Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROIs) in mammalian tissue, with no requirement for genetic manipulation. Methanol or formalin-fixed specimens are stained with fluorescent dyes or antibodies to visualize ROIs, then soaked in solutions containing the photo-tag: 4-benzoylbenzyl-glycyl-hexahistidine. Confocal imaging along with two photon excitation are used to covalently couple photo-tags to all proteins within each ROI, to a resolution of 0.67 µm in the xy-plane and 1.48 µm axially. After tissue solubilization, photo-tagged proteins are isolated and identified by mass spectrometry. As a test case, we examined amyloid plaques in an Alzheimer's disease (AD) mouse model and a post-mortem AD case, confirming known plaque constituents and discovering new ones. STOMP can be applied to various biological samples including cell lines, primary cell cultures, ex vivo specimens, biopsy samples, and fixed post-mortem tissue.
Two-Photon Excitation (TPE) microscopy is a powerful tool for the imaging of biological specimen. The most important advantages compared with conventional confocal microscopy are: intrinsic optical sectioning, reduced phototoxcicity and photobleaching, and an increased penetration into the specimen. The increased penetration arises from the use of the longer wavelength excitation light, in the red or near infra-red.Here, a quantitative in-depth TPE imaging study is presented on dental biofilm. The biofilm contains a mixed culture of a large number of oral bacteria with a typical size of ≈ 1 μm diameter. They exhibit a complex microstructure and heterogeneous physiological and biochemical properties. The high density of the biofilms limits the depth range over which confocal images can be acquired to 25-40 μm.Using a two-photon excitation microscope fluorescence intensity images could be recorded over the whole thickness (100 μm) of the biofilm sample.
Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, stimulating proliferation in single HeLa cells.
Protein Kinase A Potentiates Adrenal 4 Binding Protein/Steroidogenic Factor 1 Transactivation by Reintegrating the Subcellular Dynamic Interactions of the Nuclear Receptor with Its Cofactors, General Control Nonderepressed-5/Transformation/ Transcription Domain-Associated Protein, and Suppressor, Dosage-Sensitive Sex Reversal-1: a Laser Confocal Imaging Study in Living KGN Cells