Temperature Control Methods in a Laser Tweezers System Hanbin Mao, J. Ricardo Arias-Gonzalez, Steven B. Smith, Ignacio Tinoco, Carlos Bustamante  Biophysical Journal  Volume 89, Issue 2, Pages 1308-1316 (August 2005) DOI: 10.1529/biophysj.104.054536 Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 1 Laser-heating results. Black circles represent the temperature measurements obtained by heating the fluid chamber with a 975nm diode laser at different distances with respect to the trapping lasers position. The black curve displays the theoretical agreement with the logarithmic temperature decay that has been analytically calculated in the Appendix. The red squares correspond to temperatures at the focus of the trapping laser obtained with various powers of the heating laser, whose focus is made coincident with that of the trapping laser. Linear behavior is shown by the regressive red line. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 2 Convection in chambers of different thickness, expressed as fluid velocity. The small chamber has a thickness of 90μm, whereas the big chamber has a thickness of 180μm. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 3 (a) Schematic drawing of the temperature control element incorporated in the dual-beam laser tweezers system (view from side). Drawing is not to scale. (b) Isometric view of the copper jacket used in a. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 4 Power spectrum density distribution of force fluctuations (expressed in volts squared per Hertz since the voltage measured is proportional to the force) for 2.1μm mean diameter polystyrene beads trapped in DI water by the dual-beam laser tweezers when (green) the water circulator was not used and (red) the circulator was turned on. Both lasers are 200mW, 830nm diode lasers. Only fluctuations in the vertical direction of the trapped bead were measured. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 5 Plot of inverse of corner frequency of the trapped beads (2.1μm mean diameter) versus viscosity (in micropascal seconds) in DI water. The corner frequency was obtained by fitting the power spectrum density distribution using Eq. 1 (see the text for details). The viscosity of DI water at the corresponding temperature was obtained from the literature (43). (Inset) Plot of corner frequency versus temperature; error bars show the standard deviation of 10 individual experiments. Temperature was measured by a thermocouple fixed inside the chamber. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 6 Force-extension curves of λDNA at low temperatures. Each curve was offset 1μm to increase the clarity of the figure. Pulling was performed at a constant speed of 1000nm/s for all three temperatures (8.4°C, 15.2°C, and 21.3°C). Both forward and reverse curves are shown for each temperature. Note the increase of the hysteresis with rising temperature. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 7 Force-extension curves of λDNA at high temperatures (28.1°C, 33.8°C, 37.2°C, and 45.6°C). Pulling was performed at a constant rate of 1000nm/s. Notice two plateaus are clearly seen at stretching transitions at 37.2°C and 45.6°C. (Dashed red line) Top plateau for curve at 45.6°C. (Dashed black line) Top plateau for curve at 37.2°C. (Dotted red line) Bottom plateau for curve at 45.6°C. (Dotted black line) Bottom plateau for curve at 37.2°C. All curves show major hysteresis with the 45.6°C curve indicating that only single-stranded DNA exists after the transition. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 8 Hysteresis area between stretching and relaxing force-extension curves versus temperature. Error bars represent standard deviation of at least 13 individual experiments at each temperature. Pulling speed was 1000nm/s. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 9 Geometry for the modeling of the temperature profile inside the microchamber due to the heat reradiated after laser absorption by water. See Appendix for details. Biophysical Journal 2005 89, 1308-1316DOI: (10.1529/biophysj.104.054536) Copyright © 2005 The Biophysical Society Terms and Conditions