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Integrated imaging instrument for self-calibrated fluorescence protein microarrays.

ABSTRACT: Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

SUBMITTER: Reddington AP

PROVIDER: S-EPMC3799691 | biostudies-other | 2013 Oct

REPOSITORIES: biostudies-other

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Project description:Spatial light modulation using cost efficient digital micromirror devices (DMD) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, precise illumination by DMDs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMD and camera coordinate systems caused by optical components in the excitation and emission path. Here we develop a fast and easy to implement calibration procedure that determines these distortions and matches the DMD and camera coordinate system with a precision below the optical diffraction limit. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to a rigid transformation allowed by manual alignment of the DMD. We first demonstrate the application of our precisely calibrated light modulation by performing a proof of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP in budding yeast (S. cerevisiae). Next, we develop a spatially informed photoactivation approach for SMLM in which only regions of the cell that contain photoactivatable fluorescent proteins are selected for photoactivation. The reduced exposure of the cells to 405 nm light increased the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in cell morphology. As a result, the mean number of reliable single-molecule localizations was also significantly increased by 28%. Since the localization precision and the ability for single-molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatially informed photoactivation significantly improves the quality of SMLM images and single-molecule tracking data. Our precise calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.

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Integrated imaging instrument for self-calibrated fluorescence protein microarrays.

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