Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts

In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real‐time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa‐660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co‐expressing melanin and green fluorescent protein (GFP), C8161‐GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near‐infrared iRFP protein, and quantum dot‐carbon nanotube conjugates. Negative contrast flow cytometry provided label‐free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time‐resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

TdTomato and EGFP identification in histological sections: insight and alternatives

Abstract

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.     

Diet and abdominal autofluorescence detected by in vivo fluorescence imaging of living mice

We investigated the effect of diet on abdominal autofluorescence detected by in vivo fluorescence imaging (FLI) of living mice. Groups of mice were fed a regular, alfalfa-free, or purified diet, and whole-body FLI was performed without the administration of fluorescent probes. In addition, quantum dots were injected intravenously into mice fed one of the three diets, and FLI was performed 3 and 24 hours later. Intense autofluorescence originating from the animals' intestinal contents was observed in mice fed the regular diet. Intestinal autofluorescence decreased substantially after feeding with the alfalfa-free diet and further after feeding with the purified diet. The decline was rapid and took only 1 to 2 days; however, it may have been affected by an intake of feces. The reticuloendothelial system was clearly delineated using a low dose of quantum dots in mice fed the purified diet. On the other hand, intestinal autofluorescence was visible 24 hours postinjection in mice given the alfalfa-free diet and definitely impaired the image quality in mice fed the regular diet. The use of a low-fluorescence diet, especially a purified diet, rapidly reduces intestinal autofluorescence and is expected to enhance the potential of in vivo FLI.     

Imaging of cell surface antigens on tumor lymph-node sections using fluorescence quantum dots

In this work we explored the potential of quantum dots for fluorescent detection of lymphoid surface antigens. To optimize detection with quantum dots, we upgraded a fluorescent microscope that allowed us obtaining multiple images from different quantum dots on a single section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under the mercury lamp during scores of minutes. Double staining of frozen sections with direct conjugates of quantum dots with primary mouse monoclonal antibodies demonstrated direct conjugate high specificity and sensitivity. High stability of quantum dots’ fluorescence allows their use in diagnostics to analyze antigen coexpression on lymphoid tissue sections. “Spillover” of fluorescent signals from quantum dots into adjacent fluorescent channels maximally separated by 40 nm did not exceed 8%, which renders spectral compensation unnecessary.     

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (P<0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.     

Quantification of fluorescence in thick specimens, with an application to cyclin B-GFP expression in starfish oocytes

BACKGROUND INFORMATION: Fluorescence imaging of living cells is widely used in cell biology. It is now being extended to thick specimens such as large cells or tissues where it is important to establish methods for obtaining quantitative fluorescence data due to the increasing importance of computational and systems biology approaches. RESULTS: Fluorescent solutions were used as a calibration standard for determining cellular fluorescence concentrations from z series image sequences. The accuracy of the measurements was evaluated using quantitatively injected cells. Different fluorescence attenuation rates of the cytoplasm and nucleoplasm were documented, and autofluorescence levels were determined. This method was used to characterize the effect of cyclin B overexpression on cell-cycle timing in starfish oocytes. The time interval between application of maturation hormone and germinal vesicle breakdown decreased with increasing cyclin B-GFP (green fluorescent protein) concentration to a level of 100-300 nM, beyond which there was no effect. CONCLUSIONS: Conditions for determining fluorescent probe concentrations in large cells or multicellular tissues were established, which will facilitate the collection of data for quantitative studies. This method was used to characterize the effect of cyclin B-GFP expression levels on cell-cycle timing in starfish oocytes.     

Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently‐labeled cells in highly autofluorescent tissue

Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP‐expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single‐band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Functionalized quantum dots to quantify NADPH and their use for NADP+-dependent biocatalyzed transformations

Quantum dots are semiconducting nanoparticles that can be prepared with interesting optical properties. The fluorescent properties of quantum dots are one of the key advantages for their use as optical labels for biorecognition events and biocatalytic processes. We have prepared semiconductor quantum dots conjugated with Nile Blue (NB), and demonstrate that NB-functionalized quantum dots can act as versatile probes to analyze different biocatalyzed transformations, and can be used for the quantitative detection of NADPH as well as NADH. This approach provides a new path for the optical detection of NAD(P)H and for the quantitative analysis of NAD(P)(+)-dependent biotransformations.     

A sensitive fluorescent nanosensor for chloramphenicol based on molecularly imprinted polymer‐capped CdTe quantum dots

A novel fluorescent nanosensor using molecularly imprinted silica nanospheres embedded CdTe quantum dots (CdTe@SiO₂@MIP) was developed for detection and quantification of chloramphenicol (CAP). The imprinted sensor was prepared by synthesis of molecularly imprinting polymer (MIP) on the hydrophilic CdTe quantum dots via reverse microemulsion method using small amounts of solvents. The resulting CdTe@SiO₂@MIP nanoparticles were characterized by fluorescence, UV–vis absorption and FT‐IR spectroscopy and transmission electron microscopy. They preserved 48% of fluorescence quantum yield of the parent quantum dots. CAP remarkably quenched the fluorescence of prepared CdTe@SiO₂@MIP, probably via electron transfer mechanism. Under the optimal conditions, the relative fluorescence intensity of CdTe@SiO₂@MIP decreased with increasing CAP by a Stern–Volmer type equation in the concentration range of 40–500 µg L^–1. The corresponding detection limit was 5.0 µg L^–1. The intra‐day and inter‐day values for the precision of the proposed method were all <4%. The developed sensor had a good selectivity and was applied to determine CAP in spiked human and bovine serum and milk samples with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

A new infrared fluorescent-labeling agent and labeled antibody for diagnosing microcancers

PURPOSE: We have developed infrared fluorescent labeling agents and infrared-ray fluorescence endoscopes to establish a novel diagnostic technique. Since the fluorescence intensity of the initial labeled antibody (ICG-sulfo-OSu-labeled antibody) was not sufficient for practical use, we synthesized indocyanine green acylthiazolidinethione (ICG-ATT), which was expected to label various target molecules having amino groups efficiently. MATERIALS AND METHODS: To confirm imaging of infrared fluorescence intensity of ICG-ATT- and ICG-sulfo-OSu-labeled anti-MUC1 antibodies, cotton thread was soaked in various concentrations of the antibody solution in 0.1M PBS, and observed under the epi-illumination infrared fluorescence microscope. Localization and the intensity of infrared fluorescence and DAB coloring was compared in paraffin sections of human gastric mucosa. RESULTS: In the study of cotton threads, both labeled antibodies showed relatively clear infrared fluorescence, and significant difference was not observed between the two antibodies. ICG-ATT-labeled anti-MUC1 antibody produced stronger staining than that by ICG-sulfo-OSu-labeled antibody. Localization pattern of infrared fluorescent staining was in good agreement with that by the conventional method with oxidized DAB staining. CONCLUSION: ICG-ATT is useful as a fluorescent-labeling agent for diagnosis of microcancers by infrared fluorescence endoscopes.     

Automated selection of DAB-labeled tissue for immunohistochemical quantification.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.     

Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.     

Comparison of quantum dots immunofluorescence histochemistry and conventional immunohistochemistry for the detection of caveolin-1 and PCNA in the lung cancer tissue microarray

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide ranges of applications in cell imaging. In this study, we evaluated the capability of QDs immunofluorescence histochemistry (QDs-IHC) for detecting antigens of caveolin-1 and PCNA in the lung cancer tissue microarray (TMA) in comparison with the conventional immunohistochemistry (IHC) technique. Both methods revealed consistent antigen localization and statistically non-significant detection rates of caveolin-1 and PCNA expressions in our study. However, the sensitivity of QDs-IHC was higher than IHC. The positive detection rates of caveolin-1 and PCNA by QDs-IHC were 57% (40/70) and 86% (60/70), respectively, which were higher than the detection rates of 47% (33/70) and 77% (54/70), respectively, by IHC. Moreover, QDs exhibited a much better photostability, a broader excitation spectrum and a longer fluorescence lifetime. We showed here the advantages of QDs-IHC over IHC for the detection of caveolin-1 and PCNA in lung cancer TMA.     

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.     

Labeling of BSA and imaging of mouse T‐lymphocyte as well as mouse spleen tissue by l‐glutathione capped CdTe quantum dots

l ‐glutathione capped highly fluorescent CdTe quantum dots (QDs) were prepared by an aqueous approach and used as fluorescent labels to link albumin bovine serum (BSA) and rat anti‐mouse CD4, which was expressed on mouse T‐lymphocyte and mouse spleen tissue. The sharp and narrow emission peaks showed that the as‐prepared QDs have desirable dispersibility, uniformity and good fluorescence properties. Both CdTe–BSA and CdTe–CD4 conjugates showed an enhancement of fluorescence intensity over that of bare CdTe QDs. The experimental result of gel electrophoresis confirmed the successful conjugation of CdTe–BSA and CdTe–CD4. The fluorescent microscopic images of CdTe–CD4 labeled mouse T‐lymphocyte cells and mouse spleen tissue were compared with that obtained from fluorescein isothiocyanate labeling. It was demonstrated that the CdTe QDs‐based probe exhibited much better photostability and fluorescence intensity than fluorescein isothiocyanate, showing a good application potential in the immuno‐labeling of cells and tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Luminescence nanomaterials for biosensing applications

Due to their capacity to immobilize more bioreceptor parts at reduced volumes, nanomaterials have emerged as potential tools for increasing the sensitivity to specific molecules. Furthermore, carbon nanotubes, gold nanoparticles, polymer nanoparticles, semiconductor quantum dots, nanodiamonds, and graphene are among the nanomaterials that are under investigation. Due to the fast development of this field of research, this review summarizes the classification of biosensors using the main receptors and design of biosensors. Numerous studies have concentrated on the manipulation of persistent luminescence nanoparticles (PLNPs) in biosensing, cell tracking, bioimaging, and cancer therapy due to the effective removal of autofluorescence interference from tissues and the ultra-long near-infrared afterglow emission. As luminescence has a unique optical property, it can be detected without constant external illumination, preventing autofluorescence and light dispersion through tissues. These successes have sparked an increasing interest in creating novel PLNP types with the desired superior properties and multiple applications. In this review, we emphasize the most recent developments in biosensing, imaging, and image-guided therapy whilst summarizing the research on synthesis methods, bioapplications, biomembrane modification, and the biosafety of PLNPs. Finally, the remaining issues and difficulties are examined together with prospective future developments in the biomedical application field.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Quantum dots – a versatile tool in plant science?

An optically stable, novel class of fluorophores (quantum dots) for in situ hybridisation analysis was tested to investigate their signal stability and intensity in plant chromosome analyses. Detection of hybridisation sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide. Comparison of quantum dots (QDs) with conventional detection systems (Alexa 488) in immunolabeling experiments demonstrated greater sensitivity than the conventional system. In contrast, detection of QDs in in situ hybridisation of several plant chromosomes, using several high-copy sequences, was less sensitve than Alexa 488. Thus, semiconductor nanocrystal fluorophores are more suitable for immunostaining but not for in situ hybridisation of plant chromosomes.     

Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.     

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their ⑴broad absorption spectra allowing visualization with single light source, ⑵exceptional photo-stability allowing long term studies and ⑶narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.