Photoelectron microscopy and photoelectron quantum yields of the fluorescent dyes fluorescein and rhodamine.

Photoelectric properties of the dyes fluorescein and rhodamine were determined with the aim of assessing the usefulness of these compounds as labels in photoelectron microscopy. The photoelectron quantum yields were measured over the wavelength range 180-230 nm. At 230 nm the quantum yields for fluorescein disodium salt, rhodamine B free base and rhodamine B HCl salt are approximately 10(-5) electrons per incident photon. At 180 nm these values rise to approximately 10(-3) electrons per incident photon. All forms of fluorescein do not have the same quantum yield. The neutral form of fluorescein has a quantum yield an order of magnitude lower than the disodium salt. Beam current measurements were performed on labeled and unlabeled proteins to determine the effect of the high light intensity employed in the photoelectron microscope. The initial beam current measurements and the quantum yield curves are consistent and demonstrate that there is significant contrast between labeled and unlabeled proteins. However, after several minutes in the photoelectron microscope, the proteins become more photoemissive and the contrast diminishes. This change in contrast explains several puzzling observations in the literature.

Depth of information in photoelectron microscopy.

The depth of information is defined as the distance below the surface of a specimen from which information is contributed at a specific resolution. A simplified model of photoemission is used to explore the relationship between electron escape depths and depth of information in photoelectron microscopy (PEM or photoemission electron microscopy). The depth of information is equal to the escape depth when the escape depth is small relative to the instrument resolution. When the escape depth is large compared to the instrument resolution or when information is carried for example by reflected light, the image consists of well resolved surface detail at the instrument resolution and dimmer, more diffuse, images of detail below the surface. Thus the same sample can exhibit different depths of information depending on the image details of interest. Other mechanisms of transmitting information to the surface, for example induced topography, are discussed, and experimental examples are given.

Photoelectric properties and detection of the aromatic carcinogens benza[a]pyrene and dimethylbenzanthracene.

The absolute photoelectron quantum yield spectra for benzo[a]pyrene and dimethylbenzanthracene are presented in the wavelength range 180--230 nm. These polycyclic aromatic carcinogens have photoelectron quantum yields of approximately 2 x 10(-3) electrons per incident photon at 180 nm. The quantum yields fall off quickly and monotonically at wavelengths longer than 210 nm (5.9 eV). Threshold values for benzo[a]pyrene and dimethylbenzanthracene are 5.25 +/- 0.06 cV and 5.27 +/- 0.04 eV, respectively. The photoelectron quantum yields of benzo[a]pyrene and dimethylbenzanthracene are several orders of magnitude greater than typical components of biological membranes (amino acids, phospholipids, and polysaccharides). Preliminary micrographs of benzo[a]pyrene and dimethylbenzanthracene sublimed onto poly(L-lysine) and onto dimyristoyl phosphatidylcholine demonstrate the high contrast of small crystallites of carcinogens against a background of membrane components. These results and calculations involving relative contrast factors suggest that the distribution of these carcinogens in biological membranes can be determined by using photoelectron microscopy.