Laser tweezers and multiphoton microscopes in life sciences

Near infrared (NIR) laser microscopy enables optical micromanipulation, piconewton force determination, and sensitive fluorescence studies by laser tweezers. Otherwise, fluorescence images with high spatial and temporal resolution of living cells and tissues can be obtained via non-resonant fluorophore excitation with multiphoton NIR laser scanning microscopes. Furthermore, NIR femtosecond laser pulses at TW/cm2 intensities can be used to realize non-invasive contact-free surgery of nanometer-sized structures within living cells and tissues. Applications of these novel versatile NIR laser-based tools for the determination of motility forces, coenzyme and chlorophyll imaging, three-dimensional multigene detection, non-invasive optical sectioning of tissues ("optical biopsy"), functional protein imaging, and nanosurgery of chromosomes are described.     

Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking

Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.     

Synthesis of a quinazoline derivative: a new α₁-adrenoceptor ligand for conjugation to quantum dots to study α₁-adrenoceptors in living cells

Quantum dots (QDs) that are conjugated to small molecule derivatives of drugs and endogenous ligands may be useful tools to study the distribution and dynamic of membrane bound receptors, ion channels and transporters in live cells. In order to use these tools, it is necessary to functionalize QDs with bioactive ligands. In this paper, we successfully synthesized a ligand of α(1)-adrenoceptor that could be conjugated to QDs. In addition, the conjugation of the ligands to QDs and their biological activity were evaluated through binding assay with 30 nM QD conjugates in living human embryonic kidney 293 cells.     

Laser surgery of zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) embryos using femtosecond laser pulses: Optimal parameters for exogenous material delivery,and the laser's effect on short- and long-term development

Background  

Femtosecond (fs) laser pulses have recently received wide interest as an alternative tool for manipulating living biological systems. In various model organisms the excision of cellular components and the intracellular delivery of foreign exogenous materials have been reported. However, the effect of the applied fs laser pulses on cell viability and development has yet to be determined. Using the zebrafish (Danio rerio) as our animal model system, we address both the short- and long-term developmental changes following laser surgery on zebrafish embryonic cells.     

Untargeted quantum dots in confocal microscopy of living cells

The problem of the nonspecific binding of quantum dots (QDs) with cells is very important, but not fully understood taking into account the possible application of QDs in medical and fundamental studies. The interactions of untargeted CdSe/ZnS QDs with isolated frog muscle fibers, HeLa cells, and J774 cells were investigated. The observations were made on living cells using laser confocal microscopy (Leica TCS SL). QDs covered with polyethylene glycol without any functional reactive groups with an emission maximum at 565 nm were used in the study. This type of QD is suggested to prevent the interaction of QDs with biological molecules. It has been shown that QDs do not enter HeLa cells, the T-system, or the sarcoplasm of skeletal muscle fibers. However, during long-term incubation, J774 cells can take up QDs. The obtained data demonstrated the diversity of interactions of untargeted QDs with different cell types and are important for understanding problems of nonselective uptake and cytotoxicity of QDs.     

Quantum dots targeted to the assigned organelle in living cells

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.     

Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death

Two-photon excitation-based near-infrared (NIR) laser scanning microscopy is currently emerging as a new and versatile alternative to conventional confocal laser scanning microscopy, particularly for vital cell imaging in life sciences. Although this innovative microscopy has several advantages such as highly localized excitation, higher penetration depth, reduced photobleaching and photodamage, and improved signal to noise ratio, it has, however, recently been evidenced that high-power NIR laser irradiation can drastically inhibit cell division and induce cell death. In the present study we have investigated the cellular responses of unlabeled rat kangaroo kidney epithelium (PtK2) cells to NIR femtosecond laser irradiation. We demonstrate that NIR 170-fs laser pulses operating at 80-MHz pulse repetition frequency and at mean power of > or = 7 mW evoke generation of reactive oxygen species (ROS) such as H2O2 that can be visualized in situ by standard in vivo cytochemical analysis using Ni-3,3'-diaminobenzidine (Ni-DAB) as well as with a recently developed fluorescent probe Jenchrom px blue. The formation of the Ni-DAB reaction product as well as that of Jenchrom was relatively more pronounced when irradiated cells were incubated in alkaline solution (pH 8) than in those incubated in acidic solution (pH 6), suggesting peroxisomal localization of these reaction products. Two-photon time-lapse imaging of the internalization of the cell impermeate fluorescent dye propidium iodide revealed that the integrity of the plasma membrane of NIR irradiated cells is drastically compromised. Visualization of the nuclei with DNA-specific fluorescent probes such as 4',6-diamidino-2-phenylindole 24 h postirradiation further provided tangible evidence that the nuclei of these cells undergo several deformations and eventual fragmentation. That these NIR irradiated cells die by apoptosis has been established by in situ detection of DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Because the reactive oxygen species such as H2O2 and OH* can cause noxious effects such as cell membrane injury by peroxidation of polyunsaturated lipids and proteins and oxidative phosphorylation, and alterations of ATP-dependent Ca2+ pumps, these ROS are likely to contribute to drastic cytological alterations observed in this study following NIR irradiation. Taken together, we have established that NIR laser irradiations at mean power > or = 7 mW delivered at pulse duration time of 170 fs generally used in two- and multiphoton microscopes cause oxidative stress (1) evoking production of ROS, (2) resulting in membrane barrier dysfunction, (3) inducing structural deformations and fragmentation of the nuclei as well as DNA strand breaks, (4) leading to cell death by apoptosis.     

Cu‐doped quantum dots: a new class of near‐infrared emitting fluorophores for bioanalysis and bioimaging

Transition metal ion‐doped quantum dots (QDs) exhibit unique optical and photophysical properties that offer significant advantages over undoped QDs, such as larger Stokes shift to avoid self‐absorption/energy transfer, longer excited‐state lifetimes, wider spectral window, and improved chemical and thermal stability. Among the doped QDs emitters, Cu is widely introduced into the doped QDs as novel, efficient, stable, and tunable optical materials that span a wide spectrum from blue to near‐infrared (NIR) light. Their unique physical and chemical characteristics enable the use of Cu‐doped QDs as NIR labels for bioanalysis and bioimaging. In this review, we discuss doping mechanisms and optical properties of Cu‐doped QDs that are capable of NIR emission. Applications of Cu‐doped QDs in in vitro biosensing and in in vivo bioimaging are highlighted. Moreover, a prospect of the future of Cu‐doped QDs for bioanalysis and bioimaging are also summarized.     

Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting

Here, we report the facile preparation of tunable magnetic Ni-doped near-infrared (NIR) quantum dots (MNIR-QDs) as an efficient probe for targeting, imaging, and cellular sorting applications. We synthesized the MNIR-QDs via a hot colloidal synthesis approach to yield monodisperse and tunable QDs. These hydrophobic QDs were structurally and compositionally characterized and further functionalized with amino-PEG and carboxyl-PEG to improve their biocompatibility. Since QDs are known to be toxic due to the presence of cadmium, we have evaluated the in vitro and in vivo toxicity of our surface-functionalized MNIR-QDs. Our results revealed that surface-functionalized MNIR-QDs did not exhibit significant toxicity at the concentrations used in the experiments and are therefore suitable for biological applications. For further in vitro applications, we covalently linked folic acid to the surface of amino-PEG-coated MNIR-QDs through NHS chemistry to target the folate receptors largely present in the HeLa cells to demonstrate the specific targeting and magnetic behavior of these MNIR-QDs. Improved specificity has been observed with treatment of HeLa cells with the folic acid-linked amino PEG-coated MNIR QDs (FA-PEG-MNIR-QDs) compared to the one without folic acid. Since the synthesized probe has magnetic property, we have also successfully demonstrated sorting between the cells which have taken up the probe with the use of a magnet. Our findings strongly suggest that these functionalized MNIR-QDs can be a potential probe for targeting, cellular sorting, and bioimaging applications.     

Bioconjugated quantum dots for cancer research: Present status,prospects and remaining issues

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.     

Synthesis of near‐infrared‐emitting CdTeSe and CdZnTeSe quantum dots

We exploited the synthesis of near‐infrared (NIR) emitting ternary‐alloyed CdTeSe and quaternary‐alloyed CdZnTeSe quantum dots (QDs) with rod and tetrapod morphologies, which have tunable emission in the NIR electromagnetic spectrum. The morphologies of the QDs depended strongly on their growth kinetics, probably due to the coordinating ligands used in the preparation. Using oleic acid, stearic acid and hexadecylamine as ligands and keeping the same reaction parameters, QDs with tetrapod and rod morphologies were created. Not only had the capping ligands influenced the morphologies of QDs, but also they influenced the optical properties of QDs. The molar ratios of Cd/Zn and Te/Se upon preparation were adjusted for investigating the effect of composition on the properties of resulting QDs. By varying the composition of QDs, the photoluminescence (PL) wavelength of QDs was tuned from 650 nm to 800 nm. To enhance PL efficiency and stability, QDs were coated with a CdZnS shell. As NIR PL has numerous advantages in biological imaging detection, these QDs hold great potential for application. Copyright © 2012 John Wiley & Sons, Ltd.     

Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.     

Formulation, characterization, and in vitro evaluation of quantum dots loaded in poly(lactide)-vitamin E TPGS nanoparticles for cellular and molecular imaging

We developed a novel system of poly(lactide acid)-d-alpha-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) nanoparticles (NPs) for quantum dots (QDs) formulation to improve imaging effects and reduce side effects as well as to promote a sustainable imaging. The QDs-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method, which were then characterized by laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and transmission electron microscope (TEM) for surface morphology. Surface chemistry of the QDs-loaded PLA-TPGS NPs was analyzed by X-ray photoelectron microscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR). Encapsulation efficiency of the QDs in the polymeric nanoparticles was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The photostability of the QDs formulated in the PLA-TPGS nanoparticles was investigated as changes in the florescence intensity versus the irradiation time. Confocal laser scanning microscopy (CLSM) was used to image the cellular uptake of the QDs-loaded NPs by MCF-7 cells. Methylthiazolyldiphenyl-tetrazolium (MTT) assay was employed to assess the viability of MCF-7 cells incubated with the QDs formulated by the PLA-TPGS NPs versus the mercaptoacetic acid (MAA)-coated QDs. It was found that the QDs formulated in the PLA-TPGS NPs can result in higher fluorescence intensity and higher photostability than the bare QDs as well as lower cytotoxicity than the MAA-coated QDs.     

量子点细胞毒性及其作用机制

纳米粒子(NPS)在工业和研究中的使用急剧增加,因而这种材料面临一个其潜在毒性的问题。不幸的是,对纳米颗粒与纳米/生物界面可能发生的相互作用没有足够的了解。广大科技工作者正在积极寻求日益关注的纳米技术对人类的影响答案。我们将从NPS在生物媒体中的浓度,尺寸大小,电荷,和配位体的稳定性方面来了解纳米粒子的性质和他们在生物环境中对细胞毒所起的作用;并初步探讨已知的机制,量子点可以破坏细胞,包括氧化应激引起的活性氧(ROS)。微小浓度量子点足以造成长期持久的,甚至是跨代的影响。本文讨论了从纳摩尔到皮摩尔浓度的诱导细胞损伤的量子点(QDS)的浓度,这意味着含镉量子点可以发挥表观遗传毒性,纳米基因毒性,重金属基因的毒性。在此为评估包括量子点的在内的纳米毒性的的纳米材料,我们采用量子点作为一个例证,来阐述以科学为基础的发展到纳米毒理学的相关的问题。     

Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor

Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.     

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots

The fluorescent labeling of biological materials usingsmall-molecule organic dyes is widely employed in bio-logical imaging and clinical diagnosis. Organic fluoro-phores, however, have certain characteristics that limittheir advantages in some applications. These limitationsinclude narrow excitation bands and broad emissionbands with red spectral tails, which make the simultaneousevaluation of several light-emitting probes difficult due tospectral overlap. Also, many organic dyes exhibit highp…     

Laser-beam-triggered microcavitation: a novel method for selective cell destruction

We describe a new method of cell destruction that may have potential for use in antitumor therapy. Cells are loaded by phagocytosis with microparticles (<1 microm) and irradiated with short laser pulses. Absorption of laser energy by the microparticles causes localized vaporization of the fluid surrounding the microparticles, leading to the generation of transient vapor bubbles (microcavitation) around the microparticles. Using cultures of bovine aortic endothelial cells, we demonstrate that induction of intralysosomal microcavitation is an efficient, rapid and selective method of cell killing that is dependent on the number of microparticles, the number of laser pulses, and the fluence of the laser pulses. Cell killing by microcavitation is a very selective process that is restricted to cells containing microparticles, leaving other cells unaffected. Intracytoplasmic release of lysosomal hydrolases is, in part, responsible for cell death, because the protease inhibitors E64d and TLCK diminished cell killing. Using the broad-specificity caspase inhibitor Z-VAD-fmk, we determined that lysosomal hydrolases could induce apoptosis in a caspase-independent manner. We also examined the possibility of microcavitation-induced delayed effects in the cells that survived the treatment. Using flow cytometry, we determined that there was no delayed cell death between 1 and 4 days after microcavitation. Moreover, we did not observe changes in the cell cycle, in expression of the proteins BCL2, HSP70 and HSP27, or in PARP degradation. In conclusion, microcavitation induces rapid and specific cells death (limited only to cells containing microparticles), without producing delayed effects among the surviving cells.     

In vivo cancer targeting and imaging with semiconductor quantum dots

We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.     

Near Infrared,Highly Efficient Luminescent Solar Concentrators

The fabrication of a low reabsorption emission loss, high efficient luminescent solar concentrator (LSC) is demonstrated by embedding near infrared (NIR) core/shell quantum dots (QDs) in a polymer matrix. An engineered Stokes shift in NIR core/shell PbS/CdS QDs is achieved via a cation exchange approach by varying the core size and shell thickness through the refined reaction parameters such as reaction time, temperature, precursor molar ratio, etc. The as‐synthesized core/shell QDs with high quantum yield (QY) and excellent chemical/photostability exhibit a large Stokes shift with respect to the bare PbS QDs due to the strong core‐to‐shell electrons leakage. The large‐area planar LSC based on core/shell QDs exhibits the highest value (6.1% with a geometric factor of 10) for optical efficiency compared to the bare NIR QD‐based LSCs and other reported NIR QD‐based LSCs. The suppression of emission loss and the broad absorption of PbS/CdS QDs offer a promising pathway to integrate LSCs and photovoltaic devices with good spectral matching, indicating that the proposed core/shell QDs are strong candidates for fabricating high efficiency semi‐transparent large‐area LSCs.     

Involvement of ABC transporters in the efflux and toxicity of MPA‐COOH‐CdTe quantum dots in human breast cancer SK‐BR‐3 cells

This paper aimed to study the possible involvement of adenosine triphosphate‐binding cassette (ABC) transporters in the detoxification of quantum dots (QDs) in human breast carcinoma (SK‐BR‐3) cells. The effects of QD sizes on such interactions were also evaluated. For this purpose, we used monodispersed MPA‐COOH‐CdTe QDs with different diameters (emission length at 560 and 625 nm, named as QD‐560 and QD‐625). Such QDs tended to accumulate in cells and cause significant toxicity. Using specific inhibitors of ABC transporters, the cellular accumulation and toxicity of QDs in SK‐BR‐3 cells were significantly affected. Moreover, treatment of QDs caused concentration‐ and time‐dependent induction of ABC transporters. Furthermore, the induction effects of smaller QDs were found to be greater than larger ones at equivalent concentrations, suggesting a size‐dependent recognition of substrates by ABC transporters. Overall, these results provided important support for the modulation of QDs toxicity by ABC transporters.