Time-Resolved Synchronous Fluorescence for Biomedical Diagnosis.

This article presents our most recent advances in synchronous fluorescence (SF) methodology for biomedical diagnostics. The SF method is characterized by simultaneously scanning both the excitation and emission wavelengths while keeping a constant wavelength interval between them. Compared to conventional fluorescence spectroscopy, the SF method simplifies the emission spectrum while enabling greater selectivity, and has been successfully used to detect subtle differences in the fluorescence emission signatures of biochemical species in cells and tissues. The SF method can be used in imaging to analyze dysplastic cells in vitro and tissue in vivo. Based on the SF method, here we demonstrate the feasibility of a time-resolved synchronous fluorescence (TRSF) method, which incorporates the intrinsic fluorescent decay characteristics of the fluorophores. Our prototype TRSF system has clearly shown its advantage in spectro-temporal separation of the fluorophores that were otherwise difficult to spectrally separate in SF spectroscopy. We envision that our previously-tested SF imaging and the newly-developed TRSF methods will combine their proven diagnostic potentials in cancer diagnosis to further improve the efficacy of SF-based biomedical diagnostics.

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

High-sensitivity rod photoreceptor input to the blue-yellow color opponent pathway in macaque retina.

Small bistratified cells (SBCs) in the primate retina carry a major blue-yellow opponent signal to the brain. We found that SBCs also carry signals from rod photoreceptors, with the same sign as S cone input. SBCs exhibited robust responses under low scotopic conditions. Physiological and anatomical experiments indicated that this rod input arose from the AII amacrine cell-mediated rod pathway. Rod and cone signals were both present in SBCs at mesopic light levels. These findings have three implications. First, more retinal circuits may multiplex rod and cone signals than were previously thought to, efficiently exploiting the limited number of optic nerve fibers. Second, signals from AII amacrine cells may diverge to most or all of the approximately 20 retinal ganglion cell types in the peripheral primate retina. Third, rod input to SBCs may be the substrate for behavioral biases toward perception of blue at mesopic light levels.