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Pulsed temperature-jump (T-jump) spectroscopy with infrared (IR) detection has been widely used to study biophysical processes occurring from nanoseconds to ∼1 ms with structural sensitivity. However, many systems exhibit structural dynamics on time scales longer than the millisecond barrier that is set by the time scale for thermal relaxation of the sample. We developed a linear and nonlinear infrared spectrometer coupled to an intensity-modulated continuous wave (CW) laser to probe T-jump-initiated chemical reactions from &lt;1 ms to seconds. Time-dependent modulation of the CW laser leads to a &lt;1 ms heating time as well as a constant final temperature (±3%) for the duration of the heating time. Temperature changes of up to 75 °C in D 2 O are demonstrated, allowing for nonequilibrium measurements inaccessible to standard pulsed optical T-jump setups. T-jump linear absorption, pump-probe, and two-dimensional IR (2D IR) spectroscopy are applied to the unfolding and refolding of ubiquitin and a model intercalated motif (i-motif) DNA sequence, and analysis of the observed signals is used to demonstrate the limits and utility of each method. Overall, the ability to probe temperature-induced chemical processes from &lt;1 ms to many seconds with 2D IR spectroscopy provides multiple new avenues for time-dependent spectroscopy in chemistry and biophysics.

Temperature-Jump 2D IR Spectroscopy with Intensity-Modulated CW Optical Heating