Auto-align - multi-modality fluorescence microscopy image co-registration.

Multi-modality microscopes incorporate multiple microscopy techniques into one module, imaging through a common objective lens. Simultaneous or consecutive image acquisition of a single specimen, using multiple techniques, increases the amount of measurable information available. In order to benefit from each modality, it is necessary to accurately co-register data sets. Intrinsic differences in the image formation process employed by each modality result in images which possess different characteristics. In addition, as a result of using different measurement devices, images often differ in size and can suffer relative geometrical deformations including rotation, scale and translation, making registration a complex problem. Current methods generally rely on manual input and are therefore subject to human error. Here, we present an automated image registration tool for fluorescence microscopy. We show that it successfully registers images obtained via total internal reflection fluorescence (TIRF), or epi-fluorescence, and confocal microscopy. Furthermore, we provide several other applications including channel merging following image acquisition through an emission beam splitter, and lateral stage drift correction. We also discuss areas of membrane trafficking which could benefit from application of Auto-Align. Auto-Align is an essential item in the advanced microscopist's toolbox which can create a synergy of single or multi-modality image data.

Genetic diversity affects the nanoscale membrane organization and signaling of natural killer cell receptors.

Genetic diversity in human natural killer (NK) cell receptors is linked to resistance and susceptibility to many diseases. Here, we tested the effect of this diversity on the nanoscale organization of killer cell immunoglobulin-like receptors (KIRs). Using superresolution microscopy, we found that inhibitory KIRs encoded by different genes and alleles were organized differently at the surface of primary human NK cells. KIRs that were found at low abundance assembled into smaller clusters than those formed by KIRs that were more highly abundant, and at low abundance, there was a greater proportion of KIRs in clusters. Upon receptor triggering, a structured interface called the immune synapse assembles, which facilitates signal integration and controls NK cell responses. Here, triggering of low-abundance receptors resulted in less phosphorylation of the downstream phosphatase SHP-1 but more phosphorylation of the adaptor protein Crk than did triggering of high-abundance receptors. In cells with greater KIR abundance, SHP-1 dephosphorylated Crk, which potentiated NK cell spreading during activation. Thus, genetic variation modulates both the abundance and nanoscale organization of inhibitory KIRs. That is, as well as the number of receptors at the cell surface varying with genotype, the way in which these receptors are organized in the membrane also varies. Essentially, a change in the average surface abundance of a protein at the cell surface is a coarse descriptor entwined with changes in local nanoscale clustering. Together, our data indicate that genetic diversity in inhibitory KIRs affects membrane-proximal signaling and, unexpectedly, the formation of activating immune synapses.

The physical basis of total internal reflection fluorescence (TIRF) microscopy and its cellular applications.

Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.