Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins.

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process.

Time-shifted mean-segmented Q data of a luminal protein measured at the nuclear envelope by fluorescence fluctuation microscopy.

Fluorescence fluctuation microscopy is a widely used method to determine the mobility and oligomeric state of proteins in the live cell environment. Existing analysis methods rely on statistical evaluation of data segments with the implicit assumption that no significant signal fluctuations occur on the time scale of a data segment. Recent work on extending fluorescence fluctuation methods to the nuclear envelope of living cells identified a slow fluctuation process that is associated with the undulations of the nuclear membranes, which lead to intensity fluctuations due to local volume changes at the nuclear envelope.

Cross-correlation registration algorithm enhancements for multipixel inverse synthetic aperture LiDAR.

The accurate registration and realignment of complex signal volumes is critical for cross-range aperture gain in 3D LiDAR aperture synthesis. For targets at long range, only a limited number of diffraction-limited pixels will be projected on the target, resulting in low cross-range support. In addition, the signal-to-noise ratio (SNR) is typically low.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude.

Identifying Heteroprotein Complexes in the Nuclear Envelope.

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations.