The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

and observe more fluctuations in the same time interval. Spatial intensity distribution analysis (SpIDA) was developed to adapt analysis of the photon counting histogram (PCH) for laser scanning microscopy images to determine molecular brightnesses. We have extended SpIDA for multicolor analysis that allows for sensitive detection of heteromeric fluorescent species. Optical systems in FFS typically encode spectral information by using dichroic filters to direct emitted light onto two or more photodetectors. In contrast, our imaging system (Zeiss LSM 880) uses a diffraction grating to disperse emitted light onto an array of 32 PMTs. The increased spectral resolution afforded by this geometry has the potential to be powerful in FFS, however new challenges are introduced including intrinsic crosstalk between adjacent detectors due to their physical proximity. We introduce an approach for addressing these non-ideal detector effects. Multicolor SpIDA with non-ideal detector corrections is employed to study the interactions of D2-like dopamine receptors with signaling partners including G-proteins and G-protein coupled inwardly rectifying K þ (GIRK) channels. Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

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