Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging

In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 μm diameter field of view through a 710 μm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 μm.     

A fast tumor‐targeting near‐infrared fluorescent probe based on bombesin analog for in vivo tumor imaging

Bombesin (BBN), an analog of gastrin‐releasing peptide (GRP), of which the receptors are over‐expressed on various tumor cells, is able to bind to GRP receptor specifically. In this study, a near‐infrared fluorescent dye (MPA) and polyethylene glycol (PEG) were conjugated to BBN analog to form BBN[7–14]–MPA and BBN[7–14]–SA–PEG–MPA. The successful synthesis of the two probes was proved by the characterization via sodium dodecylsulfate–polyacrylamide gel electrophoresis, infrared and optical spectra. Cellular uptakes studies indicated that BBN‐based probes were mediated by gastrin‐releasing peptide receptors (GRPR) on tumor cells and the PEG modified probe had higher affinity. The dynamic distribution and clearance investigations showed that the BBN‐based probes were eliminated by the liver–kidney pathway. Furthermore, both of the BBN‐based probes displayed tumor‐targeting ability in GRPR over‐expressed tumor‐bearing mice. The PEG modified probe exhibited faster and higher tumor targeting capability than BBN[7–14]–MPA. The results implied that BBN[7–14]–SA–PEG–MPA could act as an effective fluorescence probe for tumor imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo tomographic imaging of red-shifted fluorescent proteins

We have developed a spectral inversion method for three-dimensional tomography of far-red and near-infrared fluorescent proteins in animals. The method was developed in particular to address the steep light absorption transition of hemoglobin from the visible to the far-red occurring around 600 nm. Using an orthotopic mouse model of brain tumors expressing the red-shifted fluorescent protein mCherry, we demonstrate significant improvements in imaging accuracy over single-wavelength whole body reconstructions. Furthermore, we show an improvement in sensitivity of at least an order of magnitude over green fluorescent protein (GFP) for whole body imaging. We discuss how additional sensitivity gains are expected with the use of further red-shifted fluorescent proteins and we explain the differences and potential advantages of this approach over two-dimensional planar imaging methods.     

A novel ratiometric fluorescent probe for rapid detection of hydrogen peroxide in living cells

In this work, we present a new ratiometric fluorescent probe JNY-1 for rapid and convenient detection of H₂O₂. The probe could selectively and sensitively respond to H₂O₂ within 10 min. In addition, this probe was successfully applied for monitoring and imaging of H₂O₂ in liver cancer HepG2 cells under physiological conditions.

A new ratiometric fluorescent probe JNY-1 for sensitive detection of H₂O₂ is presented with selectivity over other reactive oxygen species, reactive nitrogen species, and biologically relevant species. Imaging of H₂O₂ in liver cancer HepG2 cells was achieved.     

自身猝灭探针定量PCR检测HCV方法的建立

目的 建立自身猝灭荧光探针定量PCR技术检测HCV方法.方法 构建重组质粒pMD18-T-HCV5'NCR作为标准品,并通过Sigma在线软件设计自身猝灭探针,优化定量PCR体系,并进行方法学评价.结果 建立了自身猝灭荧光探针的定量PCR方法,线性为101～1010copies/μl;灵敏度达50 copies/μl;批内CV为6.1%,批间CV为6.5%,日间CV为6.8%;特异性为100%,与其他肝炎病毒无交叉反应.与紫外定量方法比较,结果差异无显著性,且高度相关.结论 以自身猝灭荧光探针建立的HCV荧光定量PCR检测方法,灵敏度高、特异性强,为新型试剂盒的开发奠定了基础.     

Wide-field imaging of fluorescent deoxy-glucose in ex vivo malignant and normal breast tissue

Rapid in situ determination of surgical resection margins during breast cancer surgery would reduce patient time under anesthesia. We present preliminary data supporting the use of a fluorescent glucose analog (2-NBDG) as an optical contrast agent to differentiate freshly excised breast tissue containing cancerous cells from normal breast tissue. Multi-spectral images of 14 breast cancer specimens acquired before and after incubation with 2-NBDG demonstrated increased fluorescent signal in all of the malignant tissue due to increased 2-NBDG consumption. We demonstrate that 2-NBDG has potential as an optical contrast agent to differentiate cancerous from non-cancerous tissue.     

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored beta-glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.     

Near-infrared optical imaging of proteases in cancer

Near-infrared optical imaging is a newer imaging technique that, coupled with sensitive enzymatically specific fluorescent beacons, shows much promise for earlier detection of many cancers and their in situ characterization. On the basis of animal studies demonstrating visualization of micrometastasis-sized tumors and the ability to evaluate therapeutic enzyme inhibition real-time, such imaging may be incorporated in the clinical imaging paradigm in the future, both to improve cancer screening as well as for monitoring therapy in individual patients. This review details some of the related biology, optical probe design, and required hardware, with in vivo cathepsin and matrix metalloprotinease imaging used as examples.     

荧光定量PCR检测梅毒螺旋体方法的建立及初步应用

目的采用二聚体蝎型探针技术建立一种高敏感性和特异性的检测梅毒螺旋体(TP)的荧光定量聚合酶链反应(PCR)方法。方法根据TP特异性外膜蛋白Gpd的基因序列设计引物和荧光探针,采用基因工程技术构建可用于TP定量的标准品,优化荧光定量PCR的反应体系和反应条件,建立检测TP的二聚体蝎型探针定量PCR。采用本研究建立方法和商品化荧光定量PCR试剂盒检测40例临床标本,对比分析检测结果的统计学差异。结果成功构建了TP重组质粒标准品和TP的二聚体蝎型探针定量PCR,该方法线性范围为101~108 copy/mL,灵敏度为10copy/mL,特异性和敏感性均为100.0%;二聚体蝎型探针定量PCR对疑似梅毒病例阳性检出率显著高于目前商品化Taqman荧光定量PCR试剂盒(82.5%vs 62.5%,P0.05)。结论成功建立了二聚体蝎型探针荧光定量PCR快速检测TP的方法,为临床上TP的早期诊断和防控奠定了基础。     

Bioluminescence imaging in vivo - application to cancer research

Molecular events promoting tumourigenesis and anticancer therapeutic strategies have been intensively studied in tumour cell culture models. In the past few years, non-invasive bioluminescence imaging (BLI) has emerged as a powerful strategy for the validation of cell culture findings in animal models of cancer. BLI allows for repetitive and exceptionally sensitive real-time monitoring of a disease course, as well as of tumour response to therapeutic interventions in an individual animal. This review discusses the application of BLI to cancer research in general and to the area of experimental neuro-oncology in particular.     

荧光定量聚合酶链反应对生殖器疱疹病毒感染的检测

目的 探讨荧光定量聚合酶链反应（FQ-PCR）在诊断生殖器疱疹病毒感染中的应用。方法 本研究共分两组，检测组即就诊的疑似生殖器疱疹患者347例，对照组12例。分别用FQ-PCR方法检测两组送检标本中的单纯疱疹病毒（HSV）DNA。结果 347例疑似生殖器疱疹患者送检标本中，HSVDNA阳性139例，阳性率为40．1％（139／347），其中阳性标本中有皮损组织液75例、男性尿道拭子31例、女性宫颈分泌物33例，各类标本的阳性率依次为91．5％（75／82）、21．1％（31／147）、28．0％（33／118）。HSVDNA阳性人群中性别差异无统计学意义（P〉O．05，X^2检验）。12例对照标本中没有检出HSVDNA。结论 分泌物中检出的HSVDNA能直观地反映患者当前病毒感染状况，在有皮损组织的患者中，HSVDNA的检出率更高，FQ-PCR能准确、快速地对生殖器疱疹病毒感染做出诊断。     

Flow cytometric detection of human red cells labeled with a fluorescent membrane label: potential application to in vivo survival studies

In vivo survival studies of human red cells (RBCs) are commonly carried out by using chromium-51 (51Cr), a gamma-emitting radionuclide, as the cell label. The effects of labeling human RBCs with PKH-2, a nonradioactive lipophilic fluorescent dye that binds to the cell membrane, and the feasibility of detecting the labeled cells by flow cytometric analysis were investigated. Optimal labeling, defined as maximum mean fluorescence intensity with minimal cell-to-cell variability in fluorescence intensity and minimal cell loss, was achieved with the use of 15.0 x 10(-6) M (15.0 x 10(-6) mol/L) PKH-2 and a cell concentration of 4.0 x 10(9) RBCs per mL. Both freshly drawn and stored RBCs could be labeled, but RBCs stored for more than 20 days did not take up the label as uniformly as fresher cells. Although labeling with PKH-2 did not interfere with the detection of ABO, Rh(D), or common minor RBC antigens by routine serologic methods, it resulted in a morphologic appearance resembling echinocytosis and an increased resistance to osmotic lysis by hypotonic saline. RBCs labeled by this method could be quantitated accurately in blood samples in which their proportion was 0.01 percent, or 1 labeled cell in 10,000 cells. This method holds promise as a simple, reliable, and sensitive method for the detection of labeled human RBCs, but the in vivo significance of the label's effects on cell morphology and osmotic fragility is not known. Further studies directly comparing PKH-2-labeled and 51Cr-labeled RBCs will be necessary to establish the accuracy of the former method in determining the in vivo survival of human RBCs.     

A highly sensitive and selective fluorescent probe for quantitative detection of Al3+ in food,water, and living cells

Three novel β-pinene-based fluorescent probes 2a–2c were designed and synthesized for the selective detection of Al³⁺. Probe 2a showed higher fluorescence intensity toward Al³⁺ than the other two compounds. Probe 2a determined the concentration of Al³⁺ with a rapid response time (45 s), wide pH range (pH = 1–9), excellent sensitivity (LOD = 8.1 × 10⁻⁸ M) and good selectivity. The recognition mechanism of probe 2a toward Al³⁺ was confirmed by ¹H NMR, HRMS and DFT analysis. Probe 2a was successfully used as a signal tool to quantitatively detect Al³⁺ in food samples and environmental water samples. Furthermore, probe 2a was successfully utilized to label intracellular Al³⁺, indicating its promising applications in living cells.

Probe 2a exhibiting high sensitivity, good selectivity, wide pH range, lower detection limit, and rapid detection for Al³⁺, probe 2a was applied for the successful detection of Al³⁺ in water samples, food samples and HeLa cells.     

终点法荧光定量PCR检测HBV-DNA的临床应用

目的运用终点法荧光定量PCR检测HBV-DNA,探讨其临床应用价值。方法运用本研究组制备的引物和荧光探针分别将HBV-DNA质粒及临床标本进行普通PCR和实时荧光定量PCR反应,再将普通PCR产物在荧光测定仪上测定荧光强度,与实时荧光定量PCR结果相比较,观察其符合程度。另外,运用相同方法比对本研究制备的PCR体系与市售荧光定量PCR试剂检测HBV-DNA的符合程度。结果终点法荧光定量PCR与实时荧光定量PCR检测HBV-DNA质粒及临床标本结果一致(P>0.05),与市售荧光定量PCR试剂比较,其符合程度较高(P>0.05)。结论终点法荧光定量PCR检测临床HBV-DNA标本稳定可靠,可作为荧光定量PCR的应用方法之一在基层医院推广应用。     

Near-infrared spectroscopy for the detection of lipid core coronary plaques

Lipid core coronary plaques (LCPs), which cannot be reliably detected by conventional diagnostic measures, are widely considered to be the cause of most acute coronary syndromes. Accumulating evidence also indicates that LCPs may increase the risk of stenting complications. A catheter-based near-infrared spectroscopy (NIRS) system is now available for the detection of LCPs in the arteries of patients undergoing coronary angiography. The system, which uses the well-documented ability of NIRS to determine the chemical composition of unknown substances, has been validated in an autopsy study and a clinical trial. The system has now been used in more than 300 patients and has provided novel information for use in assessment of coronary disease. Multiple studies are in progress to assess the full clinical benefit of NIRS for the goals of 1) improving the safety of stenting, 2) preventing a second coronary event in patients with known coronary disease, and 3) use as a possible component in a strategy for the primary prevention of coronary events.     

A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin‐D detection

Currently there are no approved biomarkers for the pre‐symptomatic diagnosis of Alzheimer's disease (AD). Cathepsin‐D (Cat‐D) is a lysosomal protease that is present at elevated levels in amyloid plaques and neurons in patients with AD and is also elevated in some cancers. We have developed a magnetic resonance imaging (MRI)/fluorescent contrast agent to detect Cat‐D enzymatic activity. The purpose of this study was to investigate the cellular and tissue uptake of this MRI/fluorescent contrast agent. The agent consists of an MRI probe [DOTA–caged metal ion (Gd³⁺ or Tm³⁺)] and a fluorescent probe coupled to a cell‐penetrating‐peptide sequence by a Cat‐D recognition site. The relaxivity of Gd³⁺–DOTA–CAT_(cleaved) was measured in 10% heat‐treated bovine serum albumin (BSA) phantoms to assess contrast efficacy at magnetic fields ranging from 0.24 mT to 9.4 T. In vitro, Tm³⁺–DOTA–CAT was added to neuronal SN56 cells over‐expressing Cat‐D and live‐cell confocal microscropy was performed at 30 min. Tm³⁺–DOTA–CAT was also intravenously injected into APP/PS1‐dE9 Alzheimer's disease mice (n = 9) and controls (n = 8). Cortical and hippocampal uptake was quantified at 30, 60 and 120 min post‐injection using confocal microscopy. The liver and kidneys were also evaluated for contrast agent uptake. The relaxivity of Gd³⁺–DOTA–CAT_(cleaved) was 3.3 (mM s)^?1 in 10% BSA at 9.4 T. In vitro, cells over‐expressing Cat‐D preferentially took up the contrast agent in a concentration‐dependent manner. In vivo, the contrast agent effectively crossed the blood–brain barrier and exhibited a distinct time course of uptake and retention in APP/PS1‐dE9 transgenic mice compared with age‐matched controls. At clinical and high magnetic field strengths, Gd³⁺–DOTA–CAT produced greater T₁ relaxivity than Gd³⁺–DTPA. Tm³⁺–DOTA–CAT was taken up in a dose‐dependent manner in cells over‐expressing Cathepsin‐D and was shown to transit the blood–brain barrier in vivo. This strategy may be useful for the in vivo detection of enzyme activity and for the diagnosis of Alzheimer's disease. Copyright © 2012 John Wiley & Sons, Ltd.     

Feasibility,sensitivity, and reliability of laser-induced fluorescence imaging of green fluorescent protein-expressing tumors in vivo

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal–spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.     

In vivo and in situ cellular imaging full-field optical coherence tomography with a rigid endoscopic probe

Full-field OCT has proved to be a powerful high-resolution cellular imaging tool for biological tissues. However the standard bulk full-field OCT setup does not match the size requirements for most in situ and in vivo imaging applications. We adapted its principle into a rigid needle-like probe using two coupled interferometers and incoherent illumination: an external processing interferometer is used for in-depth scanning, while a distal common-path interferometer at the tip of the probe collects light backscattered from the tissue. Our experimental setup achieves an axial and transversal resolution in tissue of 1.8 μm and 3.5 μm respectively, for a sensitivity of -80 dB. We present ex vivo images of human breast tissue, and in vivo images of different areas of human skin, which reveal cellular-level structures.