Laser-Driven Microsecond Temperature Cycles Analyzed by Fluorescence Polarization Microscopy

Abstract

We demonstrate a novel technique to achieve fast thermal cycles of a small sample (a few femtoliters). Modulating a continuous near-infrared laser focused on a metal film, we can drive the local temperature from 130 to 300 K and back, within a few microseconds. By fluorescence microscopy of dyes in a thin glycerol film, we record images of the hot spot, calibrate its temperature, and follow its variations in real time. The temperature dependence of fluorescence anisotropy, due to photophysics and rotational diffusion, gives a steady-state temperature calibration between 200 and 350 K. From 200 to 220 K, we monitor temperature more accurately by fluorescence autocorrelation, a probe for rotational diffusion. Time-resolved measurements of fluorescence anisotropy give heating and cooling times of a few microseconds, short enough to supercool pure water. We designed our method to repeatedly cycle a single (bio)molecule between ambient and cryostat temperatures with microsecond time resolution. Successive measurements of a structurally relevant variable will decompose a dynamical process into structural snapshots. Such temperature-cycle experiments, which combine a high time resolution with long observation times, can thus be expected to yield new insights into complex processes such as protein folding.

Local viscosity of supercooled glycerol near T g probed by rotational diffusion of ensembles and single dye molecules

“…For this analysis, it was crucial to reliably identify the same molecules at each temperature, which requires a mechanically very stable insert (30). Another obvious requirement is that a reasonable fraction of the molecules survive the accumulated illumination.…”

“…This setup is described in detail in ref. 30. An electro-optical modulator (ConOptics, Danbury, CT) in the excitation beam path allowed us to modify the polarization of the excitation laser and a polarization-dependent beam-splitter in the detection path separated the collected fluorescence light into two orthogonal polarization components, which were detected by two single-photon counting avalanche photodiodes.…”

Hybridization kinetics is different inside cells

“…1). Infrared laser light from below is absorbed by a chromium layer on a highly thermally conductive substrate, similar to previous approaches (22). HeLa cells can be seeded directly onto these object slides and kept in standard cell culture.…”

Temperature-cycle microscopy reveals single-molecule conformational heterogeneity

“…To follow microsecond dynamics on a same molecule for long times without the complication of immobilization, our group proposed a temperature-cycle technique 22 . This temperature-cycle method is closely related to the well established temperature-jump method at room temperature [23][24][25] .…”

“…This temperature-cycle method is closely related to the well established temperature-jump method at room temperature [23][24][25] . In this method, the time resolution is limited only by the heating and cooling times, which are typically around 3-4 microseconds for the diffractionlimited focal spot of a microscope 22 . A dynamical process can thus be studied as a consecutive series of snapshots of frozen states with controllable time steps.…”