The invention relates to a molecular manipulation apparatus for investigating molecules attached on one side to a surface of a sample and on another side to a bead.

The ability to observe the properties of molecules may be a great tool in biology, chemistry and physics. Early methods relied on electronic signals such as the patchclamp and electron microscopy. With the discovery of the green fluorescence protein (GFP), single biomolecules at work may be visualized in both in vitro and in vivo. A whole field of single molecule biophysics has emerged, dedicated to exploring the physical details of the cell at one molecule at a time. These single molecule experiments have helped us to create insight into the molecular structure and mechanism of protein folding, motor proteins, and DNA and protein interactions.

In order to learn about the physical properties of molecules, only observing may not be enough, often physical manipulation of the biomolecule of interest can yield additional information. Several techniques allow the manipulation by applying forces to the molecule, such as atomic force microscopy (AFM), and magnetic and optical tweezers. Each of these techniques has its own advantages and drawbacks. For example, in most of these techniques only one molecule is manipulated at the time.

It may be beneficial to manipulate a plurality of molecules and investigate their behavior. Quantitative analysis of the molecules may better help to understand more about the molecules.

It is an objective of the invention to provide a molecular manipulation apparatus for investigation of a plurality of molecules.

According to an embodiment there is provided a molecular manipulation apparatus for investigating molecules, wherein the apparatus is being provided with:

a radiation system for providing a radiation beam of radiation;

a sample comprising a plurality of molecules each attached on one side to a transparent surface of the sample and on the other side each molecule is attached to an individual bead of a plurality of beads; a sample holder for holding the sample with its transparent surface within the beam of radiation such that the plurality of beads is pushed by the radiation; and,

a detector to detect the positions of the plurality of beads.

By manipulating a plurality of molecules in the apparatus quantitative analyses of the molecules becomes possible. The apparatus according to claim 1 , wherein the apparatus comprises a computer programme to calculate a physical property of the plurality of molecules as a function of the position of the plurality of beads.

In an embodiment the detector comprises a microscope objective constructed and arranged to provide an image of the plurality of beads in the sample. The image may provide the researcher with an straightforward way to investigate the molecules. The apparatus may comprise an illumination system constructed and arranged to provide an illumination beam to illuminate the sample. Using the microscope objective, an image will be made of the illuminated sample with the camera.

The radiation system is constructed and arranged to provide a radiation beam with a power between 1 MilliWatt to 1 KiloWatt. The power should be sufficient to exert a pushing force on the beads. The radiation may have a wavelength between 150 and 1500 nm. The radiation system may comprise a laser for providing a laser beam of radiation. The laser may be a Nd:YAG laser, a fibre laser or a diode laser for providing a radiation beam of, for example 1064 nm.

The radiation system is constructed and arranged to vary radiation intensity. The radiation system may be operably connected with a computer system and the computer system controls the radiation intensity and uses the intensity of the beam of radiation to investigate the plurality of molecules.

The apparatus comprises a collimator lens group for collimating the beam of radiation before directing it to the sample. The beam may have parallel rays at the sample to push the beads.

A redirector between the sample holder and the detector can be used to direct the radiation beam away from the detector.

In-coupling optics can be used for overlapping the illumination beam with the radiation beam and direct both beams to the sample holder. The detector may comprise a CMOS or CCD camera.

According to a further embodiment there is a method for investigating a plurality of molecules is provided comprising:

providing a plurality of molecules to a sample;

connecting the molecules with one side to a transparent surface of the sample;

connecting the other side of the molecules to an individual bead of a plurality of beads; irradiating the sample with a radiation beam such that the plurality of beads is pushed by the radiation while detecting the position of the plurality of beads.

The method comprises increasing a radiation intensity of the radiation system and calculating a physical property of the plurality of molecules by using the radiation intensity in the calculations. Detecting the positions of the plurality of beads may comprise measuring a displacement of the plurality of beads after the molecules are broken by the increasing radiation intensity and a physical property which may be calculated is a breaking force of the molecules.

The method may comprise:

providing an additional molecule to the sample for reaction with the plurality of molecules,

reacting the additional molecule with the plurality of molecules, and

detecting the position of the plurality of beads comprises detecting a change in the position of the plurality of molecules. Very low concentrations of additional molecule may be detected in this way.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Figure 1 depicts a schematic drawing of the molecular manipulation apparatus according to an embodiment;

Figures 2a and 2b depict how the molecular manipulation apparatus is working;

Figure 3 depicts the resulting force for different commercial materials and available bead sizes in the molecular manipulation apparatus of figure 1 ;

Figure 4 depicts the diameter bead contour length of a DNA molecule;

Figure 5 depicts Brownion motion in 3 dimensions of a bead with increasing laser power; and,

Figure 6 depicts the manipulating force as a function of the laser power.

Figure 1 depicts a schematic drawing of the molecular manipulation apparatus according to an embodiment. A radiation system 1 may be provided with a laser source 3 (e.g. a Nd:YAG laser of 1064 nm, a fiber laser or a laser diode) for providing a radiation beam of radiation 5 with a radiation energy of 1 MilliWatt to 1 KiloWatt and a wavelength between 150 and 1200, or even 1500 nm. The wavelength is dependent on the transmission of the sample. The sample may have water in it and therefore the wavelength used may advantageously be transmitted by water.

The radiation beam 5 may be colliminated by lens group 7. The 4 mm radiation beam 5 (f = 150 mm) from the laser 3 may be focused in the focal point of an aspheric lens (e.g Thorlabs, focal length f = 4 mm) of the lens group 7, resulting in a radiation beam (Gaussian shaped) with a final diameter of 166 μηι at the sample 9. The sample 9 may be held by a sample holder 10 for holding the sample with its transparent surface 12 within the beam of radiation 5. The sample may be held in a position which is not in focus of the radiation beam.

The transparent surface 12 may be made from any heat resistant transmissive material, for example quartz. Quartz may offer good resistance against high power radiation. It may be difficult to image the sample 9 using the same lenses through which the radiation beam passes, due to thermal expansion of the lenses when the high energy laser source 3 is on. Instead a microscope objective 1 1 may be used in the detector for imaging. To prevent heating effects in the detector, the laser radiation may be redirected after interacting with the sample 9, and before it may hit the objective 1 1. Therefore, a redirecter, for example a hot mirror 13 (e.g. Thorlabs FM01) may be placed between the sample 9 and the objective 1 1. The hot mirror 13 may reflect the laser beam 5 but transmits lower wavelengths for imaging purposes in the detector.

If the beam is divergent the hot mirror may reflect the laser beam back to the sample and due to the divergence of the beam, the redirected beam may interact with the sample at only a fraction of the original intensity. To allow the hot mirror 13 to be placed between the objective 1 1 and the sample 9 a 'long working distance' objective 1 1 may be used (e.g. 50 X Mitutoyo Plan Apo SL, working distance 20.5 mm). Illumination of the sample 9 may be accomplished by an illumination system provided with a LED 15 (e.g. Thorlabs M455L2, 455 nm) and illuminator lens 19. The illumination beam 21 may be coupled into the aspheric lens of the lens group 7 using in-coupling optics, for example a dichroic mirror 17. The illumination beam may illuminate the sample 9 and may traverse through the hot mirror 13 because of its smaller wavelength. The light collected by the objective 1 1 may pass through an IR neutral density filter to get rid of any remaining laser light, and the image may be formed on a detector, for example a CMOS camera 23 (e.g. DCC1545M Thorlabs). The CMOS camera 23 may be operable connected to a computer 24 for analyzing the image and/or calculating a physical property of the molecule.

A sample 9, e.g. a simple flow cell may be made by separating a cover glass 27 (0.20x25x25mm fused quarts Vitrosil 077®) and microscope slide 29 (1x75x25mm fused quarts Vitrosil 077) with a layer of parafilm 31 (See figure 2a and 2b). A plurality of molecules e.g. DNA molecules (1000 bps) M are at one end attached (e.g. tethered) to the surface 12 via a first connector e.g. a dig 33 connecting to a second connector e.g. anti-dig 35. The other end of the molecule M may be attached (e.g. tethered) to a microrobead B of a plurality of beads via a third connector e.g. biotin 39 binding with a fourth connector e.g. streptavadin 37. To prevent sticking of the DNA, the surface of the flow cell may be coated with casein. The bead B tethered to the DNA molecule may serve a double purpose: first as a handle to apply a force 25 to the DNA with the radiation beam 25 and ii. as a probe to detect the DNA's response to that applied force 25. The physical property of the DNA molecules M may be determined by detecting the position of the bead B in 3 dimensions over time with the detector comprising objective 11 and camera 23 (in figure 1) and in response to the force 25 generated by the radiation beam 5. The beads B and molecules M may be surrounded by water in the sample 9. An advantage of the apparatus is that it may be used with a large number of different beads. In magnetic manipulation apparatus one may be bound to the use of magnetic beads whereas in our apparatus there is more freedom of the used beads of different materials.

The center position of the bead B may be calculated by the computer 24 for every image of the camera 23 to track the end-to-end length of the DNA molecule M over time. Since the size of the bead B may be in the order of a wavelength the point spread function (PSF) may be imaged on the camera 23. To detect the center of the PSF (x, y) a cross correlation algorithm may be used, that achieves sub pixel resolution even when the PSF center is not more than a few pixels. Although a single measurement of the bead position may directly yield information on the state of the DNA, multiple measurements may lead to a distribution that may be used to investigate the physical properties of the DNA molecule e.g. the length as well as length changes (for example induced by proteins). A typical value that describes the size of the distribution in relation to the average positions x and y may be the root mean square motion (RMS) and is given by equation 1.1 (x-x)2 + ( _K _y)2 (1.1)

In addition to the position of the bead in x and y, the changes in height of the bead (z) may also be recorded. And in contrast to the x, y position, the bead's height may be a direct measurement of the DNA's length. The height may be determined by

determining the shape of the PSF that is dependent on the distance from the sample to the camera. Hence, a change in height of the bead leads to a change in the PSF. In order to relate the measured PSF to the changes in height, a look up table may be used (LUT, a library that contains the radial profile as a function of the beads height). The LUTs may be made by moving the sample over a known distance, using a piezo stage, and storing the radial profile of the bead. To obtain the changes in height during a measurement, the measured radial profile may be compared to the profiles stored in the LUT. By interpolating between and averaging over multiple radial profiles, a detection accuracy of < 5 nm may be achieved at an effective frame rate of 25 Hz.

To maximize the exerted force and minimize the amount of laser power necessary to achieve desirable forces, selecting the proper material and bead size may be very important. Double stranded DNA starts to melt and convert into single stranded molecules at a force of around 65 piconewtons (pN) (depending on salt concentrations). Therefore, the molecular manipulation apparatus may ideally be able to achieve forces of several tens of pico newtons to be able to fully stretch double stranded DNA.

What kind of forces are achievable with this technique, using a maximum intensity of 20 Watts? Since the size of the beads may be in the order of the lasers wavelength, three different theories may be used to calculate the theoretical achievable optical manipulation force 25 in the apparatus: the Raleigh regime (r« X), Generalized Lorenz-Mie calculations, (r~ X) and the ray optics regime (r »X). The complex refractive indexes used for the calculations are those of the bulk material and might slightly differ for the bead sizes which may be used. Due to the Gaussian shape of the beam, the beads will feel a gradient force towards the center next to the desired forward scattering force.

The Lorenz-Mie theory may connect the Raleigh and ray optics regime and for practical purposes may be used to select the bead material and size. Figure 3 shows the resulting force for different commercial materials (e.g. P is polystyrene, M is melamine, T is Titania, or G is gold), and available bead sizes. As expected in general: the higher the refractive index or the larger the bead B the larger the induced force 25 may be. As may be seen gold or silver would be the best, however, those are only commercially available up to a few hundred nanometer. Beads that are generally available for optical trapping (polystyrine) and with forces of several tens of piconewton still reachable may be

advantageously.

Several methods may be used to calibrate the forces on the bead. For example, pushing freely diffusing beads upwards, comparing their final velocity (vs) with Stokes' law (F= -θπμΡνε). The relation between the amount of Brownian motion (i.e. bead movement) and the applied force may be used as well. By using the equipartition theorem, and solving the Lagevin equation the force for magnetic tweezers may be calibrated using the time average motion of the bead in the plane (x or y) (equation 1.2). However, in magnetic tweezers the beads align its permanent magnetic moment with the field lines of the magnetic field, restricting the rotational movement of the bead. Therefore the torsion movement of the bead is neglected in the derivation. Similar, in optical tweezers the rotational part in the equation of motion is also omitted as bead translation dominates over rotation due to the stiffness of the optical trap. In optical trapping the power spectrum rather than the time averaged movement of a bead is used to calibrate the forces involved (equation 1.3). For non-magnetic tethered particles, however, the bead is free to swivel around the anchor point of the DNA, leading to an apparent increase of the Brownian motion. See for the diameter bead contour length of the DNA molecule figure 4.

.

Introducing a second Langevin equation that may describe the torsion of the bead may help. We may include a term for the torsion drag, no slip boundary condition for interactions close to the surface, and extension behavior of the DNA . Resulting in two coupled differential equations that describes the full behavior of a tethered particle with an external force (equation 1.4). Some assumptions are made to linearize the system and the equations are solved analytically, yielding an expression for the RMS motion, auto correlation time, and power spectral density. The auto correlation time is the time it takes for a bead to diffuse to a new position that is independent of its starting position. The power spectrum shows the contribution of different frequencies to the total movement of the bead in the plane of the microscope. These sets of equations can be used to calibrate, fit or calculate the forces on particles in techniques such as molecular manipulation with radiation, optical and magnetic tweezers and even under no external forces such as tethered particle motion.

(1.5) Single DNA Molecule tethers are prepared and their restricted Brownian motion is measured with increasing laser power. The results of the raw motion in 3 dimensions is plotted in figure 5. As is seen the amount of motion of the bead in x, y is quenched, indicating that the system stiffens. Furthermore, direct measurement of the beads height (z), shows the bead gets pushed upwards, and possibly even DNA extension. These two observations proof the manipulation by pushing of microbeads using radiation. How much force is the laser exerting on the DNA tethered beads?

Forces are determined by fitting the power spectra of measured tethers with the analytical theory (see Figure 6). The exerted power scales linear with the amount of laser radiation (2.1 pN/W).

The detector to detect a position of the bead may also be a detector to detect a position of multiple beads that are connected in the sample to multiple molecules. By quantitive analysis of the beads and their position in the sample the apparatus may become very sensitive for detecting concentrations of a particular molecule in the sample and/or for detecting the influence of certain proteins on the length of the molecules. The apparatus may also be used to measure the presents and or concentration of particular molecules interacting with the molecules that are located between the cover glass and the bead. The term "lens", where the context allows, may refer to any one or combination of various types of optical components, including refractive, reflective, magnetic,

electromagnetic and electrostatic optical components. While specific embodiments of the invention have been described above, it will be appreciated that the invention may be practiced otherwise than as described. For example, the invention may take the form of a computer program containing one or more sequences of machine-readable instructions describing a method as disclosed above, or a data storage medium (e.g. semiconductor memory, magnetic or optical disk) having such a computer program stored therein.

The descriptions above are intended to be illustrative, not limiting. Thus, it will be apparent to one skilled in the art that modifications may be made to the invention as described without departing from the scope of the claims set out below.