Volume 90, Issue 8, Pages 2958-2969 (April 2006) Laser-Driven Microsecond Temperature Cycles Analyzed by Fluorescence Polarization Microscopy  Rob Zondervan, Florian Kulzer, Harmen van der Meer, Jos A.J.M. Disselhorst, Michel Orrit  Biophysical Journal  Volume 90, Issue 8, Pages 2958-2969 (April 2006) DOI: 10.1529/biophysj.105.075168 Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 1 Scheme of the proposed thermal cycles between cryogenic and room temperatures. A focused near-infrared laser beam with power PNIR (middle graph) rapidly raises the local focus temperature to Thigh (top graph). The moment the NIR laser is switched off, the temperature quickly drops back to the surrounding temperature Tcryo. In future experiments, a visible laser (bottom graph) with power PVIS will excite fluorescent labels during the cold periods at Tcryo only. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 2 Scheme of the optical paths around the sample plate in the cryostat. The sample plate is a glass substrate (thickness 0.17mm), coated with a thin, absorbing metal film (thickness 50nm), itself covered with the fluorescent solution layer. Fluorescence is excited (514.5nm) and collected by the custom-made 10-lens objective (NA=0.85, represented here by a simplified five-lens scheme) beneath the sample. Above it, a mirror at 45° and an aspheric singlet lens (NA=0.68) direct and focus the NIR beam (785nm) onto the metal film right across the visible focus. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 3 Schematic layout of the temperature-cycle setup. The two optical pathways, for probing (514.5nm) and heating (785nm), are completely separated. The probing part, represented to the left of the cryostat, is a laser-scanning confocal microscope with single-molecule sensitivity. The automatized laser scanner is composed of two one-axis galvanometer scan mirrors. The excitation and detection paths are separated by a (90/10) beam splitter. Besides standard components (beam expander, telecentric system, spatial filter, and spectral filters) the microscope includes a video camera to facilitate the positioning of the sample into the focal plane of the microscope objective. Collected fluorescence photons are sorted out by a polarizer cube and sent to two independent detectors for the parallel and perpendicular polarizations. The NIR (785nm) optical path (right of the cryostat) corresponds to the excitation path only of a laser-scanning confocal microscope. A second video camera monitors the focus of the NIR beam on the sample. An acousto-optical modulator rapidly switches the NIR power. Fig. 2 shows a detail of the optical components inside the cryostat. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 4 Simplified drawing of the bottom of the homebuilt cryostat insert. The protective cover is “cut open” to show the internal part and the optical elements schematized in Fig. 2. Sample holder and objective holder are separate parts, self-locked to one another by three prongs (one of them is labeled). A piezo stack moves the sample mount in three dimensions (x, y, z of sample), and a fourth piezo actuator moves the lens-mirror assembly to focus the NIR laser onto the sample (z of lens). Additionally, two mechanical actuators coarsely control the lens-mirror assembly (x, y of lens). This mechanical movement is a backlash-free bending of two spring-loaded flexure hinges. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 5 (Solid lines) Calculated temperature dependence of the rotation (bottom curve) and translation (top curve) correlation times for rhodamine 610 in glycerol. For translational diffusion, the spot size a (cf. Eq. 7) is chosen equal to 1μm. The rotation curve is compared to experimental results obtained in this work. The circles stem from steady-state fluorescence anisotropy measurements on R6G in glycerol. The squares represent FACS measurements on PDI in glycerol, which will be presented in more detail elsewhere (R. Zondervan, J. Berkhout, F. Kulzer, and M. Orrit, unpublished data). Both data sets are in good agreement with the model curve. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 6 (a) Variations of the fluorescence anisotropy of R6G in glycerol with temperature. The anisotropy values have been averaged over a 20×20μm2 image (visible laser intensity 50W/cm2, step size 200nm, acquisition time 10ms/point). The solid line is the expected dependence of the steady-state anisotropy due to rotational diffusion; see Eq. 4. The dashed line guides the eye through a variation mainly due to photoblinking (see text). The error bars are 95% confidence intervals (±2σ=±0.007). (b) Actual temperature calibration with anisotropy in the center of the heating spot, as a function of heating power. The temperature first scales linearly with applied power with a slope of 30K/mW (solid line). The deviation above 300K is probably due to coupled matter and heat transport out of the heated spot when glycerol becomes very fluid. The accuracy of the calibration is ±5K, indicated by error bars. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 7 Intensity images. Confocal fluorescence images, 20×20μm2 of a glycerol film doped with R6G for NIR heating powers of 0mW (a), 5mW (c), or 8.5mW (e). Graphs (b, d, and f) show the cross sections of images a, c, and e through the center of the heating spot. The total fluorescence images reveal the formation of a bright spot at first (c), then of a darker center surrounded by a bright and persistent ring (e). Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 8 Anisotropy images. The data of Fig. 7 provide 20×20μm2 images of fluorescence anisotropy. Graphs (b, d, and f) show cross sections of images a, c, and e through the center of the heating spot. The images show first a high-anisotropy spot (c). Then, the anisotropy at the center decreases below the initial level, but is still above it in a (nonpersistent) ring (e). The temperature calibration curve of Fig. 6 gives the temperature in the center, 280K for 5mW (c) and 335K for 8.5mW (e). Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 9 (a) Autocorrelation functions of the fluorescence anisotropy of PDI in glycerol at the center of the heating spot for NIR powers of 1.9, 2.3, 2.7, and 3.1mW. The fluorescence anisotropy is recorded for 100s with a time resolution of 10ms. The dashed lines are fits to Eq. 5 yielding rotation correlation times of 12.5, 2.5, 0.58, and 0.20s. (b) Calibration of the temperature at the hot spot’s center with heating power. The dependence is again linear (solid line) with the somewhat weaker slope of 10.3K/mW than in Fig. 6 because of the weaker absorption of the chromium film. The typical inaccuracy (±2σ) of the fits in panel a is ∼±20%, but the steep temperature dependence of the rotational diffusion (cf. Fig. 5) leads to a very precise determination of temperature (accuracy of ±0.5K), about the size of the symbols. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 10 (a) Total fluorescence intensity response for R6G in glycerol at the center of the hot spot during heating (first 200μs) and subsequent cooling (second 200μs) at NIR powers of 5.4, 7.0, and 11mW. The time resolution is 1μs. (b) Simultaneous time dependence of the anisotropy, from the same data. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 11 Temperature response during heating (first 200μs) and cooling (second 200μs) for NIR heating powers of 7.0mW (a) and 11mW (b). The plots are derived from the anisotropy traces in Fig. 10 b. The time resolution is 1μs. The smooth lines are simulations of the temperature response according to the heat equation, cf. Eqs. 9 and 12. The fit of the data in panel a is nearly perfect, yielding a t1/2 of ∼3.5μs. The deviation of the simulation in panel b is assigned to the different regime of heat transport in fluid glycerol above 310K. Biophysical Journal 2006 90, 2958-2969DOI: (10.1529/biophysj.105.075168) Copyright © 2006 The Biophysical Society Terms and Conditions