J Physiol 585.1 Temperature-jump force generation in muscle 265 Experimental protocols and data analysis Using dark-ﬁeld illumination on the stage of a binocular microscope, single ﬁbres were dissected under parafﬁn oil. A short ﬁbre segment was attached using nitrocellulose glue between two metal hooks, one connected to the force transducer and the other to the motor. The sarcomere length was set to 2.5 μm using He–Ne laser diffraction and the ﬁbre width and length were measured under light microscopy; ﬁbre length (L0) was ∼2 mm in most experiments. The force responses to a standard T-jump of 3◦C were examined after a ﬁbre was activated to steady isometric state and then exposed to steady shortening and lengthening at different velocities. A T-jump was applied 200–500 ms after the beginning of a ramp shortening/lengthening when the force during ﬁlament sliding reached a steady level; this was approximately so for most shortening velocities, so that the pre-T-jump tension represented the steady shortening tension without a T-jump. With lengthening, however, there was a continued slow tension rise and the tension level without a T-jump was estimated by a curve (exponential) ﬁtted to the late pre-T-jump tension trace. In some experiments, tension records were made with and without T-jumps at the same velocities (see Figs 1 and 2). Fibres were regularly examined Figure 1. Tension responses to lengthening, shortening and T-jump from one ﬁbre The ﬁbre held isometric was maximally Ca2+ activated at ∼9◦C and, during the steady isometric tension plateau (P0), a ramp lengthening (A, ∼0.06 L0 s−1; B, ∼0.1 L0 s−1) or a ramp shortening (C, ∼0.05 L0 s−1; D, ∼0.065 L0 s−1) of ∼5% L0 was applied; dotted lines indicate the isometric base levels. The tension rises above P0 during lengthening and falls below P0 during shortening to approximately steady levels. The bottom trace in each frame is the position of the ﬁrst order diffraction (sarcomere length, SL (μm)) from a He–Ne laser beam that passed through a ∼0.5 mm length of the ﬁbre; the signal does not indicate marked changes due to a T-jump. As shown by the thermocouple record (the second trace from the top in each frame) in B, C, D and E, a T-jump of ∼3◦C was induced by a < 0.2 ms, near-infrared, laser pulse applied to the ﬁbre and the solution bathing it; the initial peak and slow decay are due to direct heat absorption by the thermocouple (Goldman et al. 1987). E shows the tension response to an identical T-jump when the ﬁbre was held isometric. Note that when the ﬁbre is isometric (E) or shortening (C and D), a T-jump induced a tension rise to a new steady level; on the other hand, during ramp lengthening when the tension rises to a new level, a T-jump produces little tension rise. C 2007 The Authors. Journal compilation C 2007 The Physiological Society