Subcellular distributions and excited-state processes of hypericin in neurons

Photochemistry and Photobiology, Mar 1999 by English, Doug S, Doyle, Robert T, Petrich, Jacob W, Haydon, Philip G

Time-resolved fluorescence microscopy experiments were carried out on a Nikon Diaphot 300 inverted fluorescence microscope. Excitation laser pulses (532 nm, 76 MHz, 70 ps full width at half maximum from a Coherent Antares 76s) were routed to the rear port of the microscope via a single mode fiber and beam expander. The laser light was partially polarized. The extinction between s and p was 70:1 (s with respect to the plane of the table; p with respect to the internal dichroic of the microscope). The typical laser power delivered to the microscope port was 1-5 (mu)W. The excitation light was reflected upward into a 60X 1.4 NA oil immersion objective. The emission was collected with the same objective and separated from the scattered excitation light by a dichroic beam splitter. The fluorescence (>550 nm) was routed out the side port of the microscope through a long-pass glass filter with cut-off at 550 nm to an EG&G Canada single-photon counting avalanche photodiode. No polarizers were used in front of the detector in order to allow maximum throughput. The raw photodiode signal is fed to an EG&G 1 GHz, 100X preamplifier and subsequently to a discriminator and single-photon counting apparatus described elsewhere (20).

RESULTS

Our results are consistent with association of hypericin with membranes throughout the cell. Other reports have indicated that hypocrellins, which are structurally similar to hypericin, also associate with or localize in the membrane (21). Our results also indicate that, to a lesser extent, hypericin is distributed in the cell nucleus (Fig. 1). We have identified the nucleus by staining it with DAPI and selectively exciting it in the presence of hypericin (Fig. 1). Identifying the nucleus allows us to detect hypericin subsequently without exciting DAPI and thus to determine the extent to which hypericin may exist in the nucleus of the cell. From our results it is apparent that hypericin is partitioning in the nucleus, albeit to a much lesser extent. In order to be sure that the emission from the nucleus is not from hypericin existing in the nuclear membrane, we took confocal cross sections that confirmed that hypericin does in fact partition in the nucleus (data not shown).

Living fetal rat neurons show rapid and irreversible damage to the plasma membrane when illuminated after incubation with hypericin. Figure 2 show sequential images obtained at IS s intervals that demonstrate a progressive blebbing of the cell membrane. This photodynamic damage of living neurons required the presence of hypericin, because excitation of unstained cells did not cause structural change. Morphology of stained and unstained cells was verified using phase-contrast microscopy. Because of rapid photodynamic damage, the ability to perform even short-lived experiments on living cells was significantly reduced. However, chemical fixation with formaldehyde after loading with hypericin prevented structural change, making preparations more amenable to quantitative investigations.

We measured the fluorescence decay of hypericin in the cell body of neurons with particular attention to the area of intense signal that occurs in an acentric position (arrow, Fig. 1), corresponding to the location of the Golgi complex, as seen by NBD C^sub 6^ ceramide staining. Measurements were also taken at the edge of the cell body at the plasma membrane and in the long neuritic processes of the cells (arrowhead, Fig. 1). Hypericin displays a biexponential fluorescence decay described by a short time component of 200-500 ps and a long component of (-3 ns) (Fig. 3a). We dismiss rotational diffusion as the origin of this component because hypericin in bulk solution has a fluorescence depolarization time of 90 ps (22) and in Brij micelles, a fluorescence depolarization time of 900 ps (23).