Quantitative analysis of protein stoichiometry and cellular structures deep within biological samples demands imaging systems that can overcome the limitations of inadequate depth of focus and poor signal-to-noise ratios (SNR) common to conventional platforms. Performing quantitative microscopy, such as stepwise photobleaching in deep cellular compartments such as the cell nucleus has typically presented significant technical challenges. We present the development and physical characterization of a custom-built light sheet imaging platform, engineered to address these challenges through both hardware innovation and accompanying software solutions. A key feature of the system is its vertical, and angled delivery of the light sheet, which provides compatibility with standard coverslip-adherent cells, a format typically inaccessible to light sheet instruments. The system was demonstrated to achieve excellent optical sectioning, with a minimum sheet thickness (beam waist radius, ω 0 ) of approximately 2 μm defining its axial resolution. Furthermore, the quantitative performance of the platform was validated using a stepwise photobleaching assay enabled by our custom software solution. This method was applied to fluorescently tagged NUP96 in the nuclear pore complex of U2OS cells, yielding results consistent with its known stoichiometry and demonstrating successful quantitative measurements deep within the cell nucleus. The versatility of the platform is further demonstrated by its planned extension to imaging complex multicellular spheroids. This work establishes our system as a tool for a broad range of demanding quantitative applications, particularly enabling previously challenging deep cellular quantitative microscopy through advanced optical approaches.
Alderson et al. (Sun,) studied this question.
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