[Show abstract][Hide abstract] ABSTRACT: Oral squamous cell carcinomas are among the 10 most common cancers and have a 50% lethality rate after 5 years. Despite easy access to the oral cavity for cancer screening, the main limitations to successful treatment are uncertain prognostic criteria for (pre-)malignant lesions. Identifying a functional cellular marker may represent a significant improvement for diagnosis and treatment. Toward this goal, mechanical phenotyping of individual cells is a novel approach to detect cytoskeletal changes, which are diagnostic for malignant change. The compliance of cells from cell lines and primary samples of healthy donors and cancer patients was measured using a microfluidic optical stretcher. Cancer cells showed significantly different mechanical behavior, with a higher mean deformability and increased variance. Cancer cells (n approximately 30 cells measured from each patient) were on average 3.5 times more compliant than those of healthy donors [D(normal) = (4.43 +/- 0.68) 10(-3) Pa(-1); D(cancer) = (15.8 +/- 1.5) 10(-3) Pa(-1); P < 0.01]. The diagnosis results of the patient samples were confirmed by standard histopathology. The generality of these findings was supported by measurements of two normal and four cancer oral epithelial cell lines. Our results indicate that mechanical phenotyping is a sensible, label-free approach for classifying cancer cells to enable broad screening of suspicious lesions in the oral cavity. It could in principle be applied to any cancer to aid conventional diagnostic procedures.
Cancer Research 02/2009; 69(5):1728-32. DOI:10.1158/0008-5472.CAN-08-4073 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dual-beam laser trap is a versatile tool with many possible applications. In order to characterize its thermal properties in a microfluidic trap geometry we have developed a non-intrusive fluorescence ratio technique using the temperature sensitive dye Rhodamine B and the temperature independent reference dye Rhodamine 110. We measured temperature distribution profiles in the trap with submicron spatial resolution on a confocal laser-scanning microscope. The maximum heating in the center of the trap amounts to (13 +/- 2) degrees C/W for a wavelength of lambda = 1064 nm and scales linearly with the applied power. The measurements correspond well with simulated temperature distributions.
[Show abstract][Hide abstract] ABSTRACT: A dual-beam fiber laser trap, termed the optical stretcher when used to deform objects, has been combined with a capillary-based microfluidic system in order to serially trap and deform biological cells. The design allows for control over the size and position of the trap relative to the flow channel. Data is recorded using video phase contrast microscopy and is subsequently analyzed using a custom edge fitting routine. This setup has been regularly used with measuring rates of 50-100 cells/h. One such experiment is presented to compare the distribution of deformability found within a normal epithelial cell line to that of a cancerous one. In general, this microfluidic optical stretcher can be used for the characterization of cells by their viscoelastic signature. Possible applications include the cytological diagnosis of cancer and the gentle and marker-free sorting of stem cells from heterogeneous populations for therapeutic cell-based approaches in regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: The cytoskeleton is a major determinant of the mechanical strength and morphology of most cells. The composition and assembly state of this intracellular polymer network evolve during the differentiation of cells, and the structure is involved in many cellular functions and is characteristically altered in many diseases, including cancer. Here we exploit the deformability of the cytoskeleton as a link between molecular structure and biological function, to distinguish between cells in different states by using a laser-based optical stretcher (OS) coupled with microfluidic handling of cells. An OS is a cell-sized, dual-beam laser trap designed to nondestructively test the deformability of single suspended cells. Combined with microfluidic delivery, many cells can be measured serially in a short amount of time. With this tool it could be shown that optical deformability is sensitive enough to monitor subtle changes during the progression of cells from normal to cancerous and even a metastatic state. Stem cells can also be distinguished from more differentiated cells. The surprisingly low number of cells required for this assay reflects the tight regulation of the cytoskeleton by the cell. This suggests the possibility of using optical deformability as an inherent cell marker for basic cell biological investigation, diagnosis of disease, and sorting of stem cells from heterogeneous populations, obviating the need for external markers or special preparation. Many additional biological assays can be easily adapted to utilize this innovative physical method. This chapter details the setup and use of the microfluidic OS, the analysis and interpretation of data, and the results of a typical experiment.
[Show abstract][Hide abstract] ABSTRACT: The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of ∼220 Pa for normal fibroblasts and ∼185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.
[Show abstract][Hide abstract] ABSTRACT: Even minute alterations in a cell's intracellular scaffolds, i.e. the cytoskeleton, which organize a cell, result in significant changes in a cell's elastic strength since the cytoskeletal mechanics nonlinearly amplify these alterations. Light has been used to observe cells since Leeuwenhoek's times and novel techniques in optical microscopy are frequently developed in biological physics. In contrast, with the optical stretcher we use the forces caused by light described by Maxwell's surface tensor to feel cells. Thus, the stretcher exemplifies the other type of biophotonic devices that do not image but manipulate cells. The optical stretcher uses optical surface forces to stretch cells between two opposing laser beams, while optical gradient forces, which are used in optical tweezers, play a minor role and only contribute to a stable trapping configuration. The combination of the optical stretcher's sensitivity and high throughput capacity make a cell's "optical stretchiness" an extremely precise parameter to distinguish different cell types. This avoids the use of expensive, often unspecific molecular cell markers. This technique applies particularly well to cells with dissimilar degrees of differentiation, as a cell's maturation correlates with an increase in cytoskeletal strength. Because malignant cells gradually dedifferentiate during the progression of cancer, the optical stretcher should allow, the direct staging from early dysplasia to metastasis of a tumor sample obtained by MRI-guided fine needle aspirations or cytobrushes. With two prototypes of a microfluidic optical stretcher at our hands, we prepare preclinical trials to study its potential in resolving breast tumors' progression towards metastasis. Since the optical stretcher represents a basic technology for cell recognition and sorting, an abundance of further biomedical applications can be envisioned.
[Show abstract][Hide abstract] ABSTRACT: We describe a novel microfluidic perfusion system for high-resolution microscopes. Its modular design allows pre-coating of
the coverslip surface with reagents, biomolecules, or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special molding
station, using masters made by photolithography and dry etching of silicon or by photoresist patterning on glass or silicon.
This channel system can be reused while the coverslip is exchanged between experiments. As normal fluidic connectors are used,
the link to external, computer-programmable syringe pumps is standardized and various fluidic channel networks can be used
in the same setup. The system can house hydrogel microvalves and microelectrodes close to the imaging area to control the
influx of reaction partners. We present a range of applications, including single-molecule analysis by fluorescence correlation
spectroscopy (FCS), manipulation of single molecules for nanostructuring by hydrodynamic flow fields or the action of motor
proteins, generation of concentration gradients, trapping and stretching of live cells using optical fibers precisely mounted
in the PDMS layer, and the integration of microelectrodes for actuation and sensing.
Microfluidics and Nanofluidics 01/2006; 2(1):21-36. DOI:10.1007/s10404-005-0047-6 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The measurement of the mechanical properties of individual cells has received much attention in recent years. In this paper we describe the application of optically induced forces with an optical stretcher to perform step-stress experiments on individual suspended fibroblasts. The conversion from creep-compliance to frequency-dependent complex shear modulus reveals characteristic viscoelastic signatures of the underlying cytoskeleton and its dynamic molecular properties. Both normal and cancerous fibroblasts display a single stress relaxation time in the observed time and frequency space that can be related to the transient binding of actin crosslinking proteins. In addition, shear modulus and steady-state viscosity of the shell-like actin cortex as the main module resisting small deformations are extracted. These values in combination with insight into the cells' architecture are used to explain their different deformability. This difference can then be exploited to distinguish normal from cancerous cells. The nature of the optical stretcher as an optical trap allows easy incorporation in a microfluidic system with automatic trapping and alignment of the cells, and thus a high measurement throughput. This carries the potential for using the microfluidic optical stretcher to investigate cellular processes involving the cytoskeleton and to diagnose diseases related to cytoskeletal alterations.
[Show abstract][Hide abstract] ABSTRACT: A step stress deforming suspended cells causes a passive relaxation, due to a transiently cross-linked isotropic actin cortex underlying the cellular membrane. The fluid-to-solid transition occurs at a relaxation time coinciding with unbinding times of actin cross-linking proteins. Elastic contributions from slowly relaxing entangled filaments are negligible. The symmetric geometry of suspended cells ensures minimal statistical variability in their viscoelastic properties in contrast with adherent cells and thus is defining for different cell types. Mechanical stimuli on time scales of minutes trigger active structural responses.
[Show abstract][Hide abstract] ABSTRACT: In an optical stretcher, infrared laser light is used to exert surface stress on biological cells, causing an elongation of the trapped cell body along the laser beam axis. These optically induced deformations characterize individual cells and cell lines. When integrated within a microfluidic chamber with high throughput, this enables diagnosis of diseases, on a cellular level, that are associated with cytoskeletal processes. Additionally, it allows sorting of cells with high accuracy in a non-contact manner. To determine the surface stress on the cell, ray optics calculations as well as the system transfer operator (T-matrix) approach with an appropriate incident field are used. The latter approach allows a more accurate modeling of the cell in the optical stretcher and reveals a more detailed stress profile acting on the cell surface. Analyzing the deformation behavior of normal and malignantly transformed fibroblasts, significant differences in axial elongation even for sample sizes as low as 30 cells are already measurable on a time scale of 0.1s. Here, malignant transformation of cells is discussed as an example of how any process that affects the cell's optical or mechanical properties allows classification with the optical stretcher.
Proceedings of SPIE - The International Society for Optical Engineering 10/2004; DOI:10.1117/12.556795 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transition from localized to systemic spreading of bacteria, viruses, and other agents is a fundamental problem that spans medicine, ecology, biology, and agriculture science. We have conducted experiments and simulations in a simple one-dimensional system to determine the spreading of bacterial populations that occurs for an inhomogeneous environment under the influence of external convection. Our system consists of a long channel with growth inhibited by uniform ultraviolet (UV) illumination except in a small "oasis", which is shielded from the UV light. To mimic blood flow or other flow past a localized infection, the oasis is moved with a constant velocity through the UV-illuminated "desert". The experiments are modeled with a convective reaction-diffusion equation. In both the experiment and model, localized or extinct populations are found to develop, depending on conditions, from an initially localized population. The model also yields states where the population grows everywhere. Further, the model reveals that the transitions between localized, extended, and extinct states are continuous and nonhysteretic. However, it does not capture the oscillations of the localized population that are observed in the experiment.
[Show abstract][Hide abstract] ABSTRACT: Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker.
The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells.
Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF-10, MCF-7, and modMCF-7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker.
Preliminary work suggests a deformability-based cell sorter similar to current fluorescence-based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton.
Cytometry Part A 06/2004; 59(2):203-9. DOI:10.1002/cyto.a.20050 · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By combining the Optical Stretcher, a two beam laser trap that measures the viscoelastic properties of cells, with current microfabrication techniques, we have developed a device able to differentiate cell types within a heterogeneous population. A micro-peristaltic pump controls the flow of cells through channels constructed by PDMS soft lithography. Cells are individually trapped and deformed by two divergent, counter-propagating laser beams aligned perpendicular to the flow, and are measured using videomicroscopy. Viscoelastic properties of a cell depend strongly on its cytoskeleton, which along with a cell's optical properties determine the amount of stretching, or optical deformability, for a given laser power. This serves as a new, inherent cell marker capable, for example, of detecting less elastic cancer cells among a population of healthy cells. As higher levels of integration become possible the experiment will progress towards the ultimate goal of a lab-on-a-chip. Increasingly more advanced microfluidic systems will incorporate cell sorting, medium swapping, and DNA microarrays, and will help span the gap between genomics, proteomics, and cellomics.
[Show abstract][Hide abstract] ABSTRACT: We experimentally investigate the population dynamics of a strain of E.
coli bacteria living under spatially inhomogeneous growth conditions. A
localized perturbation that moves with a well-defined drift velocity is
imposed on the system. A reaction-diffusion model of this situation^1
predicts that an abrupt transition between spatial localization and
extinction of the colony occurs for a fixed average growth rate when the
drift velocity exceeds a critical value. Also, a transition between
localized and delocalized populations is predicted to occur at a fixed
drift velocity when the spatially averaged growth rate is varied. We
create a spatially localized perturbation with UV light and vary the
strength and drift velocity of the perturbation to investigate the
existence of the different bacterial population distributions and the
transitions between them. Numerical simulations of a 250 mm by 20 mm
system guide our experiments. ^1K. A. Dahmen, D. R. Nelson, N. M.
Shnerb, Jour. Math. Bio., 41 1 (2000).
[Show abstract][Hide abstract] ABSTRACT: The cytoskeleton of an eukaryotic cell is a composite pol y- mer material with unique structural (mechanical) properties. To investigate the role of individual c yto- skeletal polymers in the deformation response of a cell to an exte rnal force (stress), we created two structural models - a thick shell model for the actin cortex, and a three-layered model for the whole cell. These structural models for a cell are based on data obtained by deforming suspended cells, where each cell is stre tched between two counter-propagating laser beams using an optical stretcher. Our models, with the data, suggest that the outer actin cortex is the main determinant of the stru c- tural response of the cell. WHILE a cell moving in the body is soft enough to squeeze through the tissue, it is also capable of withstanding high stresses such as osmotic pressure. Thus, an eukaryotic cell can respond to a large range of applied stresses, and di splays a unique deformation response (structural response). How the cell achieves such a dynamic range in its stru ctural or mechanical properties is a question that has cha llenged scientists from various disciplines. Elucidating the structural properties of this complex compound material requires understanding the individual contribution of the different structural components to the overall structural r esponse of the cell. In an eukaryotic cell, the main determinant of the structural response is the cytoskeleton - the in vivo polymer network that spans the interior of the cell. The cytoskeleton is co m- posed of three polymers - actin, microtubules and interme- diate filaments. The short actin filaments in vivo assemble into the actin cortex - a mesh -like structure just beneath the cell membrane. The rod-like microtubules are arranged in the cell in a hub -and-spoke array that extends radially outward to the actin cortex from the centrosome, which is located near the nucleus 1