Comprensión real y consciente de la finitud de la creación.

2.3.1 Building optical tweezers

Assembly of an optical tweezers system can be achieved with standard optical components. In order to expand, reflect and steer the tweezing beam at the back aperture of the delivering objective lens, sets of lenses and mirrors were arranged in the beam path of an optical tweezers system. In order to obtain the necessary diffraction-limited spot at the beam focus, as the laser beam is emitted, it must be expanded to either exactly match or slightly overfill the back aperture of the delivering objective lens. In addition, the ability to steer the beam is crucial to enable beam tilting at the back aperture. Lee et al, 2007 (14) report on the necessary guidelines on constructing and characterizing a basic single beam tweezing setup. Figure 2.4 below illustrates an optical tweezers setup which consisted of a 658 nm, 50 mW diode laser. The beam spot emitted with a 1.5 mm diameter was magnified through a simple two plano-convex lens telescope L1 and L2 (f = 50 mm and f = 150 mm respectively) to ~ 5 mm. The beam was then reflected on a flip mirror M placed at 45o to the incident laser beam path.

Mirrors M1 and M2 were used in conjunction to align the beam optimally through the microscope objective. Mirror M2 located the beam onto mirror M1, which was conjugate with the back aperture of the objective. Therefore, manipulating M1 allowed tilting of the beam in the back aperture of the objective, thus allowing lateral movement of the focused beam spot in the focal plane. M1 (the beam steering mirror) was therefore used to get the beam through the objective. A simple 1:1 telescope arrangement, the optical relay system that consist part of the optical conjugates set was used to steer the laser beam spot. This comprised of two identical plano-convex lenses, L3 and L4 (f = 100 mm each), which were displaced by the sum of their focal lengths, so that an incident parallel beam produces a parallel output of the same beam diameter (13, 18). By placing these lenses with their flat surfaces facing one another, spherical aberration was minimized without resorting to expensive aplanatic lenses.

If L3 was pushed towards L4, then, movement in the axial (z-) direction occurred because the parallel beam entering the telescope (at the back of L3) became divergent after leaving L4. This pushed the focal spot away from the objective and deeper into the specimen. Conversely, if L3 was pulled away from L4 in the axial direction, the light from the telescope became convergent, bringing the focus towards the objective. Movement of lens L3 in the x-y plane, perpendicular to the optical axis, produced a deflection in the light leaving the lens in essence rotating the beam. If the lens L4 was imaged into the back of the objective aperture, then this rotation occurred in a conjugate plane to the objective aperture, resulting in a translation of the laser beam spot (19). To give the smallest possible spot size at the focus, which is imperative for stable three dimensional trapping, a 100X oil immersion objective lens with a high NA i.e., 1.25 NA was employed (10). By translating the xyz stage on which the sample was placed, the sample can be moved. A dichroic mirror positioned at 45o reflected the incident laser beam into the microscope objective but also allowed white light to pass through and an image to be formed on the charge coupled device (CCD) camera which can be viewed on a computer monitor. The setup was filtered by mounting a neutral density filter in the mouth of the CCD camera to limit saturation as a result of excessive transmission of the laser light. Below the sample stage was a 35 W halogen lamp that provided incoherent illumination of the sample.

Figure 2.4: A display of a basic optical trapping device produced using a diode 658 nm diode laser with maximum output power of 50 mW. Such a setup is useful for colloidal trapping applications rather than biological applications since it consists of a visible laser light that may be absorbed in biological material and result in heating and therefore cause optical damage (optocution). Radiometric effects were avoided by suspending relatively transparent particles in transparent medium (water).

To image the beam a glass slide with some index matching oil was used. The filter between the objective and the CCD camera was removed during this stage. By translating a slide up and down at the sample cell plane a pattern of the focused light beam was seen. The incoherent light beam was left off during this point and once a spherical beam image was acquired at the sample stage only then could trapping be tested. After building and aligning the setup illustrated in figure 2.4, it was used for tweezing of 3 µm polymer spheres with a refractive index (n) 1.56 (see figure 2.5 below).

Figure 2.5: Pictures from the CCD camera displaying z-trapping of 3 µ m polymer micro spheres using the optical tweezers illustrated in figure 2.4, the particles experience axial guiding by the trapping forces of laser light and migrates in the z-direction of the trap.

Figure 2.5 above illustrates the simple tweezing of polymer microspheres (diameter = 3 µm) utilizing a 658 nm diode laser system. Optical tweezers have had widespread application in biological studies as they offer, “non-invasive” precise micromanipulation of a specimen in a closed sterile environment. The response of biological cells to an applied laser beam is largely dictated by the laser wavelength as well as the laser power. Thus, Ashkin in the first studies with tweezers for biological material, recognized that near infrared (NIR) laser trapping could reduce photo-induced damage in biological cells when compared to traps made using visible light (20). Therefore to prevent the photo- damage (photobiological) effects escalating; most trapping, guiding, sorting and porating lasers operate in the NIR region of the light spectrum. Since the late 1980s a huge variety of cells and intracellular structures have been trapped and manipulated using optical tweezers. Outlined below is a summary of laser wavelength influences for biological matter.