A nanoimprinted polymer chip with a thin near-infrared absorber layer that enables light-induced local heating (LILH)
of liquids inside micro- and nanochannels is presented. An infrared laser spot and corresponding hot-spot could be
scanned across the device. Large temperature gradients yield thermophoretic forces, which are used to manipulate and
stretch individual DNA molecules confined in nanochannels. The absorber layer consists of a commercially available
phthalocyanine dye (Fujifilm), with a narrow absorption peak at approximately 775 nm, dissolved in SU-8 photoresist
(Microchem Corp.). The 500 nm thick absorber layer is spin-coated on a transparent substrate and UV exposed. Microand
nanofluidic channels are defined by nanoimprint lithography in a 1.5 μm thick layer of low molecular weight
polymethyl methacrylate (PMMA, Microchem Corp.), which is spin coated on top of the absorber layer. We have used a
previously developed two-level hybrid stamp for replicating two V-shaped microchannels (width=50 μm and height =
900 nm) bridged by an array of 200 nanochannels (width and height of 250 nm). The fluidic channels are finally sealed
with a lid using PMMA to PMMA thermal bonding. Light from a 785 nm laser diode was focused from the backside of
the chip to a spot diameter down to 5 ..m in the absorber layer, yielding a localized heating (Gaussian profile) and large
temperature gradients in the liquid in the nanochannels. A laser power of 38 mW yielded a temperature of 40oC in the
center of a 10 μm 1/e diameter. Flourescence microscopy was performed from the frontside.
In a typical position detection system for optical tweezers, laser light impinges on a quadrant photodiode, and the signal from the four quadrants of the diode is used to determine the position of a trapped object. A widely used position detection system consists of a Si-PIN photodiode and an infrared laser. In previous work we have demonstrated with two distinct experimental methods how such a system may act as an unintended low-pass filter and we modeled its physical origin mathematically. Here we demonstrate that the general solution to this model can account precisely for the "parasitic" filter's effects up to as large frequencies as we can measure, approximately 100 kHz. Thus we increase the useful bandwidth of tweezers experiments by nearly two decades. This opens for investigations of phenomena in biophysics, soft matter, and polymer science at much higher frequencies than before.
We present a procedure for a precise power spectral analysis of
optical tweezers data. This procedure uses the entire frequency range of the experimental power spectrum. We apply this procedure to experimental data from a biological system, data for the motion of a microsphere attached with a biotin-streptavidin linker to a single protein, the λ-receptor of E. coli. As in [Oddershede et al, Biophys. J. 83, 3152 (2002)] and [Oddershede et al, J. Phys.: Condens. Mat. 15, S1737 (2003)], we find that the λ-receptor moves diffusively in the outer membrane, but is confined to a particular region by a harmonic potential. Here, we show that since the power spectrum is known with precision up to its Nyquist frequency of 8 kHz, one can resolve the relative motion of the λ-receptor and the microsphere attached to it. We find that the biotin-streptavidin link between them can be described as a Hookean spring. Since we can see only the microsphere, these results are based on an interpretation of the power spectrum of its motion. This interpretation is based on a model. We use the simplest model
that is necessary and sufficient to interpret the power spectrum
in its full range. This model fits the spectrum well and the fit yields the model's parameters with some precision. These results indicate that optical tweezers have the potential to become a tool of precision. We discuss improvements of experiments and analysis that are necessary in order to realize this potential.
Without exceptions, in all living cells NADH is a key metabolite linking a large number of metabolic pathways. Flux rates through such pathways are an essential component in the understanding of the functioning of living cells. Knowledge about the way these fluxes depend on the concentrations of the metabolites involved (including NADH/NAD+) allows calculation of these fluxes. Therefore, a method to determine the concentration of free NADH is necessary. A distinction between the free and protein-bound NADH can be made on the basis of fluorescence emission spectra and fluorescence lifetimes. A method for such measurements using a microscopic set-up for time-gated fluorescence spectroscopy has been introduced by Schneckenburger and co-workers (Paul RJ, Schneckenburger H. Naturwissenschaften83, pp. 32-35, 1996). We further improve this method by first characterizing NADH binding to model proteins by isothermal titration calorimetry and fluorescence. This allows a precise calculation of bound and free NADH and their respective spectra. An analysis of experimental data is advanced by applying two-component deconvolution and subsequent fitting. Using this method we can detect a significant proportion of free NADH in isolated potato tuber mitochondria respiring malate. Taken together these improvements allow a more accurate characterization of the NADH turnover in biological systems.
The non-selective voltage activated cation channel from the human red cells, which is activated at depolarizing potentials, has been shown to exhibit counter-clockwise gating hysteresis. We have analyzed the phenomenon with the simplest possible phenomenological models by assuming 2×2 discrete states, i.e. two normal open/closed states with two different states of "gate tension." Rates of transitions between the two branches of the hysteresis curve have been modeled with single-barrier kinetics by introducing a real-valued "reaction coordinate" parameterizing the protein's conformational change. When described in terms of the effective potential with cyclic variations of the control parameter (an activating voltage), this model exhibits typical "resonant effects": synchronization, resonant activation and stochastic resonance. Occurrence of the phenomena is investigated by running the stochastic dynamics of the model and analyzing statistical properties of gating trajectories.