SUPPORTING INFORMATION
Legends for SUPPLEMENTAL FIGURES:
Fig. S1. Fluorescence intensity of polymers inside cells. (A) Same images as Fig.
1(B), except that ROI #0 corresponding to the background level in Fig. 1(C) is indicated.
(B) Time course of the fluorescence intensity of the fluorescent thermosensitive
polymers in the ROIs indicated in (A). Numbers 0-3 represent the ROIs analyzed. The
large grey column shows the time during which the microheater was illuminated by the
laser beam.
Fig. S2. Response of the cells to heat pulses in the presence of 2 mM EGTA at 22
C. (A) Bright field image of cells and pipettes. Colored numbers show the cells
analyzed. The red circle indicates the position of the microheater. The blurred pipette is
the microthermometer. (B) Fluorescence image of Fluo-4. Cells are numbered as in (A).
Scale bar = 10 m. Brightness and contrast of images in (A) and (B) were artificially
enhanced for display purposes. (C) Time course of the fluorescence intensity of Fluo-4
for each cell. The level of each record was arbitrarily shifted along the y axis for display
purposes. The vertical axis shows fluorescence intensity; tickmarks indicate steps of 20
a.u., and grey columns show the time during which the microheater was illuminated by
the laser beam. Imaging and analysis were performed with the setup described in
Materials and methods, Microscopy, (B).
Fig. S3. Response of the cells to heat pulses in the presence of 2 mM CaCl2 with 2
M thapsigargin at 22 C after pretreatment with 2 mM CaCl2 and 2 M
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thapsigargin for 1 h at 22 C. (A) Bright field image of cells and a microheater.
Colored numbers show cells analyzed. The red circle indicates the position of the
microheater. (B) Fluorescence image of Fluo-4. Cells are numbered as in (A). Scale bar
= 10 m. Brightness and contrast of images in (A) and (B) were artificially enhanced
for display purposes. (C) Time course of the fluorescence intensity of Fluo-4 for each
cell. Grey columns show the time during which the microheater was illuminated by the
laser beam. Imaging and analysis were performed with the setup described in Materials
and methods, Microscopy, (B).
Fig. S4. Response of the cells to heat pulses in the presence of 100 M ryanodine at
37 C after pretreatment with 100 M ryanodine for 30 min at 22 C. (A) Bright
field image of cells and a microheater. Colored numbers show the cells analyzed. The
red circle indicates the position of the microheater. (B) Fluorescence image of Fluo-4.
Cells are numbered as in (A). Scale bar = 10 m. Brightness and contrast of images in
(A) and (B) were artificially enhanced for display purposes. (C) Time course of the
fluorescence intensity of Fluo-4 for each cell. (D and E) Magnified views of the parts of
(C). The grey columns show the time during which the microheater was illuminated by
the laser beam.
Fig. S5. Response of the cells to heat pulses and 100 M histamine at 22 C after
the injection of either Ca2+-free buffer (cells No. 1 to 4 as controls) or heparin (cells
No. 5 to 8). (A) Bright field image of the cells and a microheater. Colored numbers
show the cells analyzed. The red circle indicates the position of the microheater. (B)
Fluorescence image of Fluo-4. Colored numbers and the position of the red circle are
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the same as in (A). Scale bar = 10 m. Brightness and contrast of images in (A) and (B)
were artificially enhanced for display purposes. (C) Time course of the fluorescence
intensity of Fluo-4 for each cell. The grey columns show the time during which the
microheater was illuminated by the laser beam. Numbers inside the grey columns
indicate laser power in mW measured between the telescope lenses. The horizontal bar
indicates the presence of 100 M histamine. The average frame rate was 52.4 frames/s
(left) and 31.5 frames/s (right). In these measurements, the cells into which Ca2+-free
buffer was injected (cells No. 2 and 3) showed small Ca2+ overshoot after the first heat
pulse with a 14.9 mW laser beam. Increasing the laser power to 21.8 mW increased T
and, therefore, induced larger Ca2+ overshoot. The cells into which heparin was injected
(cells No. 5 to 8), however, did not respond even to further increase in laser power until
water boiled near the microheater. When the injection procedures were not successful,
the fluorescence intensity of Fluo-4 increased to unusual levels (such as in cells No. 1,
5, and 7). A response to histamine was never observed in these cells, even in the control
cells. They were counted as "cells not responding", as shown in Table S1. (D)
Magnified views of the parts of (C).
Fig. S6. Correlation of the amplitude of temperature change (T) with the strength
of Ca2+ overshoot (F/F0) at 37 (A), 30 (B), 27 (C) and 22 C (D). Imaging and
analysis were performed with the setup described in Materials and methods, Microscopy,
(A), except for (D), right, which was obtained with Microscopy, (B).
Fig. S7. Response of the cells to heat pulse and [Ca2+]cyt oscillation observed at 22
C. (A) Bright field image of cells and a microheater. Colored numbers show the cells
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analyzed. The red circle indicates the position of the microheater. (B) Fluorescence
image of Fluo-4. Cells are numbered as in (A). Scale bar = 10 m. Brightness and
contrast of images in (A) and (B) were artificially enhanced for display purposes. (C)
Time course of the fluorescence intensity of Fluo-4 for each cell. Cell No. 5 shows the
spontaneous increase in [Ca2+]cyt, sometimes observed in the absence of heat pulse. The
narrow grey column shows the time during which the microheater was illuminated by
the laser beam.
Fig. S8. Analysis of the rate of fluorescence intensity change. The upper and lower
panels show the time course of the fluorescence intensity of Fluo-4 and the rate of
fluorescence intensity change of Fluo-4, respectively, for cell No. 1 in Fig. 2. For the
rate of fluorescence intensity change, the time course of the fluorescence intensity was
first median-filtered using a 3-point moving window, then the derivative of two
consecutive data points was calculated and plotted. The grey column shows the time
during which the microheater was illuminated by the laser beam.
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Legends for SUPPLEMENTAL VIDEOS:
Video 1. Response of cells to single heat pulse at 37 C played at 2 real time.
Video 2. Response of cells to 11 heat pulses at 37 C played at 5 real time.
A microheater on the right became bright as a result of the scattering of the incident
infrared laser beam when the shutter was opened. In these movies, brightness and
contrast of images have been artificially enhanced. Raw recordings of these two movies
were analyzed (see Fig. 2).
Video 3. Convection of the water flow during heating with a laser power of 8.7
mW, in real time (top) and the temperature field profile obtained by a
microthermometer (bottom).
Video 4. Convection of the water flow during heating with a laser power of 3.0
mW, in real time (top) and the temperature field profile obtained by a
microthermometer (bottom).
Video 5. Convection of the water flow during heating with a laser power of 0.7
mW, in real time (top) and the temperature field profile obtained by a
microthermometer (bottom).
Convection of the water flow was observed through the use of fluorescent microspheres
( = 0.2 m). In the temperature field profiles, the amplitude of the temperature change
(T) on the y axis was plotted against the distance (d) between the microheater and the
microthermometer along the x axis. Laser power was measured between the telescope
lenses. Since the temperature field profiles depended on the laser power and the shape
of the microheater, they were always obtained immediately after each set of the
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application of heat pulse. Under conditions in these movies, many cells located outside
the region where the convection of the water flow was observed, showed Ca2+
overshoot. Therefore, we conclude that the convection of the water flow is not the cause
of Ca2+ release.
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