Figure 1.
Sketch of the experimental setup.
The experimental arrangement for time-resolved X-ray measurements at the Austrian SAXS–beamline at the ELETTRA synchrotron light source is shown. For T-jump experiments, an erbium laser beam (IR), wavelength λ = 1.5 µm, was directed via a prism onto the sample capillary which was thermostated with a Peltier unit. Laser pulse energy was 2 J within 2 ms resulting in an average T-jump amplitude of 10–12°C. The exposure time was 10 ms per frame. For T-drop experiments, the empty X-ray capillary was pre-cooled in a stream of nitrogen adjusted to −20°C. LDL samples, preheated to approx.10°C above the melting transition, were injected by a motor-driven syringe. A drop in temperature of about 20°C could be induced in about 3–4 s. The exposure time was 250 ms per frame.
Figure 2.
(A) Schematic of the thermotropic crystalline to isotropic transition in the apolar core lipids of LDL. Below Tm, the cholesteryl ester and triglyceride molecules (shown in blue and green, respectively) are arranged in two layers with a 3.6 nm repeat distance (left panel). Above Tm, the core lipids are in a fluid, oily state (right panel). The core lipids are surrounded by a monolayer of phospholipids and a single copy of apolipoprotein B100 (drawn in black and red, respectively). (B) Tm is typically determined by differential scanning calorimetry. The thermotropic transition is directly reflected in the small angle X-ray scattering curves. Static measurements performed at the SAXS – beamline at ELETTRA at 10°C and at 40°C are shown in (C). Upon heating above Tm, the intensity of the 1st side-maximum increases, and the 5th side-maximum vanishes. All scattering curves were recorded in the q-range of 0.09<q<2.30 nm-1 (q = 4π sin(θ)/λ, where 2θ is the scattering angle and λ the wavelength, λ = 0.15 nm).
Figure 3.
Time-resolved nanophase transition in LDL.
The rise in the integrated intensity of the 1st side-maximum upon laser jump is shown as a function of time (A). The time slicing was 10 ms per image. The time point of laser flash is set to zero seconds. The error function of statistical variation displays a maximum inaccuracy in time of about 5 ms. Thus, the offset in transition is much shorter than the sampling time of 10 ms and the 2 ms of laser flash. The integrated intensities of the 1st side-maximum obtained by static measurements within a temperature range of 0°C and 50°C with a step width of 5°C (B, left panel) are correlated to the time-course of integrated intensities of the 1st side-maximum obtained by dynamic measurements (B, right panel). For static measurements, a measuring time of 30 s and an equilibration time of 10 minutes at each temperature was chosen. For dynamic measurements, the measuring time per frame was 250 ms. A half-time of 2 seconds, corresponding to a temperature drop of about 10°C, could be achieved to pass through the transition temperature. The decline in integrated intensity strictly followed the drop in temperature. Tm for the LDL sample shown was about 22°C, as determined by microcalorimetry.