Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Photographs of a dye-injected photoreceptor cell. ( a ) Left eye of a white-eyed mutant blowfly Calliphora vicina photographed with a dry objective. The facet lens overlying the dye-injected cell clearly shines up (scale bar = 0.2 mm). ( b ) Diagram of the photoreceptor organization and the recording situation after optically neutralizing the cornea with a water immersion objective . ( c ) Blue-induced green fluorescence photographed through a water-immersion objective, showing that the rhabdomere and the cell body of the stained cell fluoresce. ( d ) Green-induced red fluorescence of the same eye region showing the characteristic pattern of the rhabdomeres; one rhabdomere, belonging to the stained cell, is brighter than the others ( arrow ), allowing us to identify the stained cell as an R5 photoreceptor (scale bar in c and d = 20 μm).

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Injection, Mutagenesis, Fluorescence, Staining

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Simultaneous recording of membrane potential ( a and b ) and Ca 2+ -induced fluorescence ( c and d ) from a dark-adapted photoreceptor illuminated with saturating light. The same data are shown with high ( a and c ) and low ( b and d ) temporal resolution. The cell was injected with the low affinity dye OG5N. The fluorescence signal increases very fast after opening the shutter and peaks after ∼100 ms before levelling off towards a plateau. The depolarization of the membrane, however, displays still faster kinetics. All traces are averages of nine recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Injection

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: The effects of dye filling on the membrane potential. The photoreceptor cell was impaled with an electrode containing OG2 and then filled without applying current. Traces 1–3 were measured 1, 4, and 6 min, respectively, after impalement by a 5-s stimulus of saturating intensity. While the fluorescence signal ( c and d ) increases with time of impalement, indicative for an ongoing dye-loading, the membrane potential response ( a and b ) of the cell during the light stimulus is hardly changed. Only after turning the stimulus off can a distinct prolongation of the depolarizing after-potential be seen, due to an increase in the concentration of the dye ( b ). The fluorescence traces in d are smoothed by adjacent averaging with a window size of 20 sample points (equivalent to 4 ms) and therefore the rising phase that is visible in c is not discernible in d . The traces in e and f were obtained from a different preparation in which no cell had been dye-filled; these traces are averages of five recordings. They show that the tissue autofluorescence is essentially constant, except for a small, transient increase in fluorescence signal (*) that can be attributed to a light-induced change in redox state of mitochondrial flavoproteins .

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Membrane potential ( a and b ) and fluorescence ( c and d ) signals from two cells injected with the high affinity dyes OG1 ( a and c ) and OG2 ( b and d ), respectively. The dark-adapted cells were illuminated for 5 s with light of saturating intensity. The fluorescence signal increases monotonically upon stimulation and does not display the peak that can be seen when using the low affinity dye OG5N (Fig. ). This indicates that the high affinity dyes OG1 and OG2 are saturated by the levels of Ca i reached under bright illumination. This implies that in these conditions Ca i exceeds 10 μM, the concentration where OG2 saturates . Traces in a and c are averages of five recordings, traces in b and d are averages of seven recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence, Injection, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Example of the double pulse experiments used to determine Ca i as a function of light intensity. Membrane potential traces are given in the left hand column. The dye-injected (OG1) and dark-adapted cell was stimulated with an adapting light (5 s), the intensity of which is indicated for each experiment. Intensities are expressed relative to the light intensity that caused a half maximal peak depolarization; this intensity was assigned the value log I = 0. After adapting for 5 s, the level of Ca i was probed with a bright test flash (500 ms). The fluorescence signal measured during this test flash is shown in the right-hand column on an expanded time scale. The horizontal line at the beginning of the fluorescence traces indicates the initial fluorescence value when no adapting light was given. With increasing adapting intensity, the initial value of the fluorescence ( arrows ) increases. At the highest adapting intensities used, no increase in fluorescence can be observed during the test flash due to saturation of the high affinity dye OG1. All traces shown are averages of five recordings.

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Injection, Fluorescence

Journal: The Journal of General Physiology

Article Title: Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

doi:

Figure Lengend Snippet: Summary of the double pulse experiments. ( a ) Normalized depolarization of the peak ( filled symbols ) and plateau ( open symbols ) of the membrane potential are given as a function of adapting light intensity. The smooth curves are obtained by fitting the logistic function V = V max * I n /( I n + 1) to the experimental data (exponent n = 0.45 for peak values and 0.47 for plateau values; the normalized V max for plateau values was fitted to be 0.43). The light intensity I is taken relative to the light intensity that causes a half maximal peak depolarization; this intensity was assigned the value log I = 0. ( b ) Normalized fluorescence increase caused by the adapting light for the three dyes used ( symbols connected by thin lines ); data from three different cells are shown for each dye. Normalization procedures for all panels are explained in materials and methods . The membrane potential and fluorescence data obtained with a specific dye are indicated by the same symbol. The fluorescence increase reported by OG1 and OG2 rises with increasing adapting light intensity and saturates at bright light. The signal that was obtained with OG5N continues to increase up to the highest intensities. This shows that Ca i is regulated in a graded fashion over the whole intensity range and that Ca i levels exceed 10 μM at high light intensities. The bold lines are fits to the experimental data obtained by calculating the fluorescence ( F ) as a function of Ca i according to F (Ca i ) = Ca i h /(Ca i h + K d h ) and normalizing, as detailed in materials and methods . Ca i was assumed to increase with a simple power function of adaptation light intensity yielding the curve shown in c (see results ).

Article Snippet: A 50% mirror combined the beams, which then passed the microscope's fluorescence cube (Leitz DM 510; i.e., blue excitation causing green emission).

Techniques: Membrane, Fluorescence