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. Author manuscript; available in PMC: 2013 Feb 28.
Published in final edited form as: Biochemistry. 2012 Feb 16;51(8):1625–1637. doi: 10.1021/bi201682q

Figure 8.

Figure 8

Characterization of tetragonal lysozyme crystals exposed to different fluorescent dyes. (A) Fluorescence of lysozyme crystals increases with time after exposure to solutions of fluorescent dyes, NBD PC, fluorescein and DAPI. Fluorescence from drops of the solutions did not change over the same period of time. Total fluorescence from crystals was normalized to that measured immediately before adding dye solutions to the crystals. Fluorescence from solution drops was normalized to the time immediately after placing the solution drop on the cover slip-bottomed dish in front of microscope objective. (B) Fluorescence of a lysozyme crystal exposed for 20 min to 60 μM DAPI. Inset shows TPM images of a lysozyme crystal immediately after exposure to DAPI (top) and 9 min after exposure (bottom). (C) Spectral characteristics of a lysozyme crystal exposed to 50 μM fluorescein. The normalized spectrum from the crystal obtained before adding fluorescein overlaps the spectrum from an area inside the crystal (indicated by a square in the inset) obtained after 15 min exposure to fluorescein. The spectrum from fluorescein in solution closely resembles the spectrum taken from the edge of the crystal (indicated by a rectangle in the inset) after exposure to fluorescein. (D) Fluorescence of a lysozyme crystal exposed for 15 min to 133 μM of fluorescently labeled phospholipid NBD PC. The normalized spectrum from the crystal exposed to solution, obtained with 730 nm, indicated with open circles, overlaps with the spectrum obtained at 910 nm, indicated with open triangles. Spectrum obtained from NBD PC in solution with 730 nm, indicated with filled circles, is shifted 20 nm towards red. Inset shows data obtained from a series of z-slice images of a lysozyme crystal exposed to NBD PC obtained along the axis perpendicular to the crystal face. Main box shows image half way through the crystal thickness. The two cross-sectional images, shown at the bottom and at the right, indicate that the highest fluorescence is at the edges of the crystal. Scale bars in all images represent 150 μm.