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1 Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG UK
2 Departments of Physiological Science and Ophthalmology, University of California, Los Angeles, CA 90095-1606, USA
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Abstract |
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(Received 4 September 2006;
accepted after revision 21 November 2006;
first published online 23 November 2006)
Corresponding author H. R. Matthews: Physiological Laboratory, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge CB2 3EG, UK. Email: hrm1{at}cam.ac.uk
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Introduction |
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In cones, there is reason to believe that the relationship between channel closing and [Ca2+]i may not be so simple. The operating range of the cone spans a much greater range of light intensities than a rod (see for example Burkhardt, 1994). Furthermore, light-dependent release of Ca2+, which is known also to occur in cones (Brockerhoff et al. 2003; Cilluffo et al. 2004), has been shown to be more sensitive to illumination than in rods (Cilluffo et al. 2004). It is therefore conceivable that light-dependent release of Ca2+ from a cytosolic store or buffer site might occur within the normal operating range of the cone and be sufficiently large to affect the [Ca2+]i.
We have therefore examined the relationship between photocurrent and [Ca2+]i in the cones of zebrafish. The zebrafish retina contains four morphologically distinct cone classes (Branchek & BreMiller, 1984; Robinson et al. 1993), one of which contains a UV-sensitive pigment with its spectral maximum of absorption (
max) at about 360 nm (Robinson et al. 1993; Cameron, 2002). These cones are easily identifiable in preparations of dissociated photoreceptors, and they provide a particularly favourable preparation for measurement of [Ca2+]i. The reason for this is that the sensitivity of the UV-sensitive cone to the 514.5 nm line of an argon laser is expected to be approximately six orders of magnitude less than at the wavelength of peak UV cone pigment sensitivity (362 nm). Therefore the intensities normally used to measure [Ca2+]i bleach only a small fraction of the UV-sensitive cone photopigment during each fluorescence measurement. Consequently, multiple measurements of fluorescence had a much smaller effect on the long-term sensitivity and [Ca2+]i, allowing repeated measurements to be made for the first time from the same cell.
We have used UV-sensitive zebrafish cones to explore the relationship between [Ca2+]i and photocurrent. We find that for flashes of moderate intensity, the [Ca2+]i declines during the flash and then recovers with a time course predicted from the change in circulating current and time-dependent extrusion of Ca2+ from the cell. The [Ca2+]i also varies in parallel with the photocurrent in steady background light provided the light is dim, but in brighter backgrounds the [Ca2+]i is significantly greater than would be expected at short times after the presentation of the background. This anomalous increase in [Ca2+]i is caused by light-dependent Ca2+ release. Preliminary results of this study have been reported both to the Physiological Society (Leung et al. 2002, 2004) and to the Association for Research in Vision and Ophthalmology (Fain et al. 2004).
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Methods |
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Zebrafish (Danio rerio) 4–5 cm in length were purchased from a local supplier, maintained at 28°C on a 12 h light–12 h dark cycle, and fed tropical fish food. Animals were dark-adapted for 2–3 h and killed in dim red light by stunning by cranial concussion followed by decapitation and pithing, according to Schedule I of the Animals and Scientific Procedures Act. An eye was torn open with fine forceps and the retina peeled away. Photoreceptors were dissociated mechanically by trituration from the isolated retina under infrared illumination in Ringer solution of the following composition (mM): NaCl 111, KCl 2.5, MgCl2 1.6, CaCl2 1.0, EDTA 0.01 and HEPES 3; pH adjusted to 7.7–7.8 with NaOH. In experiments in which Ca2+ concentration was measured, dissociated cells were incubated for 30 min in darkness with 10 µM fluo-4 acetoxymethyl ester (fluo-4 AM; Invitrogen (Molecular Probes), Carlsbad, CA, USA) as previously described (Brockerhoff et al. 2003; Matthews & Fain, 2003; Cilluffo et al. 2004).
An isolated cone photoreceptor was drawn inner segment first into a suction pipette so that the outer segment was exposed to the bathing solution. Cells were normally superfused with Ringer solution, as we found no improvement in amplitude of circulating current or recording stability with prepared tissue culture media. In some experiments, rapid solution changes from Ringer solution to 0Ca2+–0Na+ solution were made by translating the photoreceptor between two rapidly flowing streams of solution, as previously described (Matthews & Fain, 2003). The 0Ca2+–0Na+ solution was identical in composition to the 0Ca2+–0Mg2+–0Na+ solution employed previously (Matthews, 1995, 1996) and consisted of (mM): choline chloride 111, KCl 2.5, EGTA 2.0 and HEPES 3.0, pH adjusted to 7.7–7.8 with tetramethylammonium hydroxide. BAPTA-AM was obtained from Calbiochem (Nottingham, UK). All experiments were performed at 22°C.
Ca2+ concentration measurement, recording and light stimulation
Ca2+ concentration was measured as previously described (Brockerhoff et al. 2003; Matthews & Fain, 2003; Cilluffo et al. 2004). In brief, dye fluorescence was excited by an air-cooled argon ion laser (Model 60, American Laser Corporation, Salt Lake City, UT, USA), tuned to 514.5 nm. The absolute intensity of the laser was adjusted with a reflecting neutral density filter (Newport Corporation, Irvine, CA, USA) to a nominal intensity of 1.4 x 1011 photons µm–2 s–1, in order to prevent excessive dye bleaching (Matthews & Fain, 2002, 2003); measurements of beam intensity were made with a calibrated silicon photodiode optometer (Model S370, Graseby Optronics, Orlando, FL, USA), whose detector was placed at the position normally occupied by the recording chamber. Fluorescence was collected with a 525 nm dichroic mirror and a 530 nm long-pass emission filter (Types 525DRLP and 530ALP, Omega Optical, Brattleboro, VT, USA). A beam stabilizer was used to decrease noise from laser intensity fluctuations (Noise Eater, Model CR-200 A, Thorlabs, Newton, NJ, USA). Fluorescence intensity was measured with a photomultiplier tube (PMT, Model 9130/100 A, Electron Tubes Ltd, Ruislip, UK). The signal from the PMT was amplified with a low-noise current-to-voltage converter (Model PDA-700, Terahertz Technology Inc., Oriskany, NY, USA), low-pass filtered at 500 Hz with an active eight-pole Bessel filter (Kemo Ltd, Beckenham, UK), and digitally sampled at 2 kHz. Electrical responses were normally low-pass filtered at 40 Hz. Calculations and curve fitting were carried out with the programs MathCad (Mathsoft, Inc., Cambridge, MA, USA) and Origin (OriginLab Corp., Northampton, MA, USA).
Light stimuli were delivered as previously described (Matthews & Fain, 2003) via a dual-beam optical bench, whose light source was a high-intensity mercury discharge lamp (Lumatec SUV-DC, Ultrafine Technology, Brentford, UK). The two beams passed through narrow-band interference filters (Comar Instruments, Cambridge, UK), calibrated neutral density filters (New Focus, San Jose, CA, USA), and high-speed shutters (Vincent Associates, Rochester, NY, USA). The absolute intensity of the light from the bench was measured as for the laser. Rhodopsin bleaching was calculated from the photosensitivity of a vitamin A1-based pigment in solution (Dartnall, 1972), corrected for the difference in dichroism in free solution and in disk membranes (Jones et al. 1993).
Multiple fluorescence measurements from the same cone
We chose UV-sensitive cones for our study so that a visible laser could be used to make successive measurements of fluorescence without undue bleaching of the cone photopigment or alteration in the resting Ca2+ concentration. To demonstrate the feasibility of this approach, multiple measurements were made repeatedly from the same cone. These data are summarized in Fig. 1, which shows the mean photomultiplier currents for successive fluorescence measurements from each of 20 cones, normalized for each cell to the current of the first measurement. Laser flashes were delivered at > 1 min intervals, a period sufficient to permit the circulating current and Ca2+ concentration to stabilize between measurements.
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Results |
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max of about 360 nm (Robinson et al. 1993; Cameron, 2002). These cones can be readily identified in preparations of dissociated photoreceptors (see Fig. 2A, inset) and have responses that resemble those of the visible cones (Brockerhoff et al. 2003; Cilluffo et al. 2004); the maximum amplitude of these responses was variable but could be as large as 15–20 pA (Fig. 2A). In subsequent experiments designed to measure dye fluorescence, the cone was typically drawn less far into the suction pipette so as to allow the entire outer segment to be visualized. Consequently, a considerably smaller proportion of the photocurrent was recorded in these experiments.
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max of 362 nm (Robinson et al. 1993): the zebrafish, although a freshwater fish, has been shown to use the A1 instead of the A2 chromophore (Nawrocki et al. 1985; Taylor et al. 2000; Cameron, 2002).
Although the fit at the shorter wavelengths is good, the cones are of the order of 100 times more sensitive at 546 nm than predicted by the 362 nm nomogram. As UV-sensitive cones in other species have been shown to contain small quantities of visible photopigments (see for example Makino & Dodd, 1996; Lyubarsky et al. 1999; Applebury et al. 2000), it seemed likely that the extra sensitivity at long wavelengths in Fig. 2B is produced by co-expression in the short single cones of one or more visible opsins. To address this possibility, the equations of Lamb (1995) were used with the parameters of Govardovskii et al. (2000) to fit the data to two alternative expressions, both consisting of a sum of two nomograms. In both cases, the majority of the action spectrum could be accounted for by a pigment with max of 362 nm, but the sensitivity at 546 nm required the addition of either 0.013% of a pigment with max of 480 nm, or 0.0027% of a pigment with max of 570 nm. The satisfactory fit of these expressions demonstrates that the amount of visible pigment in the UV-sensitive cones is likely to be quite small in proportion to the amount of UV-sensitive pigment. In our subsequent experiments to measure calcium indicator dye fluorescence, this small proportion of visible photopigment will have been almost completely bleached by the first laser exposure, with only negligible effects on subsequent Ca2+ measurements (see Fig. 1).
In Fig. 2C, sensitivity measurements are summarized from eight cones in the presence of steady background light. Sensitivity was measured by presenting for each background a flash which evoked a just-detectable response and then dividing the response amplitude by the flash intensity (Baylor et al. 1974). The mean sensitivity values have been fitted with the Weber-Fechner law. The data in Fig. 2C show that zebrafish UV-sensitive cones adapt to background lights in a manner similar to cones in other species (turtle: Baylor & Hodgkin, 1974; salamander: Matthews et al. 1990; primate: Schnapf et al. 1990; Burkhardt, 1994).
Light-dependent changes in fluorescence
Previous experiments with calcium indicator dyes have shown that light produces a decrease in photoreceptor [Ca2+]i in rods in a variety of species (see Fain et al. 2001) and in the outer segments of visible cones (Sampath et al. 1999; Cilluffo et al. 2004). The light-dependent decrease is accompanied by a rapid fluorescence increase (Matthews & Fain, 2002; Woodruff et al. 2002; Cilluffo et al. 2004), which is unaffected by incorporation of BAPTA and is therefore not produced by a change in [Ca2+]i but rather by some interaction between the indicator dye and the visual pigment. As this initial increase is superimposed upon the light-dependent decrease, it is important to determine its time course before measurements are made of changes in fluorescence during the light response.
In the experiment of Fig. 3A, UV-sensitive cones were illuminated in Ringer solution with seven consecutive flashes from the laser, the first of 50 ms and the remaining of 20 ms duration. Even though the laser was tuned to 514.5 nm, a wavelength to which the cone was relatively insensitive, the intensity of the laser was sufficiently bright so that the first laser flash was equivalent to a light delivering about 6.1 x 103 photons µm–2 at the wavelength of maximum sensitivity of the UV-cone pigment. Comparison with Fig. 2A demonstrates that this was sufficiently bright largely to suppress the circulating current, which will have been held near saturation by subsequent flashes, thereby suppressing Ca2+ influx through the cGMP-gated channels of the outer segment. The progressive decline of fluorescence induced by this sequence of laser flashes averaged from six cones resembles that from rods and cones in other species (for example Gray-Keller & Detwiler, 1994; Sampath et al. 1998, 1999; Woodruff et al. 2002; Brockerhoff et al. 2003; Cilluffo et al. 2004) and is indicative of a light-dependent decline of [Ca2+]i.
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Change in [Ca2+]i during the flash response
As control measurements demonstrated the feasibility of making repeated [Ca2+]i determinations from the same cell (see Fig. 1), we sought to measure the change in [Ca2+]i simultaneously with the change in current during the response to a brief flash. The design of these experiments is illustrated in Fig. 4 (see light monitor traces). An isolated UV-sensitive cone was illuminated with a series of flashes at 405 nm, which were 20 ms in duration and delivered 1.4 x 105 photons µm–2, the equivalent of about 1.5 x 104 photons µm–2 at the max of the UV-sensitive photopigment, based on the measured ratio of sensitivity at the two wavelengths (see Fig. 2B). Figure 4A shows the response of the cone to this flash averaged from presentations before and after the fluorescence measurement. This flash intensity was chosen to fall short of response saturation, and will have suppressed the majority of the dark current, much as in the earlier study of Gray-Keller & Detwiler (1994).
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In Fig. 5, results from this protocol are summarized from 20 UV-sensitive zebrafish cones. Figure 5A shows the mean photocurrents from these cells normalized to the maximum response amplitude of each cell for this flash intensity. The peak of the averaged response is less than 1.0 because the responses in different cells reached peak amplitude at slightly different times. Two responses are shown, the first recorded before the sequence of fluorescence measurements, and the second directly afterward. These are very similar, showing that the fluorescence measurements (and the presentation of the repeated flashes before each of the measurements) did not significantly cause light adaptation of the cell.
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We therefore adopted the following protocol to isolate the underlying time constant for Ca2+ extrusion alone (see Matthews & Fain, 2001; Cilluffo et al. 2004). The cone outer segment was first rapidly moved into 0Ca2+–0Na+ solution, and after 1 s the outer segment was exposed to steady illumination from the laser, which in these experiments was tuned to 488 nm instead of 514.5 nm to maximize bleaching of the photopigment. During the 5 s of laser exposure, there was a gradual increase in fluorescence in 0Ca2+–0Na+ solution, which we will show below is blocked by incorporation of BAPTA (see Fig. 8) and therefore represents light-dependent Ca2+ release. After most of the Ca2+ had been released, the cone outer segment was then rapidly moved back into Ringer solution. The time course of the change in fluorescence after the return to Ringer solution, which reflects the time course of decline of [Ca2+]i, largely free of interference from Ca2+ release, could be fitted by a single exponential decay with an average time constant of 255 ms (n = 5 cells), which is similar to the time constant that dominates the decline of [Ca2+]i in zebrafish visible cones (Cilluffo et al. 2004).
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In a further series of experiments, we attempted to investigate the time course of [Ca2+]i decline during the flash in greater detail, using a protocol similar to that of Fig. 4 but with 13 fluorescence measurements after the delivery of the flash instead of just four, and a higher flash intensity. The average time course of the fluorescence change evoked by the flash is shown from 10 cones in Fig. 6A. The flash resulted in an initial decrease in fluorescence followed by recovery towards the dark level qualitatively similar to the results of Fig. 5. However, comparison of the averaged normalized photocurrent responses before and after the fluorescence measurements revealed that the waveform of the flash response was accelerated over the course of the experiment (Fig. 6B), most probably as the result of adaptation resulting from cumulative photopigment bleaching by the repeated optical bench and laser flashes. Nevertheless, the dye fluorescence fell following the flash in a qualitatively similar manner to the rising phase of the light response. Furthermore, the return of the fluorescence signal towards its original level took place monotonically after the response peak, reaching 94% of its pre-existing value in darkness 1 s after the flash, a time at which the circulating current had mostly, but not completely, recovered.
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The experiments of Figs 4–6 show that the change in [Ca2+]i follows the change in photocurrent, although with a delay caused by Ca2+ removal by the Na+–Ca2+–K+ exchanger and buffering within the outer segment. We next investigated whether a similar correlation between photocurrent and [Ca2+]i occurs at steady state, during the presentation of constant background illumination.
To investigate the relationship between circulating current and [Ca2+]i, an isolated cone was exposed to a series of backgrounds of different intensity, and a flash from the laser was superimposed on each of these, in order to measure the dye fluorescence from the outer segment. Fig. 7 shows collected average measurements of photocurrent (Fig. 7A) and dye fluorescence (Fig. 7B and C). The photocurrent responses, measured after stabilization of the response to the background, were normalized to the current response for each cone evoked by a saturating flash in darkness, and the dye fluorescence evoked by a laser flash superimposed on each background was normalized cell by cell to the fluorescence evoked in darkness. The axis for photocurrent has been inverted, so that the decrease in response can be correlated with the decrease in fluorescence.
When the laser flash was presented 5 s after the onset of steady background illumination (Fig. 7B), a time sufficient for the response to stabilize, the progressive decline in dye fluorescence paralleled the monotonic suppression of the dark current by increasing background light intensity. This result is consistent with the notion that in UV-sensitive cones, as in rods, the steady level of [Ca2+]i is determined by the balance of Ca2+ fluxes across the outer segment membrane over the full range of background light intensities. However, when measured 600 ms after background onset (Fig. 7C), although dye fluorescence decreased proportionally with current for dim backgrounds, on the two brightest backgrounds it showed a surprising further increase.
Light-dependent Ca2+ concentration increase in UV-sensitive cones
We hypothesized that the increase in bright backgrounds at early times might be produced by a transient light-dependent release of Ca2+ in the cone outer segment similar to that previously described in salamander rods (Matthews & Fain, 2001, 2002, 2003) and zebrafish visible cones (Cilluffo et al. 2004). This possibility was tested in the following way. The cone outer segment was first rapidly moved to 0Ca2+–0Na+ solution, then 1.5 s later exposed to a series of 50 ms laser flashes, and finally returned to Ringer solution (not shown) for a 35–40 s recovery period before subsequent presentations. The dye fluorescence excited by these flashes in a typical cone is shown in Fig. 8A. The slow increase in fluorescence in 0Ca2+–0Na+ solution is similar to that previously seen in other photoreceptors and is indicative of light-dependent Ca2+ release evoked by the laser exposures.
Next, the laser flashes were delivered again in 0Ca2+–0Na+ solution to the same cone after exposure for 1 s to the brightest background used for the experiments of Fig. 7 (6.4 x 106 photons µm–2 s–1 at 405 nm, equivalent to about 6.9 x 105 photons µm–2 s–1 at the max of the photopigment). The corresponding dye fluorescence evoked by the laser flashes is shown in Fig. 8B, which reveals that the slow increase in fluorescence no longer occurs after the background exposure. Finally, for each cone, the fluorescence measurements were repeated again without prior background illumination, as in Fig. 8A.
Mean measurements of dye fluorescence with and without the background are given for 12 cones in Fig. 8C. The triangles show the fluorescence measured as in Fig. 8A in the absence of a background, normalized for each cell to the fluorescence evoked by the first laser flash. Two series of measurements are shown in darkness (as in Fig. 8A), before (upright triangles) and after (inverse triangles) the measurements made with a prior background light exposure. In both cases, the normalized fluorescence increased to a peak and then slowly decayed thereafter. The similarity of these two sets of data confirms our ability to make multiple fluorescence recordings from the same cell and shows that background exposure did not have an irreversible effect on the control of outer segment [Ca2+]i.
The filled circles show mean measurements from the same cells with prior background light exposure as in Fig. 8B, again normalized for each cell to the fluorescence evoked by the first laser flash. It can be seen that immediately following background exposure the slow increase in fluorescence is no longer present. To show that the increase in fluorescence was produced by an increase in [Ca2+]i, this protocol was repeated on a further nine cones that had been preincubated with 50 µM BAPTA-AM. No background light was delivered. These results have been plotted as the open circles of Fig. 8C. Exposure to BAPTA also eliminated the slow increase in fluorescence, demonstrating that in the UV-sensitive cones, as in zebrafish visible cones (Cilluffo et al. 2004) and salamander rods (Matthews & Fain, 2001), the slow increase in fluorescence in 0Ca2+–0Na+ solution represents a rise in [Ca2+]i resulting from light-induced Ca2+ release evoked by the laser flashes. As prior background illumination also abolishes this rise in [Ca2+]i, we infer that this intensity of background light is sufficient to evoke Ca2+ release, and can explain the rise in [Ca2+]i in bright light in Fig. 7C.
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Discussion |
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We have used the UV-sensitive cones to compare the time course of the change in Ca2+ concentration with the waveform of the flash response. Because even in UV-sensitive cells visible light can cause a small bleaching of photopigment, repeated laser exposure and repeated exposure to the light used to evoke the flash response can eventually cause the cell to adapt and change the response kinetics. We therefore used two different approaches. In the first (Fig. 4), we employed a sufficiently small number of measuring time points so that the waveform of the flash response after all of the Ca2+ measurements had been made was not significantly different from that before (Fig. 5A). The mean waveform convolved with the dominant time constant for Ca2+ removal from the cell was then in excellent agreement with the change in normalized dye fluorescence around the peak of the flash response (Fig. 5B). These measurements have the weakness, however, that they do not exclude the possibility of a small and slowly recovering component of reduced [Ca2+]i at later times, like that reported previously in Gekko rods (Gray-Keller & Detwiler, 1994). We therefore redesigned our protocol with a larger number of measurements, and a somewhat higher flash intensity. Although a change in response kinetics was now evident over the course of the experiment (Fig. 6B), the initial decline in dye fluorescence paralleled closely the normalized rising phase of the light response, which is not influenced by light adaptation (Pugh et al. 1999). Furthermore, the fluorescence signal returned to within a few percent of its pre-existing level in darkness by 1 s after the flash, when the circulating current had also nearly recovered. These results provide no support for a maintained component of Ca2+ decline following the responses of flashes of two different intensities, and like previous measurements from salamander rods (Matthews & Fain, 2003) are consistent with the notion that changes in [Ca2+]i during the flash response can be accounted for by Ca2+ fluxes across the outer segment membrane.
In the presence of steady background light, the [Ca2+]i of the outer segment also varied in proportion to the circulating current (Fig. 7A and B) except at early times after the onset of bright backgrounds (Fig. 7C). The results of Fig. 8 show that for a sufficiently bright background a significant amount of Ca2+ can be released transiently within the outer segment. This has the result that even in Ringer solution, the Ca2+ concentration briefly rises upon exposure to background light before falling to a value proportional to the circulating current (compare Fig. 7B and C). Cones in this respect behave differently from rods. In a rod (Matthews & Fain, 2003), a background light bright enough to completely saturate the rod and close all of its cyclic-nucleotide gated channels is still not bright enough to produce a significant release of Ca2+ in the outer segment. In cones, on the other hand, there is substantial release of Ca2+ even at physiological light levels (Brockerhoff et al. 2003), both because the release mechanism is paradoxically more sensitive in cones than in rods (Cilluffo et al. 2004) and because cones do not saturate in bright backgrounds as rods do. As a consequence, the Ca2+ concentration in the outer segment can transiently exceed that expected from the value of the outer segment current in bright light, before declining to a steady-state value.
The physiological significance of this transient increase in [Ca2+]i is unclear. It would have the effect of retarding activation of guanylyl cyclase and slowing recovery of the cone circulating current from saturation (see for example Pugh et al. 1999; Fain et al. 2001); it might therefore produce a dazzling effect due to delay of sensitivity recovery during a short interval after exposure to the background. It would be interesting to compare the sensitivity of a cone with Ca2+ concentration as a function of time in bright light. This experiment is unfortunately not feasible in the UV-sensitive zebrafish cone, because the small flash response amplitude in the presence of bright backgrounds severely limits the accuracy of sensitivity measurements.
Light-dependent Ca2+ release has now been observed in salamander (Matthews & Fain, 2001) and mouse (Woodruff et al. 2004) rods, and in salamander (H. R. Matthews and G. L. Fain, unpublished observations) and zebrafish visible (Brockerhoff et al. 2003; Cilluffo et al. 2004) and now UV-sensitive cones. In cones, this release of Ca2+ takes place within the normal physiological range of light intensities, as cones can adapt to light over a much wider range than rods (Burkhardt, 1994). It is of sufficient magnitude that it is likely to play some role in the function of the receptor in bright illumination, although the precise nature of this role remains unknown. Nevertheless, our results demonstrate that both for brief flashes and over the majority of the range of steady light intensity, changes in cone outer segment [Ca2+]i can be fully accounted for in terms of the Ca2+ fluxes across the outer segment membrane. Our study finally resolves the uncertainty regarding the possibility of additional contributions to Ca2+ homeostasis from other processes within the physiological intensity range, and allows modelling of this important feedback signal in phototransduction to be carried out with confidence.
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References |
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)], where i0 and A describe the instantaneous and rapid component of fluorescence, with time constants (
) and after (
). Measurements without background light from a further nine cones were preincubated with 50 µM BAPTA-AM (
,see text). Laser flashes were 50 ms in duration, and the value of the fluorescence for each flash was averaged between 15 and 45 ms to avoid the rapid increase in fluorescence shown in 