Confocal microscopy is an excellent tool to gain structural information from deep within a biological sample. The depth from which information can be extracted as well as the resolution of the detection system are limited by spherical aberrations in the laser pathway. These spherical aberrations of the visible light can be efficiently canceled by optimizing the refractive index of the immersion media. Another way of canceling spherical aberrations is by changing tube length, or alternatively, by changing the objective from infinite correction to finite correction, or vice versa, depending on which microscope is used. A combination of these two methods allows for confocal imaging at continuous depths. Presently, confocal microscopes typically operate at a maximum depth of 40 µm in the sample, but with the methods presented here, we show that information can easily be gained from depths up to 100 µm. Additionally, the precision of localization of a single fluorophore in the axial direction, limited by spherical aberrations, can be significantly improved, even if the fluorophore is located deep within the sample. In principle, this method can improve the efficiency of any kind of microscopy based on visible light.
Optical tweezers constitute an obvious choice as the experimental technique for manipulation and trapping of
organelles in living cells. For quantitative determination of the forces exerted in such in vivo systems, however,
tools for reliable calibration of the optical tweezers are required. This is complicated by the fact that the viscoelastic
properties of the cytoplasm are a priori unknown. We elaborate on a previously reported theoretical
calibration procedure and verify its authenticity experimentally. With this approach, we may at the same time
determine the trapping characteristics of the optical tweezers and the viscoelastic properties of the cytoplasm.
The method employs the fluctuation-dissipation theorem (FDT) which is assumed valid for the situations considered.
This allows for extracting the requested properties from two types of measurements that we denote
as passive and active. In the passive part, the Brownian motion of a particle inside the trap is observed. In
the active part, the system is slightly perturbed and the response of the trapped particle is tracked. Gently
oscillating the stage on which the sample is mounted allows the delay between the position of the stage and the
response of the trapped bead, using a quadrant photodiode, to be quantified. No assumptions about the particle
radius or geometry or about the frequency-dependent friction coefficient are needed.
The paper contains the theoretical background of the method in terms of convenient formulations of the
fluctuation-dissipation theorem and application of the method in two types of experiments. Further we discuss
experimental concerns which are i) the choice of driving characteristics in the active part of the calibration
procedure and ii) statistical errors.
By increasing the axial trap stiffness, we demonstrate an increase of at least 50% in the maximum lateral trapping
force that can be applied using optical tweezers. It has previously been shown that, using a novel method of
compensating for spherical aberrations, the axial trap stiffness at any particular chosen depth within a sample
can be increased. However, to our knowledge, the present paper is the first time this method has been used in
combination with the drag force method for the purpose of more accurately determining the maximum lateral
trapping force applicable by optical tweezers.
Previous studies have substantially shown that before the actual maximum lateral trapping force can
be reached, the particle escapes in the axial direction. Using a conventional setup, our studies support this
conclusion. However, by employing the above mentioned method for improving the axial trap stiffness, we
observed that the displacement of the bead in the lateral direction is increased by approximately 10%. This
allows progress towards a more accurate determination of the maximum lateral force that can be applied using
optical tweezers and could also permit a mapping of the trapping potential further from the trap's central region.
Theoretical predictions made, show that the point where the maximum lateral force could be applied is at
0.9 a, where a is the radius of the trapped particle. However, the experimentally measured limit 0.55 a has
until now been far lower than that theoretically predicted 0.9 a. In this proceeding, we demonstrate that the
experimental limit can be extended to 0.61 a because of the decreased axial displacement of the bead.
Oil immersion objectives have higher numerical aperture than water immersion objectives thus providing higher optical resolution. This is important for confocal microscopy as well as for the strength of an optical trap created by such an objective, because the efficiency of an optical trap is limited by its axial strength. However, light focused by oil immersion objectives suffer from spherical aberrations caused e.g. by a mismatch between the refractive index of the immersion and sample media. Such aberrations widen the intensity profile in the focal region thus restricting the axial resolution of the objective and decreasing the axial optical trapping strength. Objectives are typically designed such that the spherical aberrations are minimized for visible wavelengths and a few microns away from the surface. However, often optical traps are based on infrared lasers or are used further away from the surface thus introducing
considerable spherical aberrations. We have shown that a tuning of the immersion refractive index can minimize the total spherical aberrations at any desired depth, thus maximizing the trapping efficiency and giving rise to optical trapping strengths twice as large as previously reported.1 Changing the immersion media, however, is a discrete way of tuning the optimal trapping depth: An increment (decrement) of 0.01 in the refractive index of the immersion media gives rise to an increase (decrease) of ≃4 μm and ≃10 μm of the most efficient
trapping depth for infinity-tube length and finite-tube length objectives, respectively. Here, we show that combining a change of immersion media with changing tube length provides a continuous way of changing the optimal trapping depth. Also, we show how trapping conditions change with polarization.
We demonstrate an example of 'confocal-tweezers' wherein confocal images and precise optical force measurements,
using photodiodes, are obtained simultaneously in the x-y plane without moving the objective lens. The
optical trap is produced using a 1.064μm cw laser and is combined with Leica's TCS SP5 broadband confocal
microscope to trap and image living cells. The unique method by which the confocal images are created facilitates
the acquisition of images in areas far from the trapping location. In addition, because the scanning process
involves moving galvanic mirrors independently of the objective, the trap is held stable in position and is not
subject to any error in position for the x-y scan.
We have successfully trapped and confocally imaged 80nm gold colloids, 150nm gold colloids and 1μm
polystyrene beads whilst making quantitative measurements of the force applied by the trap on each bead.
To the best of our knowledge this is the first time that anyone has combined precise force measuring optical
tweezers with confocal microscopy. We also discuss some of the technical challenges involved in advancing the
experimental set up to make quantitative force measurements in combination with 3D stacking. Having proven
the potential of this system in 2D, we hope to develop it further to investigate the nano-mechanics of cell division
through the attachment of gold beads to fluorescently labelled organelles in S. pombe yeast cells.
With the evolution of single molecule techniques as force-scope optical tweezers, it has become possible to
perform very accurate measurements of the elastic properties of biopolymers as e.g. DNA. Nucleic acid elasticity
is important in the interaction of these molecules with proteins and protein complexes in the living cell. Most
experimental and theoretical effort has been aimed at uncovering and understanding of the behavior of polymers
with contour lengths significantly longer than their persistence length. The well-established Worm-Like-Chain
model has been modified such that a satisfactory description of such long biopolymers is available. However,
in many single molecule experiments, such as the unfolding of RNA stem-loops1 and RNA pseudoknots,2 one
is dealing with biopolymers whose contour lengths are comparable to persistence lengths. A full understanding
of such curves requires an understanding of the physics of short biopolymers. For such cases, theories are just
beginning to emerge and there is hardly any experimental data available. We target this problem by optical
tweezers quantitative force-extension measurements on short biopolymers. The biopolymers used are primarily
double stranded DNA whose total length ( 300 nm) is comparable to their persistence length ( 50 nm). As a
control of our equipment and methods, we also stretch longer dsDNA (1100 nm), the force-extension curves of
which resemble those in literature.3 For the short DNA the force-extension curves qualitatively resemble those
predicted by WLC theories, but a reasonable fit can only be made if the persistence length is allowed to be a
fitting parameter. If made a fitting parameter, the 'apparent persistence length' is found as 8.7±4 nm, a number
which is significantly lower than the real physical value.
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