Callamaras N , Parker I .

GM-48071 NIGMS NIH HHS , R01 GM048071 NIGMS NIH HHS , R37 GM048071 NIGMS NIH HHS

	Figure 2. Optical resolution of the axial scanning system. (A) Images of fluorescent pollen grains (mixed pollen grain slide; Carolina Biological Supply). The upper frame shows a z-y image, obtained using the piezo drive to scan rapidly (100 Hz) in the z axis, while the laser spot was scanned slowly in the y axis by a rotating mirror. For comparison, the lower frame shows a conventional, lateral x-y scan of a matching region of a different pollen grain, obtained using a galvanometer-driven mirror to scan rapidly in the x axis. (B) Images illustrating the lateral resolution of the microscope when focused to different depths into an oocyte. Each frame shows the tip of a fluorescein-filled micropipette that was inserted through an oocyte to different depths (indicated in micrometers) relative to the pigment granules on the lower (vegetal) side of the oocyte. See text for further details. Images are individually normalized to the peak intensity within the frame. (C) Measurements of absolute peak fluorescence from images like those in B, plotted as a function of distance into the oocyte, relative to the pigment granules. (D) Measurements of the full-width at half-maximal intensity from images like those in B.
	Figure 3. Radial distribution of calcium puffs evoked by photoreleased InsP3. (A) Radial scan images showing puffs and calcium wave evoked by brief photolysis flashes. Distance into the oocyte is depicted vertically, and time runs from left to right. The cell surface corresponds to the top of the image, and increasing calcium levels (increasing fluorescence ratio F/Fo of Oregon green-1) are depicted by increasingly “warm” colors. Each image was independently scaled so that the maximal fluorescence is depicted as red. Photolysis flashes were applied at varying times before each record. Flashes in a, b, and d evoked only calcium puffs, whereas that in c was sufficiently strong to just evoke a calcium wave. The trace in d shows fluorescence monitored at the active site in the image. (B) Photorelease of fluorescein from a caged precursor, demonstrating uniformity of photolysis at different depths in the oocyte. Radial scan image shows responses to two identical photolysis flashes, delivered when indicated by the arrows. (C) Lateral (x) scan images showing puffs evoked by sustained photorelease of InsP3 with the microscope focussed at different depths. The oocyte was continuously exposed to strongly attenuated photolysis light, beginning ∼2 min before recording. Images were acquired sequentially at the granule layer (a), at a depth 11 μm further into the cell (b), and after refocusing at the granule layer (c). (D) Examples of different puff morphologies, imaged by radial scanning. (a and b) Averaged images showing, respectively, presumptive single- and multiple-site puffs. Each frame is an average of six individual events that were selected by eye. Averages were formed after aligning events in space and time, and do not indicate the positions of the events relative to the cell surface. Both images are shown on a common pseudocolor scale, corresponding to fluorescence ratios (F/Fo) as indicated by the color bar. (c) An individual event arising through near-synchronous release at two radially separated sites. (d) A puff preceded by a blip (arrow).
	Figure 5. Calcium release at depths below the puff sites. (A) Radial linescan image in response to a suprathreshold, wide-field flash shows calcium liberation beginning independently after varying latencies at different depths. (B) Abrupt calcium release evoked with short latency at all depths by a strong photolysis flash, with an intensity ∼10× that required to just evoke a calcium wave. The image was processed by rebinning the original record to expand five times in time and two times in space, and then applying three iterations of a 3 × 3 pixel smoothing function. A faint artifact is present during the photolysis flash. (C) Diffusive spread of calcium, shown on the same scales for comparison with the InsP3-evoked response in C. The image was obtained by lateral (x) scanning, and shows the spread of calcium released by local “photoperforation” induced by a 5-ns pulse from a frequency-tripled Nd-YAG laser focused to a spot (∼1-μm diameter) at the site indicated by the asterisk. The oocyte was loaded with Oregon green-1, without caged InsP3. (D–F) Calcium release evoked by laser spot photolysis at different depths into the cell. A brief (5-ns) pulse of UV light was synchronized with the piezo-scan so that photolysis could be induced in an albino oocyte at a virtual “point” source at any desired depth throughout the scan. Axial scan images show responses at the same lateral position when the laser pulse was focussed to a spot (indicated by white arrows) near the granule layer (D) and at a depth ∼15 μm further into the cell (E). F shows an example where photorelease ∼25 μm into the oocyte evoked both a superficial puff and sustained calcium release beginning near the photolysis site.
	Figure 7. Morphology of the oocyte and correspondence of puff sites with localization of the ER and of InsP3 receptors. (A–C) Confocal sections into the vegetal hemisphere of oocytes, with the vertical axis representing depth into the cell (z axis) and the horizontal axis lateral (y) position. Images were obtained by rapidly scanning in the x axis using a galvanometer mirror while slowly advancing the microscope focus using a synchronous motor. The aspect ratio provided square pixels. (A) Resting fluorescence of an oocyte loaded with Oregon green-1. (B) Distribution of ER stained with the lipophyllic dye fast DiI. (C) Distribution of InsP3 receptors visualized using an anti–InsP3 receptor antibody and an FITC-conjugated secondary antibody. (D–F) Images derived from those in A–C, by enhancement to partially compensate for the fall off in fluorescence at increasing depths as a result of light scattering and absorption. Fluorescence in all images was scaled by a correction factor increasing linearly from 1 at the level of the pigment granules (about one third the way down each image) to 3 at the bottom of the images. (G) Profile of fluorescence (arbitrary scale) as a function of depth, averaged across a 15-μm section in the y axis of A. (H) Histogram shows frequency of puff occurrence as a function of depth into the cell, replotted from Fig. 4 A, after scaling to match the dimensions of the images. (I) Distribution of InsP3 receptors in the animal hemisphere of a physically sectioned oocyte. The image shows a lateral (x–y) confocal scan focused a few micrometers below the cut surface of the cell. Receptors were visualized using an anti–InsP3 receptor antibody and Cy3-conjugated secondary antibody. The staining in this image is more dense and less obviously punctate than that in C, probably because the animal hemisphere shows a higher density of InsP3 receptors and more closely packed functional release sites than does the vegetal hemisphere (Callamaras et al., 1998b). Diffuse fluorescence at the lower left arose primarily through incomplete blocking of autofluorescence by the barrier filter. Scale bar applies to all images in the figure.
	Figure 4. Puffs originate in a thin band close to the depth of the pigment granules, and often involve concerted activation of multiple radial sites. (A) Radial distribution of puffs evoked by photolysis flashes with strengths below the wave threshold. Measurements of axial puff locations were made from images like those in Fig. 3 A, and are plotted with respect to the position of pigment granules in each scan. In the case of presumptive multiple site puffs, locations were estimated by eye, assuming involvement of two release sites (e.g., Fig. 3 A, c, arrows). Data are from 83 events, recorded at 20 sites in the vegetal hemisphere of five oocytes. Similar results (not shown) were obtained in two further experiments on oocytes from different donor frogs. (B and C) Puffs in the vegetal hemisphere rarely involve multiple, laterally separated release sites, whereas they frequently involve synchronous activation of radially separated sites. Histograms show distributions of puff widths (full width at half-maximal amplitude) recorded in the vegetal hemisphere by axial (x) line scanning (B) and lateral (z) scanning (C). Axial measurements are from eight oocytes, and lateral measurements from five other oocytes.
	Figure 6. Lateral (x) linescan imaging of calcium release evoked at different depths by strong photolysis flashes. (A) Images show calcium release monitored along a 50-μm lateral scan line with the microscope focussed at different depths into the oocyte (indicated in micrometers to the right of each frame relative to the pigment granule layer). Identical photolysis flashes (20 ms duration) with a strength ∼10× the wave threshold were delivered when marked by the arrow. (B) Corresponding measurements of fluorescence ratio averaged across a 30-μm wide region in the center of the images in A. The timing of the photolysis flashes is evident from the small artifacts. (C) The peak fluorescence signal during calcium waves remains roughly constant at increasing depths into the oocyte. Points show measurements of peak fluorescence ratio obtained from records like those in B, plotted as a function of depth relative to the pigment granules. Data are from two oocytes loaded with Oregon green-1 (filled symbols) and one oocyte loaded with the low-affinity indicator Calcium green-5N (open symbols). (D) Variation in latency of calcium signal (measured from beginning of photolysis flash to half-maximal rise of fluorescence) with depth into cell. Filled symbols show measurements from two oocytes loaded with Oregon green-1 in response to moderate (squares) and strong (circles) photolysis flashes, with respective strengths ∼10× and 50× the wave threshold. Open symbols show measurements from an oocyte loaded with Calcium green-5N, and stimulated by strong photolysis flashes.

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