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Current Advances in Molecular Imaging: Noninvasive In Vivo Bioluminescent and Fluorescent Optical Imaging in Cancer Research
Abstract
Recently, there has been tremendous interest in developing techniques such as MRI, micro-CT, micro-PET, and SPECT to image function and processes in small animals. These technologies offer deep tissue penetration and high spatial resolution, but compared with noninvasive small animal optical imaging, these techniques are very costly and time consuming to implement. Optical imaging is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. In vivo optical imaging is the result of a coalescence of technologies from chemistry, physics, and biology. The development of highly sensitive light detection systems has allowed biologists to use imaging in studying physiological processes. Over the last few decades, biochemists have also worked to isolate and further develop optical reporters such as GFP, luciferase, and cyanine dyes. This article reviews the common types of fluorescent and bioluminescent optical imaging, the typical system platforms and configurations, and the applications in the investigation of cancer biology.
... [10][11][12][13] Fluorescence biological imaging often causes little to no disturbance of bodily tissues, has minimal effects on metabolic processes, and provides images of native biological states. [14,15] Significant improvements to increase the practicality of NIR materials in water remains possible through the design of dyes with increased molar absorptivities, longer absorption and emission wavelengths, higher quantum yields, better photostabilities, and increased ease of incorporation into biological environments. Cyanines and squaraines are ubiquitous classes of fluorescent imaging materials which address the design requirements for NIR probes. ...
... ChemPhotoChem 2022, 6, e202200127 (3 of14) © 2022 Wiley-VCH GmbH ...
... ChemPhotoChem 2022, 6, e202200127 (10 of14) © 2022 Wiley-VCH GmbH ...
The design of bright, high quantum yield (QY) materials in the near‐infrared (NIR) spectral region in water remains a significant challenge. A series of cyanine and squaraine dyes varying water solubilizing groups and heterocycles are studied to probe the interactions of these groups with albumin in water. Unprecedented, ′ultra‐bright′ emission in water is observed for a sulfonate indolizine squaraine dye (61.1 % QY) and a sulfonate indolizine cyanine dye (46.7 % QY) at NIR wavelengths of >700 nm and >800 nm, respectively. The dyes presented herein have a lower limit of detection than the most sensitive dyes known in the NIR region for albumin detection by at least an order of magnitude, which enables more sensitive diagnostic testing. Additionally, biotinylated human serum albumin complexed with the dyes reported herein was observed to function as an immunohistochemical reagent enabling high resolution imaging of cellular α‐tubulin at low dye concentrations.
... The advantage of optical imaging is that it does not use ionizing radiation or expensive equipment and does not entail high operating and maintenance costs, unlike CT, PET or MRI [5][6][7]. Optical imaging provides a highly sensitive and cost-effective imaging technique for use with tissues and diseases and is therefore worth exploring further [4,[7][8][9][10][11]. In particular, fluorescence imaging is a highly sensitive and functional imaging process that enables high contrast enhancement between tumors and the surrounding healthy tissue [8][9][10][11][12]. ...
... Optical imaging provides a highly sensitive and cost-effective imaging technique for use with tissues and diseases and is therefore worth exploring further [4,[7][8][9][10][11]. In particular, fluorescence imaging is a highly sensitive and functional imaging process that enables high contrast enhancement between tumors and the surrounding healthy tissue [8][9][10][11][12]. The principle is based on laserinduced fluorescence [13][14][15][16]. ...
... In the in vivo experiments, two tumor-specific fluorescently labeled contrast agents bearing different tumor-targeting molecules were used. These molecules were bombesin 7-14 (BBN [7][8][9][10][11][12][13][14], which binds to the gastrin-releasing peptide receptor (GRPR), and Lys-urea-Glu (LUG), which addresses the prostate-specific membrane antigen (PSMA). The molecules were assembled on the surface of gold nanoparticles (AuNPs) [46] together with SIDAG dye [absorbance: 600-800 nm/emission: 750-820 nm] to visualize GRPR-and PSMApositive tumors. ...
To date, few optical imaging systems are available in clinical practice to perform noninvasive measurements transcutaneously. Instead, functional imaging is performed using ionizing radiation or intense magnetic fields in most cases. The applicability of fluorescence imaging (e.g., for the detection of fluorescently labeled objects, such as tumors) is limited due to the restricted tissue penetration of light and the required long exposure time. Thus, the development of highly sensitive and easily manageable instruments is necessary to broaden the utility of optical imaging. To advance these developments, an improved fluorescence imaging system was designed in this study that operates on the principle of noncontact laser-induced fluorescence and enables the detection of fluorescence from deeper tissue layers as well as real-time imaging. The high performance of the developed optical laser scanner results from the combination of specific point illumination, an intensified charge-coupled device (ICCD) detector with a novel light trap, and a filtering strategy. The suitability of the laser scanner was demonstrated in two representative applications and an in vivo evaluation. In addition, a comparison with a planar imaging system was performed. The results show that the exposure time with the developed laser scanner can be reduced to a few milliseconds during measurements with a penetration depth of up to 32 mm. Due to these short exposure times, real-time fluorescence imaging can be easily achieved. The ability to measure fluorescence from deep tissue layers enables clinically relevant applications, such as the detection of fluorescently labeled malignant tumors.
... A unique advantage of bioluminescence for in vivo imaging is the lack of autoluminescence produced from mammalian cells, providing extremely low background (Mezzanotte et al., 2017;Dimond et al., 2020), whilst imaging at depth is aided by the lack of requirement for excitation by an external source. Nonetheless, as with any optical method, detection is limited by signal attenuation from deep and dark tissues; the scatter of light by cells, lipids and other components increases with depth, and substances such as haemoglobin absorb blue-green light (Choy et al., 2003;Zhao et al., 2005). These issues are being addressed through development of novel luciferase/ luciferin variants designed for deeper and more sensitive signal detection (Zambito et al., 2021). ...
... Traditional methods to investigate tumour burden in animal models involve calliper measurements of subcutaneous tumours or post-mortem weighing. Alternatively, cancer cell lines with constitutive luciferase expression can be injected into animals to visualise tumour growth in real-time, greatly reducing the number of animals needed (Choy et al., 2003;Contag et al., 2000;O'Farrell et al., 2013;Edinger et al., 1999) (Figure 1B, left panel). Using this technique, even a single cell could be detected (Kim et al., 2010;Iwano et al., 2018), with a linear relationship between cell number and signal over 5 logarithmic scales (Contag et al., 2000;Rehemtulla et al., 2000). ...
The naturally occurring phenomenon of bioluminescence has intrigued on-lookers for decades and is now being developed as a powerful tool for medical research and preclinical imaging. Luciferase enzymes emit light upon substrate encounter, enabling their activity to be visualised and dynamically tracked. By inserting luciferase genes into specific sites in the genome, it is possible to engineer reporters to monitor gene expression in its native context, and to detect epigenetic change in vivo . Endogenous bioluminescent reporters provide a highly sensitive, quantitative read-out of gene expression that is both well suited to longitudinal studies and can be adapted for high-throughput drug screens. In this article we outline some of the applications and benefits of bioluminescent reporters for epigenetic research, with a particular focus on revealing new therapeutic options for treating genetic and epigenetic disorders.
... However, a newer approach known as optical imaging has gained attention as a promising technique for the detection and diagnosis of cancer [4]. Fluorescence imaging is one of the most widely used optical bioimaging methods due to the variety of signal readouts, such as fluorescence intensity, lifetime, quenching, or Förster resonance energy transfer. ...
... It offers deep tissue penetration and has minimal obstruction by autofluorescence and photon scattering [7]. As a model of optical imaging, NIR is capable of providing information related to the biochemistry and anatomy of any cancerous tissues [4]. There are several NIR dyes, including ICG [8] and IR-1061 [9], that allow high-resolution tissue imaging. ...
The development of fluorescence dyes for near-infrared (NIR) fluorescence imaging has been a significant interest for deep tissue imaging. Among many imaging fluoroprobes, indocyanine green (ICG) and its analogues have been used in oncology and other medical applications. However, these imaging agents still experience poor imaging capabilities due to low tumor targetability, photostability, and sensitivity in the biological milieu. Thus, developing a biocompatible NIR imaging dye from natural resources holds the potential of facilitating cancer cell/tissue imaging. Chlorophyll (Chl) has been demonstrated to be a potential candidate for imaging purposes due to its natural NIR absorption qualities and its wide availability in plants and green vegetables. Therefore, it was our aim to assess the fluorescence characteristics of twelve dietary leaves as well as the fluorescence of their Chl extractions. Bay leaf extract, a high-fluorescence agent that showed the highest levels of fluorescence, was further evaluated for its tissue contrast and cellular imaging properties. Overall, this study confirms bay-leaf-associated dye as a NIR fluorescence imaging agent that may have important implications for cellular imaging and image-guided cancer surgery.
... BLI generally provides high sensitivity with a high signal-to-noise ratio when compared to fluorescent imaging [35]; moreover, its tissue penetrability in higher than that of fluorescence imaging [14,36], and has a high throughput for imaging of small animals [9,15], as also a wide temporal detection window (0 days to 1 year) [9], despite its use being limited to preclinical studies. In vivo BLI enables real-time monitoring of gene expression and cell fate through visual representation of the bioluminescence generated by oxidation of specific substrates by luciferase enzymes, providing a dynamic profile of engraftment and proliferation in live recipient animals [9,37]. ...
... Optical imaging certainly holds utility in assessing hematopoietic stem cell tracking of xenografts, allografts, autologous grafts applied in the bone marrow transplant model [35]. The same study reported that bioluminescence imaging is certainly more sensitive than fluorescence imaging. ...
The hematopoietic stem cell engraftment depends on adequate cell numbers, their homing, and the subsequent short and long-term engraftment of these cells in the niche. We performed a systematic review of the methods employed to track hematopoietic reconstitution using molecular imaging. We searched articles indexed, published prior to January 2020, in PubMed, Cochrane, and Scopus with the following keyword sequences: (Hematopoietic Stem Cell OR Hematopoietic Progenitor Cell) AND (Tracking OR Homing) AND (Transplantation). Of 2191 articles identified, only 21 articles were included in this review, after screening and eligibility assessment. The cell source was in the majority of bone marrow from mice (43%), followed by the umbilical cord from humans (33%). The labeling agent had the follow distribution between the selected studies: 14% nanoparticle, 29% radioisotope, 19% fluorophore, 19% luciferase, and 19% animal transgenic. The type of graft used in the studies was 57% allogeneic, 38% xenogeneic, and 5% autologous, being the HSC receptor: 57% mice, 9% rat, 19% fish, 5% for dog, porcine and salamander. The imaging technique used in the HSC tracking had the following distribution between studies: Positron emission tomography/single-photon emission computed tomography 29%, bioluminescence 33%, fluorescence 19%, magnetic resonance imaging 14%, and near-infrared fluorescence imaging 5%. The efficiency of the graft was evaluated in 61% of the selected studies, and before one month of implantation, the cell renewal was very low (less than 20%), but after three months, the efficiency was more than 50%, mainly in the allogeneic graft. In conclusion, our review showed an increase in using noninvasive imaging techniques in HSC tracking using the bone marrow transplant model. However, successful transplantation depends on the formation of engraftment, and the functionality of cells after the graft, aspects that are poorly explored and that have high relevance for clinical analysis.
... Due to its sensitivity, optical imaging (OI) in general and fluorescence reflectance imaging (FRI) in particular are comparable to nuclear medical imaging procedures such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) and has the advantage of lacking radioactive tracers. Moreover, OI is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo (13). In vivo OI is the result of a coalescence of technologies from chemistry, physics, and biology (13), but should be adjusted to in vivo conditions. ...
... Moreover, OI is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo (13). In vivo OI is the result of a coalescence of technologies from chemistry, physics, and biology (13), but should be adjusted to in vivo conditions. Previous studies show that imaging of green fluorescent protein (GFP)-transfected tumor cell lines is an interesting tool to monitor both tumor progression, and treatment-induced regression, and can yield prognostic information (14)(15)(16)(17)(18)(19)(20)(21). ...
Background:
Mouse models of human-malignant-melanoma (MM) are important tools to study tumor dynamics. The enhanced green fluorescent protein (EGFP) is widely used in molecular imaging approaches, together with optical scanners, and fluorescence imaging.
Purpose:
Currently, there are no data available as to whether other fluorescent proteins are more suitable. The goal of this preclinical study was to analyze two fluorescent proteins of the GFP superfamily under real-time in vivo conditions using fluorescence reflectance imaging (FRI).
Material and methods:
The human melanoma cell line MeWo was stable transfected with one plasmid: pEGFP-C1 or pDsRed1-N1. We investigated two severe combined immunodeficiency (SCID)-mice groups: A (solid xenografts) and B (xenografts as metastases). After three weeks, the animals were weekly imaged by FRI. Afterwards the mice were euthanized and metastases were imaged in situ: to quantify the cutis-dependent reduction of emitted light, we compared signal intensities obtained by metastases in vivo with signal intensities obtained by in situ liver parenchyma preparations.
Results:
More than 90% of cells were stable transfected. EGFP-/DsRed-xenograft tumors had identical growth kinetics. In vivo the emitted light by DsRed tumors/metastases was much brighter than by EGFP. DsRed metastases were earlier (3 vs. 5 weeks) and much more sensitive detectable than EGFP metastases. Cutis-dependent reduction of emitted light was greater in EGFP than in DsRed mice (tenfold). Autofluorescence of DsRed was lower than of EGFP.
Conclusion:
We established an in vivo xenograft mouse model (DsRed-MeWo) that is reliable, reproducible, and superior to the EGFP model as a preclinical tool to study innovative therapies by FRI under real-time in vivo conditions.
... Moreover, the detection of large molecules such as a-fetoproteins and IgG has been performed through obelin [135,136]. The monitoring of tumor progression in animal models of cancer using bioluminescence imaging has been well reported [137]. Animal models of prostate [138], breast [139] and colon [140] cancer has recently been used. ...
... Although several techniques exist to overcome these challenges, including radiolabels, near-infrared quantum dots, and near-infrared fluorescing carbon nanotubes these techniques are limited due to their associated toxicity [219][220][221]. Bioluminescence imaging is thus an attractive technique for tracking cells of interest in vivo [137,222,223]. Beside its role as a detector for host-pathogen interactions, it can provide monitoring of therapeutic efficacy of various therapeutics in real time [224,225]. ...
... The tracking interest cells in vivo are other unbeatable application of luminescence imaging [199][200][201]. Beside of its role as detector for host-pathogen interactions, it provide the monitoring of therapeutic effects at antibiotic therapy in real time [202,203]. ...
... The noninvasive marking and tracking of genetically modified cells in transgenic animal model is another interesting application of bioluminescence imaging [226,227]. Probably, the monitoring of tumor progression in animal models of cancer be the most interesting and popular use of bioluminescence imaging [201]. The animal models such as prostate [228], breast [229]and colon [230]cancer has recently been used from this method. ...
Bioluminescence is referred to the light emission by a living organism due to a specific biochemical reaction. This interesting feature of the organisms could highly influences behavioral and ecosystem dynamics. Luminescence, mostly observed in marine species, is generally higher in deep-living genera than in benthic or shallow organisms. However, among creatures living in land, fireflies, beetles, springtails and fungi have shown some bioluminescent activities. Classically, the emission of light is catalyzed by luciferase from a substrate. Interestingly, light-emitting organisms are more abundant and widespread in marine than terrestrial environments. Novel tools derived from understanding bioluminescent reactions have led to countless valuable applications in modern biotechnology and biochemical engineering. Here, we overview some main properties bioluminescence in marine organism from bacteria to fishes following the latest advances and new discoveries of state-of-the-art bioluminescent tools in molecular biology, bioluminescent bioassays and imaging. The overview showed available and wide biotechnological tools of bioluminescence take advantage of its high detectability, high sensitive, low toxic and quantum efficiency which make wide usage as reporter of many biological functions in different fields, such as studying bacterial pathogens, ecotoxicology, food toxicity, tracking cells of interest in vivo, protein–protein interactions, gene expression and circadian rhythms. With the recent invention of luminescent reporters, future possibilities for the development of additional reporter applications are promising.
... The signal can be either generated by bioluminescence, where the optical moiety has to be activated in vivo by an enzyme, usually luciferase, which was transfected in the target tissue [85]. Fluorescent probes on the other hand contain an optical moiety that, after excitation at a specific wavelength, emits a signal of a different wavelength [86,87]. In both cases, the optical signal with the defined wavelength can be detected with both a high spatial resolution and high sensitivity. ...
... Thus, the imaging of organs is still challenging. A large variety of fluorophores are available, ranging from low wavelengths such as 510 nm (green fluorescence protein), which are not suitable for in vivo imaging, up to 900 nm (near-infrared flourophores), providing a better penetration for imaging of deeper tissues [86]. Such near-infrared (NIR) probes are successfully used for intraoperative detection of various lesions [88][89][90]. ...
β-cells, located in the islets of the pancreas, are responsible for production and secretion of insulin and play a crucial role in blood sugar regulation. Pathologic β-cells often cause serious medical conditions affecting blood glucose level, which severely impact life quality and are life-threatening if untreated. With 347 million patients, diabetes is one of the most prevalent diseases, and will continue to be one of the largest socioeconomic challenges in the future. The diagnosis still relies mainly on indirect methods like blood sugar measurements. A non-invasive diagnostic imaging modality would allow direct evaluation of β-cell mass and would be a huge step towards personalized medicine. Hyperinsulinism is another serious condition caused by β-cells that excessively secrete insulin, like for instance β-cell hyperplasia and insulinomas. Treatment options with drugs are normally not curative, whereas curative procedures usually consist of the resection of affected regions for which, however, an exact localization of the foci is necessary. In this review, we describe potential tracers under development for targeting β-cells with focus on radiotracers for PET and SPECT imaging, which allow the non-invasive visualization of β-cells. We discuss either the advantages or limitations for the various tracers and modalities. This article concludes with an outlook on future developments and discuss the potential of new imaging probes including dual probes that utilize functionalities for both a radioactive and optical moiety as well as for theranostic applications.
... 16,17 Fluorescence imaging is a relatively new and rapidly evolving modality used in the intraoperative setting to delineate the vasculature and lymphatic drainage or demarcate between tumor and normal tissue. [13][14][15] In recent studies, its clinical application has Complete adhesiolysis was done using a monopolar hook and a harmonic scalpel, taking care not to injure any major blood vessels. Complete release with 4-cm clearance over celiac, left gastric, and common hepatic arteries was done. ...
... Optical imaging techniques offer several advantages for early diagnosis of tumors and imagingguided therapy compared to alternative approaches, such as high sensitivity, high spatial resolution, high signal to noise ratio in the near infrared spectral region and the use of low concentrations of contrast agent, [1][2][3][4][5] . However, optical imaging has been primarily limited to dermal and ocular applications, due to autofluorescence, light scattering and attenuation by tissues and the absorption of light by hemoglobin, lipids and water, leading to low penetration depths. ...
Herein, we demonstrate the potential of ultrabright fluorescent silica-coated organic nanocrystals for two-photon in vivo imaging. These unique nanoparticles (NPs) containing a crystalline core of small push–pull dipolar dye are specifically designed to exhibit two-photon absorption for fluorescence imaging. The NPs can be easily functionalized using click chemistry in pure water, with preservation of the organic core. A novel small-volume DLS technique was used to evaluate the effect of PEGylation on the colloidal stability of the NPs in complex media containing salts and proteins, mimicking the composition of blood serum. The potential of these bright red-emitting NPs for two-photon fluorescence imaging is demonstrated both in vitro and in vivo.
... Although optical imaging does have limitations due to tissue absorption and reflection of light, the inherent low cost of optical imaging methods compared to more complex imaging techniques such as MRI and PET has attracted increasing interest from cancer researchers [5], particularly with the development of inexpensive fiberoptic confocal imaging systems capable of being fed through ducts and capillaries [6]. ...
Nanoparticles, labeled with a signaling moiety for in vivo imaging, and one or more ligands for molecularly targeted specificity, hold considerable promise in oncology. Nanoparticles can serve as modular platforms, from which a wide variety of highly sensitive and specific imaging agents can be created. For example, many hundreds or thousands of atoms that provide imaging signals, such as radioisotopes, lanthanides, or fluorophores, can be attached to each nanoparticle, to form imaging agents that would provide higher sensitivity that can be obtained from agents based on small molecules. Similarly, many copies of targeted ligands can be attached to nanoparticles to markedly inrease specific binding. Drugs or therapeutic isotopes can be added to create multifunctional nanoparticles. Appropriately labeled and targeted nanoparticles could lead to a paradigm change in which cancer detection, diagnosis, and therapy are combined in a single molecular complex.
... Fluorescence imaging is a relatively new and rapidly evolving modality used in the intraoperative setting to delineate the vasculature, lymphatic drainage, or demarcate between tumor and normal tissue. [1][2][3] Indocyanine green (ICG), a fluorescence agent, is a water-soluble dye with a peak spectral absorption and emission at 800 to 810 nm in the blood or plasma. ICG has been used clinically since 1956 for angiography, to measure cardiac output or cerebral blood flow, evaluate hepatic function, and assess regional blood flow assessment. ...
Objectives:
Fluorescence imaging using indocyanine green (ICG) allows for the intraoperative mapping of the vascular supply of various tissue beds. Although generally safe and effective, rare adverse effects have been reported including anaphylactoid reactions. The current study retrospectively reviewed our experience the intraoperative administration of ICG to pediatric patients.
Methods:
The anesthetic records of patients who received ICG over a 2-year time period were retrospectively reviewed and demographic, surgical, and medication data retrieved. Objective intraoperative data before and after the administration of ICG were also recorded. These included heart rate, systolic and diastolic blood pressures, oxygen saturation, and peak inflating pressure.
Results:
The study cohort included 100 patients with a median age of 12 years (9.5 ± 7.4 years) and the median weight being 44.5 kg (45.9 ± 36.9 kg). ICG was administered intravenously to all patients. In all cases, 2.5 mg/mL ICG solution was used, with a median dose of 1.1 mL (1.79 ± 1.8 mL). Eight patients received more than 1 dose of ICG, with no adverse respiratory or hemodynamic effects related to its use.
Conclusions:
ICG fluorescence is an important imaging modality that can be safely used as an intraoperative adjunct to various surgical procedures in the pediatric population.
... Stably modified cells expressing FP under the control of promotor represent a highly sophisticated tool for studying gene regulation. The fusion of FP-encoding genes with genes of interest enables the possibility of localizing and quantifying specific proteins in vitro and in vivo, especially in cancer research [42,[57][58][59]. This approach has become a powerful tool in preclinical research. ...
Targeted drug delivery using nano-sized carrier systems with targeting functions to malignant and inflammatory tissue and tailored controlled drug release inside targeted tissues or cells has been and is still intensively studied. A detailed understanding of the correlation between the pharmacokinetic properties and structure of the nano-sized carrier is crucial for the successful transition of targeted drug delivery nanomedicines into clinical practice. In preclinical research in particular, fluorescence imaging has become one of the most commonly used powerful imaging tools. Increasing numbers of suitable fluorescent dyes that are excitable in the visible to near-infrared (NIR) wavelengths of the spectrum and the non-invasive nature of the method have significantly expanded the applicability of fluorescence imaging. This chapter summarizes non-invasive fluorescence-based imaging methods and discusses their potential advantages and limitations in the field of drug delivery, especially in anticancer therapy. This chapter focuses on fluorescent imaging from the cellular level up to the highly sophisticated three-dimensional imaging modality at a systemic level. Moreover, we describe the possibility for simultaneous treatment and imaging using fluorescence theranostics and the combination of different imaging techniques, e.g., fluorescence imaging with computed tomography.
... Absorption of photons at a certain wavelength range by the fluorescent molecule causes its electrons to get excited to a higher energy state. The loss of this energy when the electrons relax back into the ground state releases photons in an emission spectrum that has lower energy than the initial photons absorbed producing longer wavelengths than the absorption spectrum [21,22]. The increased stability of fluorescently labelled molecules [17], low hazard quotient in a clinical setting [23] and commercial availability of substrates has allowed successful implementation of fluorescent imaging in a wide array of applications including cancer typing, tracking drug metabolism and monitoring response to treatment [24,25]. ...
Tuberculosis remains one of the greatest challenges to global health, making the development of novel diagnostics and therapeutics for tuberculosis a high priority. However, the unique cause, Mycobacterium tuberculosis, demonstrates a number of characteristics that have hindered progress in tuberculosis research. These challenges include an unusually slow growth rate that makes traditional microbiological methods time consuming, a unique glycolipid-rich cell wall that causes bacterial aggregation and complicates enumeration of bacterial loads, and a highly variable disease progression including both acute and chronic stages of infection that can complicate in vivo studies due to variation between infected animals. One strategy that has proven to be remarkably successful in overcoming these challenges is the application of in vivo optical imaging to the study of M. tuberculosis. This approach allows the progress of an infection to be followed in individual animals over time, enabling researchers to better understand this important pathogen and assay new vaccines, treatments, and diagnostic tests more accurately. In this chapter, we discuss the techniques and tools that have been developed to facilitate application of bioluminescent and fluorescent in vivo imaging to tuberculosis research. We also summarize the progress and potential contributions of real-time imaging to the tuberculosis field. Based on recent progress, optical imaging has the potential to transform the field, leading to more rapid discovery of therapeutics, vaccines and mechanisms of pathogenesis.
... 1,2 To this end, an image sensor is required; however, typical image sensors are insensitive or have shown low sensitivity to the UVA radiation. 3 Thereby, increasing detection sensitivity is a priority. By coating the sensitive area with an appropriate phosphor, the image sensor response in the UV range of interest may be improved. ...
... LP SR method adopts 3 levels as decomposition scheme. NSCT FUZZY method adopts the filter [1], [1], [3] used in NSCT. NSST PCNN method performs in YUV color space. ...
... Optical imaging techniques have high-resolution and realtime modalities (Luo, Zhang, Su, Cheng, & Shi, 2011). These techniques are noninvasive and can be used to obtain information about anatomical structure, apart from the metabolism and biochemistry of disease tissues such as tumors (Choy, Choyke, & Libutti, 2003;Luo et al., 2011). ...
Developments in fluorescence imaging, brought popularity to near infrared (NIR) imaging with far‐red and NIR fluorophores applied for biosensing and bioimaging in living systems. Noninvasive NIR imaging gained popularity with the use of effective NIR dyes to obtain macroscopic fluorescence images. Several attributes of NIR dyes make them desirable agents, including high specificity, high sensitivity, minimized background interference, and the ability to easily conjugate with drug delivery systems. However, NIR dyes have some drawbacks and limitations, such as low solubility, low stability, and degradation. To overcome these issues, NIR dyes can be encapsulated in appropriate nanocarriers to achieve effective diagnosis, imaging, and therapy monitoring during surgery. Moreover, the vast majority of NIR dyes have photosensitizer features that can effectuate cancer treatment referred to as photodynamic therapy (PDT). In the near future, by combining NIR dyes with appropriate nanocarriers such as liposomes, polymeric micelles, polymeric nanoparticles, dendrimers, quantum dots, carbon nanotubes, or ceramic/silica based nanoparticles, the limitations of NIR dyes can be minimized or even effectively eliminated to form potential effective agents for imaging, therapy, and therapy monitoring of several diseases, particularly cancer.
... The in vivo determination of optical sources has been an area of extensive research [1][2][3][4][5][6][7][8][9][10][11]. One major obstacle is that when optical sources are small, weak and deep in a living tissue they cannot be effectively imaged on the body surface, due to the heterogeneous anatomy, strong absorption, scattering, and so on. ...
Acomputational fiberscope systemhas been designed and developed to detect and quantify an optical source deep in a homogeneousmediumby quickly determining tissue optical properties (absorption coefficient ma and transport scattering coefficient m).Our systemdetermined the optical properties ma and m with average difference of 2.0%and 14.8%between this study and reference. Location of source is 0.15 cmfromreference position. Diffuse light was collected by an optical fiber based fiberscope with a detectingwindowat the end of it. The position and orientation of the detectingwindowof the fiberscope were controlled by a 3Dtracking systemand a rotary stage.Duringmeasurements, the detected signals were recorded using a high-speed data acquisition system. To test the ability of this algorithmto accurately reconstruct the features of an optical source deep in a homogeneous tissuemedium, measurements were performed in a tissue simulating phantom(an aqueous suspension of 5.5 L 3% Liposyn-10%).The currentmethod is effective for samples whose optical properties satisfy the requirement of the diffusion approximation.Our results indicate that the customdesigned fiberscope systemhas a potential for tumor sensing in fluorescent applications on patients.
... It hence provides molecular information complementary to CT for tumor detection and allows evaluation of tumor treatment response. Compared with other molecular imaging modalities such as positron emission tomography and single photon emission CT, optical molecular imaging is more affordable, is portable, and is free of ionizing radiation (9). Our group has developed an image guided small animal arc radiation treatment (iSMAART) platform that integrated CBCT, bioluminescence tomography (BLT), and fluorescence molecular tomography for tumor detection and image guided RT (4,10,11). ...
Purpose
s: The image-guided SMall Animal Arc Radiation Treatment platform (iSMAART) has adopted onboard cone beam computed tomography (CBCT) and bioluminescence tomography (BLT). In this study, we used BLT to guide radiation delivery and quantitatively assess radiation-induced tumor response.
Methods and Materials
BLT was first validated on a tissue-simulating phantom, where the internal chemiluminescent liquid had a constant volume while its luminescence intensity gradually decayed. Then, in vivo experiments were performed on BALB/c mice orthotopically inoculated with 4T1 breast carcinoma cells expressing luciferase. Animals received either radiation treatment (RT group, n=9) or not (Control group, n=9). BLT was used to guide delivery of a single fraction of 5 Gy radiation dose to the tumor, and to evaluate the treatment response. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the radiation-induced DNA damage and cell apoptosis.
Results
Phantom results showed that BLT not only recovered the constant target volume with <2% deviation, but also accurately monitored the decay of the chemiluminescent molecules. For the RT animal group, there was significant reduction in both BLT-based tumor volume (21±10%, P=0.001) and bioluminescence intensity (48±17%, P=0.0008). For the control group, the significant increase was detected in the BLT tumor volume (35±12%, P<0.0001), but not in the BLT bioluminescence intensity (P=0.4). There was a significant difference in the BLT tumor volume between the RT and control group 7 days after radiation (P=0.03). Regression analysis suggests a strong correlation between the BLT and CBCT tumor volume (R²=0.93). The TUNEL staining analysis showed a significant difference in tumor cell apoptosis between the RT and control group (20.6±2.9% vs 3.2±1.7%, P<0.05).
Conclusion
BLT onboard the iSMAART can be used to accurately guide radiation delivery, and to quantitatively assess treatment response by simultaneously monitoring tumor volume and cancer cell population.
... Bioluminescence is the emission of light by a chemiluminescent reaction involving light-generating enzymes called luciferases, which are a large family of enzymes that catalyze the oxidation of a substrate, luciferin, into oxyluciferin, with the concomitant production of light [19,20]. At the cellular level, this light can be captured and detected using an extremely sensitive cooled chargecoupled device (CCD) camera or a photomultiplier. ...
Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.
... Many reporter proteins are used for optical imaging [34,35]. One such protein, Renilla luciferase, produces luminescence in the presence of substrates such as coelenterazine. ...
In a previous study, we developed an E1 monobody specific for the tumor biomarker hEphA2 [PLoS ONE (2015) 10(7): e0132976]. E1 showed potential as a molecular probe for in vitro and in vivo targeting of cancers overexpressing hEphA2. In the present study, we constructed expression vectors for E1 conjugated to optical reporters such as Renilla luciferase variant 8 (Rluc8) or enhanced green fluorescent protein (EGFP) and purified such recombinant proteins by affinity chromatography in E. coli. E1-Rluc8 and E1-EGFP specifically bound to hEphA2 in human prostate cancer PC3 cells but not in human cervical cancer HeLa cells, which express hEphA2 at high and low levels, respectively. These recombinant proteins maintained >40% activity in mouse serum at 24 h. In vivo optical imaging for 24 h did not detect E1-EGFP signals, whereas E1-Rluc8 showed tumor-specific luminescence signals in PC3 but not in HeLa xenograft mice. E1-Rluc8 signals were detected at 4 h, peaked at 12 h, and were undetectable at 24 h. These results suggest the potential of E1-Rluc8 as an EphA2-specific optical imaging agent. © 2017 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Luciferases are a large family of light-generating enzymes that catalyze the oxidation of a substrate, generically called luciferin, to yield oxyluciferin with the concomitant production of light. For in vivo bioluminescence imaging of malignancy, tumor cells or cancer-related genes are tagged with a reporter gene that encodes a light-generating enzyme, luciferase [14][15][16]. When this reporter is in the presence of the substrate it emits a blue to yellow-green light with an emission spectra peaking at a wavelength between 490 and 620 nm [14]. ...
Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.
... All these methodologies are capable of 3D imaging of tumour sites. However the radioisotopes based techniques have high spatial resolution and penetratability in the tissue but these are very costly and time consuming where optical imaging is comparatively cost effective, rapid and easy to perform (Choy et al. 2003). Also, Near Infrared (NIR) fluorescence imaging is a potential approach for the non-invasive diagnosis of cancer which makes use of various fluorescent dyes such as Cy.5 and cy75 active in the NIR region . ...
... OI allows threedimensional reconstruction of optical signals in human body using diffuse optical tomography. These technological advances in imaging helps in investigating the molecular functions of tumors [51] [58]. ...
The visualization of drugs in living systems has become key techniques in modern therapeutics. Recent advancements in optical imaging technologies and molecular design strategies have revolutionized drug visualization. At the subcellular level, super-resolution microscopy has allowed exploration of the molecular landscape within individual cells and the cellular response to drugs. Moving beyond subcellular imaging, researchers have integrated multiple modes, like optical near-infrared II imaging, to study the complex spatiotemporal interactions between drugs and their surroundings. By combining these visualization approaches, researchers gain supplementary information on physiological parameters, metabolic activity, and tissue composition, leading to a comprehensive understanding of drug behavior. This review focuses on cutting-edge technologies in drug visualization, particularly fluorescence imaging, and the main types of fluorescent molecules used. Additionally, we discuss current challenges and prospects in targeted drug research, emphasizing the importance of multidisciplinary cooperation in advancing drug visualization. With the integration of advanced imaging technology and molecular design, drug visualization has the potential to redefine our understanding of pharmacology, enabling the analysis of drug micro-dynamics in subcellular environments from new perspectives and deepening pharmacological research to the levels of the cell and organelles.
We present a methodology for a high-throughput screening (HTS) of transcription factor libraries, based on bacterial cells and GFP fluorescence. The method is demonstrated on the Escherichia coli LysR-type transcriptional regulator YhaJ, a key element in 2,4-dinitrotuluene (DNT) detection by bacterial explosives’ sensor strains. Enhancing the performance characteristics of the YhaJ transcription factor is essential for future standoff detection of buried landmines. However, conventional directed evolution methods for modifying YhaJ are limited in scope, due to the vast sequence space and the absence of efficient screening methods to select optimal transcription factor mutants. To overcome this limitation, we have constructed a focused saturation library of ca. 6.4 × 107 yhaJ variants, and have screened over 70 % of its sequence space using fluorescence-activated cell sorting (FACS). Through this screening process, we have identified YhaJ mutants exhibiting superior fluorescence responses to DNT, which were then effectively transformed into a bioluminescence-based DNT detection system. The best modified DNT reporter strain demonstrated a 7-fold lower DNT detection threshold, a 45-fold increased signal intensity, and a 40 % shorter response time compared to the parental bioreporter. The FACS-based HTS approach presented here may hold a potential for future molecular enhancement of other sensing and catalytic bioreactions.
Objective
The present research aimed to analyze the application of indocyanine green (ICG) fluorescence contrast technique in the resection of hepatoblastoma (HB) in children, and to discuss the use of ICG in the surgery of HB and the value of guidance.
Methods
We retrospectively analyzed the data of 23 children with HB resected using ICG fluorescence contrast technique at the Children’s Hospital of Nanjing Medical University from June 2020 to September 2022, including 16 boys and 7 girls, aged 5 days to 80 months. The patients were administered with an ICG injection of 0.1 mg/kg around 24–48 h before surgery. The surgical margin was detected by real-time fluorescence imaging and confirmed by postoperative pathology.
Results
All primary lesions showed bright fluorescence in 23 HB cases. 22 had clear borders with normal liver tissue, while one neonatal case showed no difference between tumor and background. 13 anatomic resection and 10 non-anatomic resection were performed with ICG fluorescence navigation. The surface of the residual liver was scattered with multiple tumor fluorescence, which was then locally enucleated according to the fluorescence. 22 isolated specimens were dissected and fluorescently visualized. Pathology identified deformed, vacuolated and densely arranged hepatocytes resembling pseudo-envelope changes without tumor residual, due to the compression of the tissue at the site of circumferential imaging.
Conclusion
The ring ICG fluorescence imaging of HB indicates the tumor resection boundary effectively, especially in multiple lesions cases.
Indocyanine green (ICG) is one of the FDA-approved near infra-red fluorescent (NIRF) probes for cancer imaging and image-guided surgery in the clinical setting. However, the limitations of ICG include poor photostability, high concentration toxicity, short circulation time, and poor cancer cell specificity. To overcome these hurdles, we engineered a nanoconstruct composed of poly (vinyl pyrrolidone) (PVP)-indocyanine green that is cloaked self-assembled with tannic acid (termed as indocyanine green-based glow nanoparticles probe, ICG-Glow NPs) for the cancer cell/tissue-specific targeting. The self-assembled ICG-Glow NPs were confirmed by spherical nanoparticles formation (DLS and TEM) and spectral analyses. The NIRF imaging characteristic of ICG-Glow NPs was established by superior fluorescence counts on filter paper and chicken tissue. The ICG-Glow NPs exhibited excellent hemo and cellular compatibility with human red blood cells, kidney normal, pancreatic normal, and other cancer cell lines. An enhanced cancer-specific NIRF binding and imaging capability of ICG-Glow NPs was confirmed using different human cancer cell lines and human tumor tissues. Additionally, tumor-specific binding/accumulation of ICG-Glow NPs was confirmed in MDA-MB-231 xenograft mouse model. Collectively, these findings suggest that ICG-Glow NPs have great potential as a novel and safe NIRF imaging probe for cancer cell/tumor imaging. This can lead to a quicker cancer diagnosis facilitating precise disease detection and management.
It is essential to be aware of the advances in the nutritional aspects of cancer for oncology practitioners. Unlike the popular myth, the term “Cachexia” more effectively defines the present status in oncology patients rather than “cancer-related malnutrition” (CRM). The consensus guidelines recommend the usage of management algorithm, staging of the CRM involving screening for the nutritional risk and also devising a management strategy consistent with a phenotype and the staging. There should be an inclusion of preventive measures for the CRM in the management algorithm like pharmacological and non-pharmacological interventions aiming both anabolic and the anti-catabolic measures as well as repeated checking of the status of Nutrition. At present, the multimodal mechanism is the optimum method to battle catabolic resulting in CRM. This method should be simultaneously given with anticancer treatments and involve pharmaconutrients, nutritional intervention and the multiple target drug treatments and physical activity. This article provides the practitioners with an awareness of the recent development in the area of nutritional care and as well as defined guidelines to regulate cancer therapy during treatment. This book chapter thus gives insightful guidelines pointing on crucial aspects of Nutrition for cancer treatment.
Near-infrared II (NIR-II) imaging at 1100–1700 nm supports deep tissue penetration, low auto-fluorescence interruption, and high imaging resolution due to advances in attenuating photon scattering and absorption. For example, semiconductor quantum dots and carbon nanotubes as pioneer molecular probes were used for hind limbs and cerebrovascular imaging, and organic small molecules and polymers were employed for ultrafast and three-dimensional tumor vasculature imaging. Compared with the reported NIR-II fluorescence probe systems, the atomic-precision metal clusters with unambiguous geometry structure can make the most of the spatial coordination of metal atoms by artificial atom manipulation, thus improving the luminescent quantum yield with tunable emission wavelength. Meanwhile, the water soluble, renal cleanable, and highly stable features make them promising candidates for in vivo imaging. In this review, we aim to summarize the molecular and electronic structure, and optical properties of NIR-II emissive metal clusters. Meanwhile, we highlight their applications in brain, kidney, gastrointestinal imaging and tumor metastasis monitoring. We particularly reviewed recent advances on the biosafety of gold clusters at ultrahigh concentration. Present work will promote the development of NIR-II imaging and make a step towards the clinical application.
Molecular imaging technology enables us to observe the physiological or pathological processes in living tissue at the molecular level to accurately diagnose diseases at an early stage. Optical imaging can be employed to achieve the dynamic monitoring of tissue and pathological processes and has promising applications in biomedicine. The traditional first near-infrared (NIR-I) window (NIR-I, range from 700 to 900 nm) imaging technique has been available for more than two decades and has been extensively utilized in clinical diagnosis, treatment and scientific research. Compared with NIR-I, the second NIR window optical imaging (NIR-II, range from 1000 to 1700 nm) technology has low autofluorescence, a high signal-to-noise ratio, a high tissue penetration depth and a large Stokes shift. Recently, this technology has attracted significant attention and has also become a heavily researched topic in biomedicine. In this study, the optical characteristics of different fluorescence nanoprobes and the latest reports regarding the application of NIR-II nanoprobes in different biological tissues will be described. Furthermore, the existing problems and future application perspectives of NIR-II optical imaging probes will also be discussed.
Purpose
Tumors exhibit areas of decreased oxygenation due to malformed blood vessels. This low oxygen concentration decreases the effectiveness of radiation therapy, and the resulting poor perfusion can prevent drugs from reaching areas of the tumor. Tumor hypoxia is associated with poorer prognosis and disease progression and is therefore of interest to preclinical researchers. Although there are multiple different ways to measure tumor hypoxia and related factors, there is no standard for quantifying spatial and temporal tumor hypoxia distributions in preclinical research or in the clinic. This review compares imaging methods utilized for the purpose of assessing spatio-temporal patterns of hypoxia in the preclinical setting. Conclusions: Imaging methods provide varying levels of spatial and temporal resolution regarding different aspects of hypoxia, and with varying advantages and disadvantages. The choice of modality requires consideration of the specific experimental model, the nature of the required characterisation and the availability of complementary modalities as well as immunohistochemistry.
In addition to critical biological roles, DNA is also considered an excellent engineering material with unique characteristics of high programmability and addressability. Recent advances in DNA nanotechnology enable the rational design and precise fabrication of various exquisite DNA nanostructures, providing many new opportunities for technological uses. Meanwhile, the integration of DNA nanostructures with molecular recognition capacity can further expand the scope of their application. Particularly suited to this goal, aptamers are short single-stranded nucleic acids selected via an in vitro strategy called systematic evolution of ligands by exponential enrichment. As analogs to antibodies, aptamers can bind to various targets, such as small molecules, proteins, viruses, bacteria, and even whole cells. Apart from comparable specificity and affinity as antibodies, aptamers have several advantages, including small molecular weight, high designability, easy synthesis, and convenient modification. Indeed, the marriage of aptamers with DNA nanostructures has generated many powerful tools for advanced biological and biomedical applications. In this review, we summarize the recent progress, application, and prospect of aptamer-integrated DNA nanotechnology in the fields of biosensing, bioimaging, targeted drug delivery, bioregulation, and biomimicry.
This review explores recent work directed toward the development of nanoparticles (NPs) for multimodality cancer imaging and targeted cancer therapy. In the growing era of precision medicine, theranostics, or the combined use of targeted molecular probes in diagnosing and treating diseases is playing a particularly powerful role. There is a growing interest, particularly over the past few decades, in the use of NPs as theranostic tools due to their excellent performance in receptor target specificity and reduction in off‐target effects when used as therapeutic agents. This review discusses recent advances, as well as the advantages and challenges of the application of NPs in cancer imaging and therapy.
Up-converting nanoparticles are dielectric crystalline particles doped with rare-earth ions such as Yb3⁺, Er3⁺, Tm3⁺, Ho3⁺, Nd3⁺, etc. When excited in infrared, they emit visible radiation. Used as markers, they present significant advantages in comparison to traditional fluorophores: sharp emission lines, superior photostability, resistence to photobleaching, no blinking and lack of toxicity. Infrared radiation is less harmful to cells avoiding tissue degradation, minimizes auto-fluorescence from endogenous biocomponents offering a good signal-to-background ratio and penetrates tissues deeply. In spite of the great advantages of using up-converting nanoparticles for biomedical applications, there are still some limitations. These refer to identification of optimal size suited for specific samples, prevention of aggregation, water stability/ dispersibility, optical efficiency and biocompatibility. This chapter reviews principal characteristics of up-converting nanoparticles and issues related to their use in biomedical applications.
In the recent years, the number of publications including small animal imaging increased. Nevertheless, most publications only report on proof of principle studies, and there is still uncertainty how small animal imaging can support preclinical research most effectively. It is also discussed controversially which imaging modality should be used for a certain demand. Thus, it is the aim of this book chapter to support the reader in choosing the optimal imaging modality for his research. First, the demands on imaging are defined, hereby distinguishing between imaging of morphology, vascular function, metabolism and molecular markers. In this regard it is also considered whether imaging of single or multiple lesions and phenotyping of animals are intended and whether the lesion lays superficially or in the abdomen, chest or brain. Next, the time effort required to scan an animal is discussed, which can be a significant obstacle in complex research projects that require a high number of study groups and animals. The last part focuses on the use of targeted molecular imaging. Here, different imaging modalities may be chosen if it is intended to evaluate binding affinity of a new ligand, to assess the biodistribution of a molecule, or to indirectly monitor therapy response. A single imaging modality will not be able to fulfil all these requirements, and as a consequence, a small animal imaging centre should host different imaging applications that can be used as complementary. Even more, for many purposes image fusion or dual-modality scanners may be required.
A facile ruthenium(II)-catalyzed regiospecific C–H/O–H oxidative annulation methodology was developed to construct isochromeno[8,1-ab]phenazines. This methodology delivers various advantages, such as scope for diverse substrates, tolerance to a range of functional groups, stability under air, and yields regioselective products. This methodology was successfully applied to synthesize far red (FR) fluorescent probes for live cancer cell imaging. The synthesized compounds displayed notable fluorescence properties in solution and thin-film. Their application in live cancer cell imaging was investigated using various cancer cell lines. The synthesized compound showed prominent FR fluorescence, with high quantum yield, and exhibited better cell-imaging properties, with excellent biocompatibility.
Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phosphoserine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.
Up-converting nanoparticles are dielectric crystalline particles doped with rare-earth ions such as Yb3+, Er3+, Tm3+, Ho3+, Nd3+, etc. When excited in infrared, they emit visible radiation. Used as markers, they present significant advantages in comparison to traditional fluorophores: sharp emission lines, superior photostability, resistence to photobleaching, no blinking and lack of toxicity. Infrared radiation is less harmful to cells avoiding tissue degradation, minimizes auto-fluorescence from endogenous biocomponents offering a good signal-to-background ratio and penetrates tissues deeply. In spite of the great advantages of using up-converting nanoparticles for biomedical applications, there are still some limitations. These refer to identification of optimal size suited for specific samples, prevention of aggregation, water stability/dispersibility, optical efficiency and biocompatibility. This chapter reviews principal characteristics of up-converting nanoparticles and issues related to their use in biomedical applications.
We report a class of multi-functional core-shell nanoarchitectures, consisting of silica nanospheres as the core and Gd2O3:Dy(3+) nanocrystals as the ultra-thin shell, that enable unique multi-color living cell imaging and remarkable in vivo magnetic resonance imaging. These types of targeted cell imaging nanoarchitectures can be used as a variety of fluorescence nanoprobes due to the multi-color emissions of the Gd2O3:Dy(3+) nanophosphor. We also proposed a strategy of modulating core-shell structure design to achieve an enhanced magnetic resonance contrast ability of Gd2O3 nanoagents, and the classical Solomon-Bloembergen-Morgan theory was applied to explicate the mechanism underlying the enhancement. The as-synthesized ligand-free nanomaterial possesses a suitable particle size for cellular uptake as well as avoiding penetrating the blood-brain barrier with good water-solubility, stability, dispersibility and uniformity. The extremely low cytotoxicity and favorable biocompatibility obtained from in vitro and in vivo bioassays of the as-designed nanoparticles indicate their excellent potential as a candidate for functioning as a targeted nanoprobe.
This chapter highlights several theranostic nanoplatforms based on different imaging modalities with great potential as cancer theranostics. It discusses the developments and applications of near-infrared (NIR) fluorescence imaging (FLI) and bioluminescence imaging (BLI) used for optical image-guided cancer diagnosis and treatment. Nuclear imaging is a noninvasive imaging technique producing images by detecting radiation from different parts of the body after a very low dose of radioactive tracer material is administered. In contrast with imaging techniques, such as MRI and CT, that mainly show anatomy information, nuclear imaging can provide quantitative functional information about normal tissues or disease conditions at the molecular and cellular levels. Molecular imaging is now playing a key role in drug discovery at the preclinical level, clinical diagnosis, and treatment of many diseases. Multimodality imaging has demonstrated promising prospects in tumor detection or therapy. The chapter introduces the application of several multimodality-imaging techniques in cancer theranostics.
The early detection of esophageal squamous cell carcinoma (ESCC), one of the most common human neoplasms, is of great importance in improving prognosis. However, there is a lack of screening methods with high sensitivity for early diagnosis of ESCC. In this study, we developed a αvβ3 integrin targeted near-infrared (NIR) fluorescence nanoprobe by conjugation of NIR dye Cy5.5 and tumor vasculature targeted cyclic RGDfK peptide onto a polyamidoamine (PAMAM) dendrimer (RGD–PEG–PAMAM–Cy5.5). The applicability of the as-prepared targeted nanoprobe for NIR fluorescence imaging of 4-NQO induced large and tiny esophageal neoplasms in mice was examined. It was demonstrated that the NIR fluorescence imaging with our targeted nanoprobe enables the identification of ESCC at an early stage.
Herein, hollow-structured Gd2O3[ ]:[ ]RE3+/Yb3+ (RE = Er, Ho, Tm) nanoparticles (NPs) were prepared via a urea-based chemical coprecipitation method followed by subsequent calcination and etching. Under 980 nm near-infrared (NIR) irradiation, upconversion (UC) emission gains the highest intensity of red, green and blue peaks for Gd2O3[ ]:[ ]Er3+/Yb3+, Gd2O3[ ]:[ ]Ho3+/Yb3+ and Gd2O3[ ]:[ ]Tm3+/Yb3+ NPs respectively. The corresponding fluorescence in-cell images exhibit bright visible light. The continuous color-tunable UC emission of each spectrum was investigated by increasing the concentration of the sensitizer Yb3+ ions from 0 to 20 mol% with the most intense red, green and blue emission achieved. The structure, morphology, components and magnetic property of Gd2O3[ ]:[ ]Ho3+/Yb3+ NPs were investigated intensively. The results show that the NPs possess a distinctly hollow structure and uniform spherical shape, and are well-crystallized and highly monodispersed with a mean diameter of 118 nm. Due to the designed hollow structure which is of benefit to water contact of a large number of Gd3+ on the surface, the measured T1 relaxivity of the NPs is nearly 5 times larger than the relaxivity of the commercial gadolinium diethylenetriaminepentaacetate (Gd-DTPA). The relaxation enhancement in the hollow-structured Gd2O3[ ]:[ ]RE3+/Yb3+ (RE = Er, Ho, Tm) NPs is addressed in the framework of Solomon–Bloembergen–Morgan theory. The biocompatibility studies in 293 and HeLa cells indicate that the hollow NPs have no observable cytotoxicity at a concentration up to 200 μg ml−1. All these results demonstrate that the hollow-structured nanocomposite has the potential to be developed into a safe and highly efficient magnetic resonance and fluorescence nanoprobe for in-cell molecular imaging of cancer.
Whole-body optical imaging of small animals has emerged as a powerful, user friendly, and high-throughput tool for assaying molecular and cellular processes as they occur in vivo. As with any imaging method, the utility of such technology relies on its ability to provide quantitative, biologically meaningful information about the physiologic or pathologic process of interest. Here we used an animal tumor model to evaluate the extent of correlation between noninvasively measured fluorescence and more traditional measurements of biomass (tumor volume and tumor weight). C57/BL6 mice were injected subcutaneously with murine colon adenocarcinoma cells that were engineered to express GFP. Serial measurements of fluorescence intensities were performed with a macroscopic in vivo fluorescence system. The progressive increases in intensity correlated strongly with growth in tumor volume, as determined by caliper measurements (R2 = 0.99). A more stringent correlation was found between fluorescence intensity and tumor weight (R2 = 0.97) than between volume and weight (R2 = 0.89). In a treatment experiment using tumor necrosis factor-alpha, fluorescence intensity (but not tumor volume) was able to differentiate between treated and control groups on day 1 post-treatment. These results validate the ability of noninvasive fluorescent imaging to quantify the number of viable, fluorescent cells in vivo.
Quantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooper et al ., 1990). A brief review of low‐light‐level imaging is presented, with particular reference to charge‐coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photoncounting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular highsensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguez et al. , 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non‐infected cells. The rapid detection of luciferase‐expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles.
This direct imaging technique is simple and versatile. It offers a rapid, non‐invasive method for the sensitive detection of luciferase activity in single, luciferase‐expressing cells. One can envisage the use of luciferase as a sensitive and convenient co‐selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and cellular biology.
Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life science. These systems use advances in camera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in imageprocessing hardware and software have increased the usefulness of these systems in the biosciences.
Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).