Near-infrared quantum dots for deep tissue imaging

Developments in nanotechnology have paved the way for the early detection, treatment, and prevention of several tumors which affect mankind. In the past few years, near-infrared (NIR) fluorescence imaging techniques have emerged that enable the in vivo imaging of physiological, metabolic, and molecular function. The NIR window, also known as the diagnostic window (700–900 nm), can be explored for sensitive detection techniques. Nanoparticles, particularly semiconductor quantum dots (QDs), can be utilized for the purpose of optical imaging. These semiconductor QDs possess novel electronic, optical, magnetic, and structural properties which are quite different from those of bulk materials. NIR QDs with these unique properties can be utilized as contrast agents for optical imaging, particularly for deep tissue imaging. Deep tissue imaging provides more information about the pathological status of the disease, which makes the treatment more effective and efficient. In this review we highlight the importance of NIR QDs as probes for optical imaging. We describe the different types of NIR QDs, their synthesis, and their application for deep tissue imaging along with recently developed self-illuminating NIR QDs.     

Visible light induced photoelectrochemical biosensing based on oxygen-sensitive quantum dots

A visible light induced photoelectrochemical biosensing platform based on oxygen-sensitive near-infrared quantum dots (NIR QDs) was developed for detection of glucose. The NIR QDs were synthesized in an aqueous solution, and characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. The as-prepared NIR QDs were employed to construct oxygen-sensitive photoelectrochemical biosensor on a fluorine-doped tin oxide (FTO) electrode. The oxygen dependency of the photocurrent was investigated at as-prepared electrode, which demonstrated the signal of photocurrent is suppressed with the decreasing of oxygen. Coupling with the consumption of oxygen during enzymatic reaction, a photoelectrochemical strategy was proposed for the detection of substrate. Using glucose oxidase (GOx) as a model enzyme, that is, GOx was covalently attached to the surface of CdTe QDs, the resulting biosensor showed the sensitive response to glucose. Under the irradiation of visible light of a wavelength at 505 nm, the proposed photoelectrochemical method could detect glucose ranging from 0.1 mM to 11 mM with a detection limit of 0.04 mM. The photoelectrochemical biosensor showed a good performance with high upper detection limit, acceptable stability and accuracy, providing an alternative method for monitoring biomolecules and extending the application of near-infrared QDs.     

Near-infrared fluorescent molecular probes for imaging and diagnosis of nephro-urological diseases

Near-infrared (NIR) fluorescence imaging has improved imaging depth relative to conventional fluorescence imaging in the visible region, demonstrating great potential in both fundamental biomedical research and clinical practice. To improve the detection specificity, NIR fluorescence imaging probes have been under extensive development. This review summarizes the particular application of optical imaging probes with the NIR-I window (700–900 nm) or the NIR-II window (1000–1700 nm) emission for diagnosis of nephron-urological diseases. These molecular probes have enabled contrast-enhanced imaging of anatomical structures and physiological function as well as molecular imaging and early diagnosis of acute kidney injury, iatrogenic ureteral injury and bladder cancer. The design strategies of molecular probes are specifically elaborated along with representative imaging applications. The potential challenges and perspectives in this field are also discussed.

Near-infrared fluorescent molecular probes with improved imaging depth and optimized biodistribution have been reviewed, showing great potential for diagnosis of nephro-urological diseases.     

Design of Photostable,Activatable Near‐Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Near‐infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water‐soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase‐labile or an H₂O₂‐labile group. The photoacoustic signals of these hydroxy‐masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H₂O₂, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.     

Design of Photostable,Activatable Near‐Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Near‐infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water‐soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase‐labile or an H₂O₂‐labile group. The photoacoustic signals of these hydroxy‐masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H₂O₂, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.     

A novel aptamer functionalized CuInS2 quantum dots probe for daunorubicin sensing and near infrared imaging of prostate cancer cells

In this paper, a novel daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS₂ quantum dot (DNR–MUC1–QDs) conjugates were developed, which can be used as a targeted cancer imaging and sensing system. After the NIR CuInS₂ QDs conjugated with the MUC1 aptamer–(CGA)₇, DNR can intercalate into the double-stranded CG sequence of the MUC1–QDs. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of DNR on these conjugates. DNR–MUC1–QDs can be used to target prostate cancer cells. We evaluated the capacity of MUC1–CuInS₂ QDs for delivering DNR to cancer cells in vitro, and its binding affinity to MUC1-positive and MUC1-negative cells. This novel aptamer functionalized QDs bio-nano-system can not only deliver DNR to the targeted prostate cancer cells, but also can sense DNR by the change of photoluminescence intensity of CuInS₂ QDs, which concurrently images the cancer cells. The quenched fluorescence intensity of MUC1–QDs was proportional to the concentration of DNR in the concentration ranges of 33–88 nmol L⁻¹. The detection limit (LOD) for DNR was 19 nmol L⁻¹. We demonstrate the specificity and sensitivity of this DNR–MUC1–QDs probe as a cancer cell imaging, therapy and sensing system in vitro.     

Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm

Compared to imaging in the visible and near‐infrared regions below 900 nm, imaging in the second near‐infrared window (NIR‐II, 1000–1700 nm) is a promising method for deep‐tissue high‐resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single‐walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long‐wavelength NIR region (1500–1700 nm, NIR‐IIb). With this imaging agent, 3–4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood‐flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR‐IIb tumor imaging of a live mouse was explored. NIR‐IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high‐performance in vivo optical imaging.     

Characterization of CdHgTe/CdS QDs for near infrared fluorescence imaging of spinal column in a mouse model

Near infrared (NIR) CdHgTe/CdS quantum dots (QDs) were successfully prepared by a green synthetic route. The characteristics such as morphology, size, spectra, stability and toxicity were investigated in detail. The fluorescence wavelength of CdHgTe/CdS QDs could be adjusted to the NIR range (812nm), which made the in vivo NIR imaging possible. The in vivo dynamic biodistribution of CdHgTe/CdS QDs in a mouse model was monitored by an NIR imaging system. Results indicated that CdHgTe/CdS QDs with a diameter of about 5.8nm targeted to spinal column effectively. Further imaging of the dissected spine disclosed that QDs targeted to vertebra rather than spinal cord. The high fluorescence intensity together with targeting effect makes CdHgTe/CdS QDs particular candidates for imaging purposes in experimental animal models of vertebral injury.     

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of β-Galactosidase

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β-galactosidase (β-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon β-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of β-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing β-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.     

FSiNPs mediated improved double immunofluorescence staining for gastric cancer cells imaging

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen-antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.     

Hadamard transform fluorescence image microscopy using one-dimensional movable mask

With a 511-slit one-dimensional (1D) Hadamard mask and a highly sensitive linear charge-coupled device (CCD), spatial multiplexing is performed and a programmable Hadamard transform (HT) microscopic fluorescence imaging system was developed. The system can generate 511×512 pixel format images for small samples. Sensitivity, signal to noise ratio, imaging speed and spatial resolution of this system were discussed. The results show that the system can be applied for single-cell imaging sensitively in a short time. Spatial resolution up to 0.24 μm/pixel, which is close to the resolution limit of the conventional optical microscope, has been obtained under oil lens. The weak native fluorescence imaging for pollen cells can be realized within 1 min. The system has been applied for multi-parameter evaluation of tumor malignancy based on nuclear DNA ploidy measurements for one breast tumor specimen. The result indicates that the system has good application prospect in cell biology and medicine.     

NIR‐II Fluorescent Self‐Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis

Fluorescence‐guided cytoreductive surgery is one of the most promising approaches for facile elimination of tumors in situ, thereby improving prognosis. Reported herein is a simple strategy to construct a novel chainlike NIR‐II nanoprobe (APP‐Ag₂S‐RGD) by self‐assembly of an amphiphilic peptide (APP) into a nanochain with subsequent chemical crosslinking of NIR‐II Ag₂S QDs and the tumor‐targeting RGD peptide. This probe exhibits higher capability for cancer cell detection compared with that of RGD‐functionalized Ag₂S QDs (Ag₂S‐RGD) at the same concentration. Upon intraperitoneal injection, superior tumor‐to‐normal tissue signal ratio is achieved and non‐vascularized tiny tumor metastatic foci as small as about 0.2 mm in diameter could be facilely eliminated under NIR‐II fluorescent imaging guidance. These results clearly indicate the potential of this probe for fluorescence‐guided tumor staging, preoperative diagnosis, and intraoperative navigation.     

Cellular uptake and subcellular localization of highly luminescent silica-coated CdSe quantum dots--in vitro and in vivo

With excellent optical properties, quantum dots (QDs) have been made as attractive molecular probes for labeling cells in biological research. The purpose of the present work is to explore the possible role of silica-coated cadmium selenide (CdSe) QDs in the in vitro and in vivo cellular uptake and their subcellular localization. The in vitro uptake characteristics of silica-coated CdSe QDs were performed in cultured New Zealand rabbit adipose tissue-derived mesenchymal stem cells (RADMSCs) and Human cervical cancer cells (HeLa) using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). The in vitro results showed that the silica-coated CdSe QDs were efficiently taken up by the cells and it was localized in the intracellular vesicles giving strong fluorescence from the cytoplasm and nearby nucleus. Subsequently, the in vivo localization and distribution of QDs were studied by the hematoxilin stained semithin cryosections of tissues (~15 μm thickness) under fluorescence microscopy and ultrathin sections of tissues (~100 nm thickness) under confocal laser scanning microscopy at the distribution maxima. Our in vivo results confirmed the effective cellular uptake and even distribution pattern of QDs in tissues. Overall, these in vitro and in vivo results are represented with focus on internalization, subcellular localization and distribution of the QDs, in view of their potential applications in biomedical field.     

Emerging applications of near-infrared fluorescent metal nanoclusters for biological imaging

Recent advances in the development of near-infrared fluorescent metal nanoclusters for bioimaging applications have been thoroughly overviewed.     

All-in-one mitochondria-targeted NIR-II fluorophores for cancer therapy and imaging

Small-molecule subcellular organelle-targeting theranostic probes are crucial for early disease diagnosis and treatment. The imaging window of these molecules is mainly focused on the visible and near-infrared region (below ∼900 nm) which limits the tissue penetration depth and therapeutic effects. Herein, a novel NIR-II small-molecule probe H4–PEG-Glu with a thiopyrylium cation was synthesized. H4–PEG-Glu not only can quickly and effectively image mitochondria in acute myeloid leukemia (AML) cells, and induce G₀/G₁ phase arrest by the intrinsic mitochondrial apoptosis pathway w/o irradiation, but also exhibit moderate cytotoxicity against AML cancer cells in a dose dependent-manner without laser irradiation. The THP-1 cells treated with H4–PEG-Glu upon NIR laser irradiation showed enhanced chemo- and photothermal therapy (CPTT) with 93.07% ± 6.43 apoptosis by Annexin V staining. Meanwhile, H4–PEG-Glu displayed high synergistic CPTT effects in vivo, as well as specific NIR-II tumor imaging in AML patient derived PDX mouse models for the first time. Our work lays down a solid foundation for designing small-molecule NIR-II mitochondria-selective theranostic probes.

Small-molecule subcellular organelle-targeting theranostic probes are crucial for early disease diagnosis and treatment.     

Near-infrared fluorescence probes for surgical navigation

Optical imaging is a promising tool for visualizing fundamental biological processes including disease progression, detection of tumors, and therapeutic monitoring non-invasively. Unlike visible light, near-infrared fluorescence (NIRF) imaging (beyond 700–1,700 nm) offers a competitive advantage to yield high-resolution images within a certain penetration depth (few millimeters to centimeters depending on NIR window). The last few years have witnessed rapid development of new NIRF probes within the span of whole NIR window, including small-molecule dyes, inorganic nanoparticles, and organic macromolecules. Benefitted by this, we observe a continual surge in the number of preclinical and clinical studies of NIRF imaging in surgery and related applications. At present, NIRF-guided imaging has emerged as a quintessential procedure to assist surgeons for intraoperative delineation and resection of tumors. Moreover, NIRF imaging is also used to improve the intraoperative staging, identify the hidden lesion in diseased organs, map lymph node metastases, detect tumor margins, and highlight vital organs intraoperatively. Considering rapid advancement of this field, we review recent progress in the development of NIRF probes, cancer-targeting strategies and their application for surgical navigation, particularly for the sentinel lymph node mapping, detection of tumors, and angiography. Moreover, we spotlight surgical navigation instrumentation that is currently used for intraoperative tumor detection.     

DNA‐Templated Assembly of a Heterobivalent Quantum Dot Nanoprobe For Extra‐ and Intracellular Dual‐Targeting and Imaging of Live Cancer Cells

Quantum dots (QDs) hold great promise for the molecular imaging of cancer because of their superior optical properties. Although cell‐surface biomarkers can be readily imaged with QDs, non‐invasive live‐cell imaging of critical intracellular cancer markers with QDs is a great challenge because of the difficulties in the automatic delivery of QD probes to the cytosol and the ambiguity of intracellular targeting signals. Herein, we report a new type of DNA‐templated heterobivalent QD nanoprobes with the ability to target and image two spatially isolated cancer markers (nucleolin and mRNA) present on the cell surface and in the cell cytosol. Bypassing endolysosomal sequestration, this type of QD nanoprobes undergo macropinocytosis following the nucleolin targeting and then translocate to the cytosol for mRNA targeting. Fluorescence resonance energy transfer (FRET) based confocal microscopy enables unambiguous signal deconvolution of mRNA‐targeted QD nanoprobes inside cancer cells.     

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core–shell particles specifically recognizing glucuronic acid (GlcA) or N‐acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP‐GlcA and red QDs conjugated with MIP‐NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non‐imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.     

Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging

Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with l-glutathione and l-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine–glycine–aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer.