Nicole M. Haralampus-Grynaviski

Spectroscopic and morphological studies of human retinal lipofuscin granules

The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.

Probing the Spatial Dependence of the Emission Spectrum of Single Human Retinal Lipofuscin Granules Using Near-field Scanning Optical Microscopy¶

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near‐field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale ∼150 nm.

A unique optical arrangement for obtaining spectrally resolved confocal images

The design for a confocal beam-scanning microscope with a unique optical configuration for the rapid acquisition of spectrally resolved images is presented. The novel aspect of the optical configuration is the location of the detection device, which is placed in an intermediate position between fully descanned detection and nondescanned detection (direct projection). This placement allows for the practical implementation of spectrally resolved confocal imaging. The device is demonstrated by the spectral resolution of a sample of fluorescently labeled microbeads.

Design and Applications of Rapid-Scan Spectrally Resolved Fluorescence Microscopy

This paper addresses the design and applications of a spectrally resolved confocal fluorescence microscope that measures spectral information for each pixel in the image with a fast multi-point approach. The combined spatial and spectral resolution of the device is demonstrated by performing two-photon imaging of mixtures of fluorescently labeled microspheres. Overlapping emission spectra of dye solutions contained in microcapillaries are readily distinguished. Images of a human skin tissue section and a mouse kidney section are presented which demonstrate that the structure and spectra of biologic samples can be resolved.

Femtosecond Pump-Probe Studies of Radiationless Decay Dynamics in Arthropod and Mollusc Hemocyanins

Femtosecond pump-probe spectroscopy has been used to measure the dynamics following excitation of the ligand-to-metal charge-transfer states in arthropod and molluscan hemocyanins isolated from Limulus polyphemus and Busycon canaliculatum, respectively. These results are discussed in terms of the thermal elimination and geminate recombination of oxygen following rapid nonradiative relaxation in the protein. The two hemocyanins exhibit the same dynamics, consistent with the structural similarity of their active sites.