Requerimientos necesarios para la puesta en marcha del Plan de Acción

Unlike the passive measurements approaches, the active microrheology methods incorporate some form of force application either locally at the site of interrogation or to the whole cell. There exist a variety of techniques to manipulate the mechanical

environment of cell population, individual living cells, and individual biomolecules. These approaches differ in three important respects: operating principles, force and displacement phenomena and extent of deformation(i.e., global vs. local; See Table2.1).

Table 2.1: Application of active rheology techniques in cell studies Application Technique Example

Cell population Substrate defor- mation

Effect of global stress on cell morphol- ogy (Banes, 2000)

Cell population and single cell

Microfabricated post array detector

Measuring inter/intracellular trac- tion(Tan et al., 2003)

Magnetic twist- ing cytometry

Characterizing frequency dependence of cellular component(Bursac et al., 2005)(Navajas et al., 1999;Fabry et al., 2001)

Magnetic tweezer

Chapter 4and 5

Single cell Cytodetacher Measure cell substrate adhesion forces Optical stretcher See below

Single molecule Atomic force mi- croscopy

Cell and cytoskeletal protein stiff- ness(Rotsch and Radmacher, 2000;

Radmacher, 2002)

Optical tweezers erythrocyte elasticity(Sleep et al., 1999)

Magnetic tweezer

Viscoelastic deformations of cells and membranes (Bausch et al., 1999) High resolution

force microscopy

See below

Cell population techniques focus on the mechanical response or mechanical manip- ulation of entire cell populations. Here, the purpose is often to understand the role that mechanics plays in regulating the structure and function of tissues that comprise organs. Substrate deformation relies on applying strain on substrates that the cells are attached to. The strains are imposed and measured via standard strain gauges or other low resolution displacement sensors. Maximum applied force and displacement are in

the Newton range and millimeter range.

Several cell manipulation technologies have been enabled by the development of thin film lithographic techniques. Micro-patterned substrates are fabricated to allow count wise control of cell adhesion and traction measurements through displacement of the pattern features(Chen et al., 1997) . Arrays of independently deforming posts onto which cells adhere have been produced using standard photolithography to create a silicon replica, then casting an elastomer(PDMS) within the replica to form a pattern of flexible micron-scale cantilever (oriented vertically) and finally microcontact print- ing the cantilever ends with ECM protein to facilitate cell adhesion. Here, the strain imposed on a particular cell cannot be modulated in a given experiment; it is set by the compliance of the cantilever array. In addition, the deformation is quantified only in the plane of the cell/post interface. Atomic force microscopy(AFM) is a specific example of scanning probe microscopy which has been exploited by the biophysics community because this technique affords Angstrom scale positioning accuracy, the ability to image and mechanically manipulate a single biological structure with bet- ter than nm/pN resolution and the potential to track the biological processes in near physiological environments over time. In particular, AFM has been used to explore the elastic deformations of cells and cellular components like the cytoskeleton. The elastic behavior of the cytoskeletal filaments can be explored via AFM as a function of position with the cell, first by creating a contact based image of the cell an then by conducting high resolution force spectroscopy measurements at particular points on that image.

2.3.1

The optical stretcher for imposing force on the whole

cell

The basic principal behind all laser based manipulation techniques is that momen- tum is transferred from light to the object, which in turn, by Newton’s second law,

exerts a force on the object. Based on this principal, the single beam laser tweezers have been an invaluable tool in cell biology: for trapping cells(Ashkin and Dziedzic, 1989), measuring force exerted by molecular motors such as myosin and kinesin(Block

et al., 1990) or the swimming forces of sperm(Tadir et al., 1990)and studying the poly-

meric properties of single DNA strand(Chu, 1991). The optical stretcher developed by Guck and Kas in contrast is based on a double beam trap in which two opposed, slightly divergent and identical laser beams with Gaussian intensity profile trap an ob- ject in the middle(Guck et al., 2005). The condition of stable trapping is fulfilled if the refractive index of the object is larger than the refractive index of the surrounding medium and if the beam sizes are larger than the size of the trapped object. In object such as cells, the momentum transfer primarily occurs at the surface. The total force acting on the center of gravity is zero because the two beam trap is symmetric and all the resulting surface forces cancer. In elastic objects, the surface forces stretch the object along the beam axis. A simple schematic of the optical stretcher is shown in Figure 2.2. Some of the advantages of this technique is that due to the incorporation of an automated flow chamber, the optical stretcher has the potential to measure elas- ticity of large number of cels in a short amount of time. This condition is ideal for circulating tumor cell measurements(CTC). The technique also bypasses issues related to localized cell stiffness heterogeneity and mechanical contact of the probe leading to adhesion and active cellular response. An application of this technique in testing for malignant transformation is shown in Chapter4.

2.3.2

Fluorescence force spectroscopy

Taking a quick diversion from techniques used to apply forces in biology, I want to quickly describe a technique to measure forces in biology.This technique ranks high in many single molecule, protein protein interaction applications and for a lack of a better

rigidity should provide information about its state and may be viewed as a new biological marker.

There are only a few experimental techniques capable of assessing cellular mechanical properties, but they consis- tently imply a correlation of cellular rigidity to cell status. Historically, the prevalent technique has been micropipette aspiration (Hochmuth, 2000). Using this technique, re- searchers found a 50% reduction in elasticity of malignantly transformed fibroblasts as compared to their normal counter- parts (Ward et al., 1991). More recently, atomic force microscopy has been used for the determination of cellular rigidity (Mahaffy et al., 2000; Rotsch et al., 1999). Lekka et al. (1999) used atomic force microscopy to investigate normal human bladder endothelial cell lines and compli- mentary cancerous cell lines and found that their rigidity differed by an order of magnitude. Other researchers have employed magnetic bead rheology (Wang et al., 1993), microneedle probes (Zahalak et al., 1990), microplate manipulation (Thoumine and Ott, 1997), acoustic micro- scopes (Kundu et al., 2000), sorting in microfabricated sieves (Carlson et al., 1997), and the manipulation of beads attached to cells with optical tweezers (Sleep et al., 1999). In general, malignant cells responded either less elastic (softer) or less viscous (less resistant to flow) to stresses applied, depending

on the measurement technique and the model employed. Metastatic cancer cells have been found to display an even lower resistance to deformation (Raz and Geiger, 1982; Ward et al., 1991). This stands to reason, because metastatic cancer cells must squeeze through the surrounding tissue matrix as they make their way into the circulatory systems where they travel to establish distant settlements (Wyckoff et al., 2000).

These findings suggest that cellular elasticity may be used as a cell marker and a diagnostic parameter for underlying disease. However, all of these techniques face major obstacles to generalized application: low cell throughput leading to poor statistics; mechanical contact of the probe leading to adhesion and active cellular response; and spe- cial preparation and nonphysiological handling leading to measurement artifacts. In this study we demonstrate that, in contrast, an optical stretcher (Guck et al., 2001) combined with microfluidic delivery can deform individual suspended cells by optically induced surface forces at rates that could eventually rival flow cytometers and thus circumvents these impediments (see Fig. 1,AandB). In addition, we show that the deformability of cells measured with a microfluidic optical stretcher is likely to be a tightly regulated inherent cell marker.

FIGURE 1 Optically induced surface forces lead to trapping and stretching of cells. Cells flowing through a microfluidic channel can be serially trapped (A) and deformed (B) with two counterpropagating divergent laser beams (an animation can be found as supplementary material online). The distribution of surface forces (small arrows) induced by one laser beam with Gaussian intensity profile incident from the left (indicated withlarge triangles) for a cell that is (C) slightly below the laser axis and (D) on axis. An integration of these forces over the entire surface results in the net force Fnetshown as large arrows. The corresponding

distributions for two identical but counter- propagating laser beams are shown for a cell on axis (E). This is a stable trapping configu- ration. When the cell is displaced from the axis (CandF), the symmetry of the resulting force distribution is broken, giving the net force a restoring component perpendicular to the laser axis. The inset shows the momenta of the various light rays and the resulting force at the surface.

3690 Guck et al.

Biophysical Journal 88(5) 3689–3698

Figure 2.2: From (Guck et al., 2005). Cells flowing through a microfluidic channel can be serially trapped (A) and deformed (B) with two counter propagating divergent laser beams. The distribution of surface forces (small arrows) by one laser beam from the left(large triangles) for a cell that is (C) slightly below the laser axis and (D) on the laser axis. Fnet is an integration of all the forces. (E) Stable trapping configuration

word, in “coolness” factor.

Mechanical probes have often suffered from the fact that they lack the ability to de- tect small scale conformational changes unless strong and persistent force is applied. At weak forces, the flexible tether connecting the mechanical probe to the biological molecule is not stretched enough to transmit small movements. Taekjip Ha’s group at University of Illinois has been involved in developing single molecule detection tech- niques for years. In 2007, they combined single molecule fluorescence resonance energy transfer(smFRET) with manipulations using optical trap. smFRET has high spatial resolution (≤ 5˚A) and can be measured at arbitrarily low forces(Hohng et al., 2007). The basic principal of such a system is use a flexible molecule that deforms or unzips at low forces, calibrate it using an optical trap and then insert FRET pairs into the molecule. SInce FRET signals inversely vary with increased stretching or deformation, one can insert this sensor into molecules of interest and detect FRET signals to get forces involved. An application of such a system relevant to this document was the development of the Vinculin tension sensor(Grashoff et al., 2010). Here, the authors designed a tension sensor module that contains a spring like protein segment, based on a sequence from a spider silk protein, that stretches in response to tension. They then positioned the fluorescent proteins at the ends of this spring like protein segment. Ap- plication of tension causes the sensor module to stretch leading to decrease in FRET signal and this way the authors were able to measure local tension in the vinculin molecule.