We have developed temperature-pulse microscopy in which the temperature of
a microscopic sample is raised reversibly in a square-wave fashion with ris
e and fall times of several ms, and locally in a region of approximately 10
mu m in diameter with a temperature gradient up to 2 degrees C/mu m, Tempe
rature distribution was imaged pixel by pixel by image processing of the fl
uorescence intensity of rhodamine phalloidin attached to (single) actin fil
aments, With short pulses, actomyosin motors could be activated above physi
ological temperatures (higher than 60 degrees C at the peak) before thermal
ly induced protein damage began to occur. When a sliding actin filament was
heated to 40-45 degrees C, the sliding velocity reached 30 mu m/s at 25 mM
KCl and 50 mu m/s at 50 mM KCl, the highest velocities reported for skelet
al myosin in usual in vitro assay systems. Both the sliding velocity and fo
rce increased by an order of magnitude when heated from 18 degrees C to 40-
45 degrees C. Temperature-pulse microscopy is expected to be useful for stu
dies of biomolecules and cells requiring temporal and/or spatial thermal mo
dulation.