closely related to the nuclear properties of radioisotopes, such as decay type, photon energy, and half-life. The use of foreign radioactive elements may cause adverse reactions in the body. In the field of molecular imaging, the effectiveness of nuclear agents is closely related to the nuclear properties of the radioisotopes used, including decay type, photon energy, and half-life. However, the use of radioactive elements can lead to negative bodily reactions.

During surgery, the surgeon relies on both touch and vision to make decisions. However, the rise of laparoscopic surgery, which is minimally invasive, has led to a decrease in the surgeon’s tactile sense. As a result, surgeons now depend heavily on intraoperative images. Fortunately, optical molecular imaging technology has advanced significantly in recent decades. As it continues to be integrated and improved upon in surgical procedures, it has paved the way for fluorescence-guided surgery, molecular image-guided surgery, and targeted surgery. These new directions have the potential to lead to further advancements in surgical techniques (Laguna, 2014; Benson et al., 2021; Lauwerends et al., 2021; Chand and Polom, 2022). Optical molecular imaging technology offers safe and convenient means to achieve high spatial and temporal resolution. This technology is useful for clinical diagnosis and analyzing biological phenomena, monitoring molecular content, observing the distribution of target tissues, and visualizing different biological tissues. It has played an irreplaceable role in revealing disease occurrence, development, and treatment and has opened up new avenues for research (SAJI, 2017; Wu et al., 2021). The advancement of this technology has resulted in the creation of various technologies including narrow-band imaging (Herr, 2014), photodynamic diagnosis (Didamson and Abrahamse, 2021), optical coherence tomography (Wang et al., 2022), and confocal laser endoscopic imaging (Pilonis et al., 2022).

The NIR fluorescence imaging system is continuously improving with the increasing utilization of various fluorescent agents in clinical applications (van Willigen et al., 2017). The researchers discovered that the fluorescent agent’s excited or emitted light could be absorbed by various cellular structures, including water, hemoglobin, oxygenated hemoglobin, skin, fat, and proteins. This absorption would significantly hinder the technology’s usability (Hemmer et al., 2016; Zhao et al., 2018). The spatial resolution of fluorescence systems is significantly reduced due to biological tissues’ strong optical scattering and absorption properties. This problem creates a significant challenge in achieving maximum imaging depth of biological tissues. In addition, the background signal produced by body tissues, known as spontaneous fluorescence, can decrease the SBR and lower the quality of the resulting image. However, adjusting parameters such as excitation duration and frequency harmonics can improve the imaging effect and mitigate the effects of scatter, ultimately resolving this issue (Chakraborty et al., 2019). In comparison to NIR-I, NIR-II fluorescence imaging offers advantages such as reduced tissue scattering and weaker tissue spontaneous fluorescence resulting in deeper penetration and higher spatial resolution in biological tissues, which can minimize background interference. However, the development of NIR-II fluorescent agents has been hindered by challenges such as poor water solubility, stability, fluorescence efficiency, biocompatibility, and metabolic rate (Frangioni, 2003; Miao et al., 2018; Liu et al., 2019). Researchers have been searching for fluorescent materials, such as probes and agents, that possess two critical characteristics at least. Firstly, these materials must effectively overcome the spontaneous fluorescence caused by thick and opaque tissue scattering to ensure the clearest possible images (Feng et al., 2013; Cao et al., 2018). Secondly, it must possess good biological stability and ideally cluster in the target tissue to produce fluorescence images that are specific to the target tissue (Feng et al., 2013).

Based on the above characteristics, researchers have become interested in quantum dots (QDs), which are inorganic semiconductor nanomaterials developed using certain metal elements like Cu, Cd, and Pb. QDs consist of a fluorescent core and a semiconductor shell. The surface of the shell is modified with specific targeting groups and other biomolecules using bio-coupling active targeting methods (Zhao et al., 2018; Yu et al., 2019). Numerous studies have confirmed that NIR QDs hold immense potential in preclinical research due to their superior brightness, sensitivity, specificity, and optical stability when compared to other fluorescent agents (Gil et al., 2021). The potential toxicity of NIR QDs in biological systems is a major concern for researchers. If the chemical stability of quantum dots in vivo is compromised, it can have fatal consequences. Therefore, low toxicity and stable biocompatibility are crucial for the clinical translation of NIR QDs (Ozkan Vardar et al., 2018; Aydemir et al., 2020). While NIR QDs have been extensively studied for their toxicity issues, it is important to note that preclinical studies may not always accurately predict the material’s behavior in clinical trials.

In the related research for Yan Wo’s group and Thomas Reiner’s group (Feng et al., 2019), it was discovered that PbS quantum dots, formed by Pb²⁺ and subjected to water-soluble surface modification, exhibit favorable biocompatibility. Through metabolic experiments in mice, researchers discovered that the presence of these quantum dots resulted in a decreased ion release rate and an increased clearance rate in the body. Furthermore, no deposition of PbS quantum dots was observed in the liver, spleen, and other organs. These findings indicate that these quantum dots exhibit extremely low cytotoxicity and a high rate of blood clearance, thus suggesting their potential for clinical applications. In research on fluorescent agents, it is worth considering modifying their molecular structure to reduce toxicity. This can be achieved by removing or altering specific functional groups. In addition to this, the research also emphasizes the exploration of non-toxic or low-toxic new peptide sequences and various small molecular substances during the nerve electrical signal transduction process (Hingorani et al., 2018; Gonzales et al., 2020), which are coupled with currently clinically approved fluorescent agents (such as ICG). It is crucial to carry out numerous experiments to ascertain the safety events and dosage range for its application before implementing clinical transformation.

Currently, NIR-I imaging is rapidly moving towards broad clinical application, while NIR-II imaging is entering a transitional period from basic research to more advanced applications. We believe that as fluorescent agents continue to develop and imaging systems are upgraded, the application of NIR-II imaging will be more extensive than that of NIR-I in the future. The use of NIR imaging for intraoperative nerve visualization offers several advantages. Real-time imaging and high SBR facilitate accurate nerve identification during surgery. Furthermore, NIR light has a stronger penetration ability compared to visible light. This non-invasive method does not cause additional harm to the patient and does not interfere with normal surgical procedures.

The development of optical molecular imaging techniques for surgical guidance is progressing rapidly. The fluorescent agents mentioned in the paper are summarized in Table 1. However, the identification of neural tissue using these techniques is still limited. Due to the unique properties of neural tissue, it is crucial to select a suitable fluorescent agent for visualization. An ideal neuro fluorescent agent should possess the following characteristics: ① excitation and emission wavelengths in the NIR window, ② easy perioperative administration, ③ log D between 0.5 and 3 at pH = 7.4, and molecular weight <500Da to ensure maximum BNB penetration, ④ long-term retention in neural tissue, ⑤ high SBR value; ⑥ high safety (Waterhouse, 2003; Pajouhesh and Lenz, 2005; Fagerholm, 2007; SAJI, 2017; Walsh et al., 2019). Then, we are faced with several problems. The issue at hand is that nerve-specific agents need to have a low enough molecular weight to pass through the BNB. However, for a NIR agent to achieve the excited state in the NIR region, it must have a sufficiently large conjugated system, which ultimately increases its molecular weight (Gibbs-Strauss et al., 2011). The structure-activity relationship (SAR) of neuron-specific agents has been proposed as a way to guide the synthesis of other fluorescent agent libraries based on the molecular configuration of the agent because changes in the surrounding environment can affect the imaging effect of agents (Gibbs et al., 2013).

Various injection methods have been found to impact the resulting fluorescence, as depicted in Figure 9. While systemic administration may decrease the SBR of vital nerves, local administration at the same dosage can enhance the fluorescence signal of the neural target. Moreover, the fluorescent agent dosage and pre-injection time required for local administration to achieve the same fluorescence intensity were significantly lower than those for systemic administration. Local administration of fluorescent agents cannot effectively absorb and eliminate them through circulation, leading to potential tissue accumulation and injury due to their cytotoxicity. Therefore, to enhance nerve-specific fluorescence intensity and minimize non-specific fluorescence background signal and tissue damage, it is crucial to carefully consider the injection technique, dosage, and pre-injection period (Barth and Gibbs, 2017).

Currently, there is no standard consensus on accurately and comprehensively evaluating the effectiveness of existing fluorescent agents in clinical reports due to the use of different fluorescent agents, a small patient sample size, and varying clinical diagnoses and preoperative interventions. To fully understand the effects of neuro-specific agents, it is crucial to investigate their physicochemical characteristics such as injection dose, injection time, cytotoxicity, sensitivity, temporal and spatial resolution, penetration depth, and pharmacokinetics. Intraoperative optical data can be used to improve the process of assessing the location of the target nerve during surgery. Currently, this process is subjective and relies on the surgeon’s judgment. By analyzing the quantitative outcomes of the optical data in real-time, the location and course of the nerve can be judged based on the changes in the optical signal feedback from the instrument.

The imaging of nerves in fluorescence-guided surgery is advancing smoothly thanks to the development of fluorescent agents. This technology is expected to further improve intraoperative nerve preservation and monitoring of nerve regeneration, as well as aid in the identification of neurological illnesses. As clinical settings continue to adopt this developing technology, we can expect significant advancements in the field. In comparison to previous studies that have summarized the principles of neuro fluorescence imaging using existing fluorophores and the imaging effects of optical molecular imaging technology in central nervous system diseases, this research focuses on discussing the future development trends and advancements in new imaging equipment and fluorescent agents (Chen et al., 2020; Cen et al., 2022). The aim is to inspire and guide future researchers in this field. Moving forward, our team will continue conducting extensive research in the hopes of obtaining neuro-specific agents with highly safe and excellent imaging effects as soon as possible.