Quantifying F-actin patches in single melanoma cells using total-internal reflection fluorescence microscopy.

Total-internal reflection fluorescence (TIRF) microscope is a unique technique for selective excitation of only those fluorophore molecules in a cellular environment, which are located at the sub-diffraction axial distance of a cell's contact-area. Despite this prominent feature of the TIRF microscope, making quantitative use of this technique has been a challenge, since the excitation intensity strongly depends on the axial position of a fluorophore molecule. Here, we present an easy-implemented data analysis method to quantitatively characterize the fluorescent signal, without considering the intensity-value. We use F-actin patches in single-melanoma cells as an example and define two quantities of elongation and surface density for F-actin patches at the contact-area of a melanoma cell. The elongation parameter can evaluate the dispersion of F-actin patches at the contact-area of a cell and is useful to classify the attaching, spreading, and expanding stages of a cell. Following that, we present the profile of the surface density of F-actin patches as a quantity to probe the spatio-temporal distribution of the F-actin patches at the contact-area of a cell. The data analysis methods that are proposed here will also be applicable in the image analysis of the other advanced optical microscopic methods.

Morphometry and Modeling of Label-Free Human Melanocytes and Melanoma Cells.

A combination of light microscopy and image processing was applied to investigate morphology of label-free primary-melanocytes and melanoma cells. A novel methodological approach based on morphology of nuclear body was used to find those single cells, which were at the same phase of cell cycle. The area and perimeter of melanocytes and melanoma cells were quantified. We found that there was a significant difference between area and perimeter of adendritic-shaped melanocytes with melanoma cells and the reason(s) of this finding was speculated. Finally, a theoretical model based on losing dendrites was proposed, which was in agreement with our experimental data.

Capture and detection of rare cancer cells in blood by intrinsic fluorescence of a novel functionalized diatom.

The ability to identify and enrich target cells can play a significant role in biosensing in general. For the separation of rare cells; a biosilica structure was extracted from "Chaetoceros sp." diatoms as a novel natural source of mesoporous materials. These diatoms had special optical capabilities, especially in fluorescence emission. Biosilica surfaces of Chaetoceros sp. were chemically modified by iron oxide nanoparticles resulting in diatom silica magnetic particles functionalized with Trastuzumab antibody to separate the breast cancer cells from normal cells. The fully characterization of magnetic biosilica structure were studied by various spectroscopic techniques. The magnetic diatom conjugated with antibody displays strong absorption and two main types of fluorescence emission with peaks centered at 493 and 650 nm (photo-excited at 405 nm). As in vitro study, SKBR3 cells (HER2 positive cells) were selectively targeted and separated with this magnetic diatom structure from the mix of HER2 negative cells using a magnetic field. These results show that Chaetoceros silica shells are promising eco-friendly biomaterials suitable for biosensing chip and the targeted delivery of drugs to the specific sites.

A novel aspect of functionalized graphene quantum dots in cytotoxicity studies.

Graphene quantum dots (GQDs) represent a new generation of graphene-based nanomaterials with enormous potential for use and development of a variety of biomedical applications. However, up to now little studies have investigated the impact of GQDs on human health in case of exposure. GQDs were synthesized from citric acid as carbon precursor by hydrothermal treatment at 160 °C for 4 h. The synthesized GQDs showed strong blue emission under UV-Irradiation with fluorescence quantum yield of 9.8%. The obtained GQDs were further carbonized, activated and functionalized by nitric acid vapor method. Nitrogen adsorption/desorption isotherms were used to analyze the surface area and porous structures of GQDs. The results revealed that compared to GQDs, the specific surface area of functionalized graphene quantum dots (fGQDs) has been increased from 0.0667 to 2.5747 m2/g and pore structures have been enhanced significantly. The potential cytotoxic effect of GQDs, fGQDs and GO suspensions was evaluated on HFF cell line using MTT assays and flow cytometry method after 24 h incubation. We have for the first time demonstrated that by carbonization, activation and functionalization of GQDs they still showed cytocompatible properties. We observed excellent biocompatibility of GQDs and fGQDs at low concentrations. Moreover, the results suggested that modification of GQDs yields product suspensions with high surface area, enhanced pore volume and loading capacities. Thus, fGQDs represent an attractive candidate for further use in drug delivery systems and bio-imaging application.

Label free phosphate functionalized semiconducting polymer dots for detection of iron(III) and cytochrome c with application to apoptosis imaging.

We report on facile synthesis and characterization of phosphate-functionalized polymer dots (PDs) by doping tributyl phosphate (TBP) in a semiconducting polymer poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,10-3}-thiadiazole)] (PFBT). Then, the prepared TBP@PFBT PDs were used to develop a very high sensitive probe for detection Fe3+, Cu2+ ions and Cytochrome c based on aggregation induced fluorescence off mechanism. The PDs exhibited a linear dynamic range for Fe3+ from 0.1 to 2 nM with a detection limit of 30 pM and for Cu2+ from 2.0 to 50.0 nM with a detection limit of 0.35 nM. Meanwhile, this probe showed a linear dynamic range for Cyt c from 175 to 1750 pM with a detection limit of 32.7 pM. The TBP@PFBT PDs is a simple, one-step, fast, non-invasive, label-free, and inexpensive probe that is capable of online apoptosis monitoring response to drugs with an ever-present opportunity to contribute in a variety of in-vitro and in-vivo biological applications. We also obtained sharp, specific 2D and 3D imaging results for early stage apoptosis in breast cancer cells. Moreover, this technique possesses the advantage of rapid determination of Fe3+ ion in biological or environmental samples. Importantly, this label-free assay provides short determination time of only a few min, easy operation and very low LOD allowing 100-4000 times increased in sensitivity over previously reported probes, together with high selectivity without need to using biorecognition elements like enzymes, antibodys and/or aptamers. Such excellent features make the TBP@PFBT PDs an excellent probe for successful apoptosis imaging in live cells.

Chemiluminescent liposomes as a theranostic carrier for detection of tumor cells under oxidative stress.

Hydrogen peroxide (H2O2) is one of the main source of oxidative stress and a typical marker of reactive oxygen species (ROS)-associated diseases. Therefore, selective detection and scavenging of overproduced H2O2 provide enormous benefits to the successful treatment of ROS-related diseases. The authors took advantage of this property to detect cancer cells using chemiluminescent peroxyoxalate reaction. Here, a new contrast agent presented for hydrogen peroxide, termed peroxyoxalate liposomes, which detect hydrogen peroxide through chemiluminescence reaction, and have the physical/chemical properties needed for imaging applications. The peroxyoxalate liposomes are composed of Bis (2, 4, 6-trichlorphenyl) oxalate (TCPO) and curcumin as fluorophore. Experimental factors such as TCPO, imidazole, hydrogen peroxide and curcumin concentration were optimized. Moreover, application of curcumin makes it possible to design a system for selective tumor destruction. In the reaction of peroxyoxalate, it acts as an oxalate activator with intracellular hydrogen peroxide and experiences excitation as a result of the reaction. In addition, curcumin also acts as a photosensitizer (PS) causing cell damage. In the optimum conditions, the measurable concentration range of 0.86-220 µM of hydrogen peroxide were evaluated from the linear calibration curve with satisfactory RSD% and corresponding detection limits of 650 nM. Therefore, it has the sensitivity needed to detect physiological concentrations of hydrogen peroxide. Moreover, cellular uptake experiments showed that the liposomes enhance extravasation into permeable membranes and significantly increased the bioavailability of curcumin.