Rapid assessment of drug resistance of cancer cells to gefitinib and carboplatin using optical imaging

The overall goal of this study was to evaluate optical molecular imaging approaches to determine the drug response of chemotherapy and molecular targeted agents in drug sensitive and drug resistant cell lines. The optical molecular imaging approaches selected in this study were based on changes in intracellular uptake and retention of choline and glucose molecules. The breast cancer cell lines were treated with a molecular targeted anti-EGFR therapy. The bladder cancer cell lines were treated with a conventional chemotherapy approach. Sensitivity of optical molecular imaging approach was also compared with conventional cell viability and cell growth inhibition assays. Results demonstrate that optical molecular imaging of changes in intracellular uptake of metabolites was effective in detecting drug susceptibility for both molecular targeted therapy in breast cancer cells and chemotherapy in bladder cancer cells. Between the selected metabolites for optical molecular imaging, changes in glucose metabolic activity showed higher sensitivity in discrimination between the drug sensitive and drug resistant cell lines. The results demonstrated that optical molecular imaging approaches more significantly sensitive as compared to the conventional cell viability and growth assays. Overall, the results demonstrate potential of optical molecular imaging of metabolic activity to improve sensitivity of in-vitro drug response assays.     

Engineered systems for the physical manipulation of single cells

Manipulating the physical location of cells is useful both to organize cells in vitro and to separate cells during screening. The quest to manipulate cells on length scales commensurate with their size has led to a host of technologies exploiting optical, chemical, mechanical, electrical, and other phenomena. Researchers interested in organizing cells are gaining the ability to pattern more than two cell types, to create dynamic surfaces, and to pattern cells in the third dimension. In the realm of cell separation for screening, there has been significant progress in miniaturized flow-based optical sorters as well as in sorting following static microscopic observation.     

Real-time fluorescence detection of multiple microscale cell culture analog devices in situ.

We investigated multiple microscale cell culture analog (microCCA) assays in situ with a high-throughput imaging system that provides quantitative, nondestructive, and real-time data on cell viability. Since samples do not move between measurements, captured images allow accurate time-course measurements of cell population response and tracking the fate of each cell type on a quantitative basis. The optical system was evaluated by measuring the short-term response to ethanol exposure and long-term growth of drug-resistant tumor cell lines with simultaneous samples. The optical system based on epi-fluorescent excitation consists of an LED and a CCD as well as discrete optical components for imaging a large number of cells simultaneously. HepG2/C3A and MESSA cell lines were cultured in two microCCA systems for continuous cell status monitoring in cell death experiments with ethanol and long-term cell growth. The experiment that tested ethanol uptake showed that ethanol immediately caused cell death. The system was applied to extracting dynamic constants in the uptake process. In the long-term cell growth experiment, growth of MESSA cells was followed by a stationary phase and eventual cell death attributed to nutrient and oxygen depletion and a change in the pH because of the accumulation of wastes by cell metabolism. HepG2/C3A cells were subject to contact inhibition and cell number did not change significantly over time. Issues related to long-term assays are also discussed. The quantitative results have been consistent with qualitative images and confirm the applicability of the portable optical system, and potential application to high-throughput analysis of cell-based assays to measure long-term dynamics.     

How should the optical tweezers experiment be used to characterize the red blood cell membrane mechanics?

Stretching red blood cells using optical tweezers is a way to characterize the mechanical properties of their membrane by measuring the size of the cell in the direction of the stretching (axial diameter) and perpendicularly (transverse diameter). Recently, such data have been used in numerous publications to validate solvers dedicated to the computation of red blood cell dynamics under flow. In the present study, different mechanical models are used to simulate the stretching of red blood cells by optical tweezers. Results first show that the mechanical moduli of the membranes have to be adjusted as a function of the model used. In addition, by assessing the area dilation of the cells, the axial and transverse diameters measured in optical tweezers experiments are found to be insufficient to discriminate between models relevant to red blood cells or not. At last, it is shown that other quantities such as the height or the profile of the cell should be preferred for validation purposes since they are more sensitive to the membrane model.     

Nondividing, Bacteroid-Like Rhizobium trifolii: In Vitro Induction Via Nutrient Enrichment

Rhizobium trifolii 0403 maintained in exponential phase via periodic dilution doubled in 210 min in mannitol-salts medium and doubled in 244 min in glycerolsalts. In both media, cell number and optical density increased in parallel. When exponentially growing cells in either medium were supplemented with a mixture of glucose, Casamino Acids, succinate, and yeast extract, optical density continued to increase but within less than the time required for one doubling, division ceased. The increase in optical density coupled with division cessation resulted in the formation of large, pleomorphic, nondividing cells. Large cells apparently increased in size as a result of swelling only at regions of most recent cell envelope synthesis. Greater than 95% of the cells in a population swelled, and commitment to swelling occurred within two doubling time equivalents. Swollen cells eventually reached a characteristic maximum size and exhibited osmotic fragility.     

Single cell viability and impact of heating by laser absorption

Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s.     

Near-field optical analysis of living cells in vitro

Near-field optical analysis (NOA) provides morphological nanoscale mappings of living cells in liquid cell culture media and nondestructive insight into cell functionality. Here we show for the first time the performance of NOA in imaging living cells. Unlabeled human endothelial cells attached to polished titanium disks were analyzed with hydrophobically coated optical biosensors mounted to a near-field scanning optical microscope (NSOM). Biosensors and titanium substrates could be simply implemented in standard NSOM and high-throughput NOA.     

Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.     

Mechanical force characterization in manipulating live cells with optical tweezers

Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach.     

Laser-guidance based cell detection for identifying malignant cancerous cells without any fluorescent markers

Laser guidance technique employs the optical forces generated from a focused Gaussian laser beam incident on a biological cell to trap and guide the cell along the laser propagation direction. The optical force, which determines the guidance speed, is dependent on the cellular characteristics of the cell being guided, such as size, shape, composition and morphology. Different cell populations or subpopulations can be detected without any fluorescent markers by measuring their guidance speeds. We found that cell guidance speeds were sensitive enough to monitor the subtle changes during the progression of mouse fibroblast cells from normal to cancerous phenotype. The results also demonstrated that this technique can effectively distinguish mouse mammary cancerous cells with different metastatic competence. Laser guidance technique can be used as a label-free cell detection method for basic cell biological investigation and cancer diagnosis.     

Morphometric analysis of B2cAMP induced reverse transformation in synchronized CHO cells.

Synchronized tranformed and reverse-transformed (by 10(-3) M B2cAMP) CHO-K1 cells, growing adherent to plastic, are characterized by means of geometric and densitometric parameters at the level of both the entire cell and of the nuclei at various time intervals after selective miotic detachment. Transformed and reverse-transformed cells triple-stained with Feulgen, Napthol Yellow S, and periodic acid-Schiff appeared very similar in terms of integrated optical density (IOD), related to either polysaccharides, protein, or DNA amount. On the other hand, a shift from a polygonal to a spindle-shaped morphology is a accompanied by a significant decrease in both form factor and average optical density (AOD) of intact cell and nuclei, which are the most conspicuous measured changes caused by B2cAMP, in addition to a lengthening of the cell cycle duration. In both control and treated cells, important and parallel cell-cycle-dependent modulations of geometric and densitometric parameters are also observed, for both the cytoplasmic (i.e., cell morphometry) and DNA space (i e., nuclear morphometry). Specifically, the modulation in nulear morphometry during G1, S, G2, and M phases confirms previous findings on synchronized HeLa cells. The optical density threshold-dependence of geometric parameters shows that, while becoming fusiform, the cytoplasm of reverse-transformed cells had a particularly low optical density precisely in the polar area. Utilization of such an approach in the development of an objective morphological classification of all cell lines grown as monolayers "in vitro" is also discussed.     

Laser-guided direct writing of living cells

To perform their myriad functions, tissues use specific cell-cell interactions that depend on the spatial ordering of multiple cell types. Recapitulating this spatial order in vitro will facilitate our understanding of function and failure in native and engineered tissue. One approach to achieving such high placement precision is to use optical forces to deposit cells directly. Toward this end, recent work with optical forces has shown that a wide range of particulate materials can be guided and deposited on surfaces to form arbitrary spatial patterns. Here we report that, when we use the light from a near-infrared diode laser focused through a low numerical aperture lens, individual embryonic chick spinal cord cells can be guided through culture medium and deposited on a glass surface to form small clusters of cells. In addition, we found that the laser light could be coupled into hollow optical fibers and that the cells could be guided inside the fibers over millimeter distances. The demonstration of fiber-based guidance extends by 2 orders of magnitude the distance over which optical manipulation can be performed with living cells. Cells guided into the fiber remained viable, as evidenced by normal cell adhesion and neurite outgrowth after exposure to the laser light. The results indicate that this particle deposition process, which we call "laser-guided direct writing," can be used to construct patterned arrays of tens to hundreds of cells using arbitrary numbers of cell types placed at arbitrary positions with micrometer-scale precision.     

Experimentally manipulating fungi with optical tweezers

A short review of the use of optical tweezers in fungal cell biological research is provided. First, we describe how optical tweezers work. Second, we review how they have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Third, we indicate how optically trapped microbeads can be used for the localized delivery of chemicals or mechanical stimulation to cells, as well as permitting measurements of the growth forces generated by germ tubes. Finally, the effects of optical trapping on fungal cell viability and growth are assessed. Parts of this review were presented at the Mycological Society of Japan (MSJ) / British Mycological Society (BMS) Joint Symposium, “The new generation mycologists in Japan and the UK” held in Chiba, Japan on June 3, 2006.     

高斯光束中细胞横向受力分析

按照几何光学的原理建立了高斯光束中细胞受力的力学模型,并利用数值计算,得到了细胞偏离光轴受到的向轴回复力大小与细胞的离轴距离X0、直径2R等的关系.结果表明高斯光束对大小不同的细胞有相同的光学势阱宽度,对较大细胞势阱较深所以较容易稳定俘获.讨论了光钳设计中注意的问题.     

Probing Mechanical Properties of Jurkat Cells under the Effect of ART Using Oscillating Optical Tweezers

Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effect of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an oscillating optical tweezer was employed to characterize the frequency-dependent mechanical properties of Jurkat cells exposed to the chemotherapeutic agent, artesunate (ART). A motion equation for a bead bound to a cell was applied to describe the mechanical characteristics of the cell cytoskeleton. By comparing between the modeling results and experimental results from the optical tweezer, the stiffness and viscosity of the Jurkat cells before and after the ART treatment were obtained. The results demonstrate a weak power-law dependency of cell stiffness with frequency. Furthermore, the stiffness and viscosity were increased after the treatment. Therefore, the cytoskeleton cell stiffness as the well as power-law coefficient can provide a useful insight into the chemo-mechanical relationship of drug treated cancer cells and may serve as another tool for evaluating therapeutic performance quantitatively.     

Free and centrosome-attached microtubules: quantitative analysis and the modeling of two-component system

In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult.     

Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Cell wall lipopolysaccharide damage in Escherichia coli due to freezing

Freezing and thawing of Escherichia coli in water suspensions produce uninjured, nonlethally injured, and lethally injured cells as determined by their ability to multiply under different conditions. These treatments do not affect the microscope count or the optical density of the suspensions. The nonlethally injured cells develop extreme sensitivity to deoxycholate, lauryl sulfate, actinomycin D, and lysozyme. Lethally injured cells can be lysed by lysozyme as measured by the reduction in microscope count and optical density. These results have suggested that the outer membrane of the cell wall, which acts as a protective barrier in normal cells, has been damaged during freezing. In nonlethally injured cells, the damage can be repaired in K₂HPO₄ solutions. Reduction in the adsorption efficiency of the T-series phages indicated that the lipopolysaccharide, and not the lipoprotein of the outer membrane of the cell wall, is damaged in the frozen cells.