Person: Richardson, Douglas
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Richardson, Douglas
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Publication SRpHi ratiometric pH biosensors for super-resolution microscopy(Nature Publishing Group UK, 2017) Richardson, Douglas; Gregor, Carola; Winter, Franziska R.; Urban, Nicolai T.; Sahl, Steffen J.; Willig, Katrin I.; Hell, Stefan W.Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy. Therefore, to aid in investigations of pH dynamics during endocytosis at the nanoscale, we have specifically designed a family of ratiometric endosomal pH probes for use in live-cell STED nanoscopy.Publication Tutorial: Avoiding and Correcting Sample-Induced Spherical Aberration Artifacts in 3D Fluorescence Microscopy(Springer Science and Business Media LLC, 2020-07-31) Diel, Erin E.; Lichtman, Jeff W.; Richardson, DouglasSpherical Aberration (SA) occurs when light rays entering at different points of a spherical lens are not focused to the same point of the optical axis. SA that occurs inside the lens elements of a fluorescence microscope is well understood and corrected for. However, SA is also induced when light passes through an interface of refractive index (RI) mismatched substances (i.e. a discrepancy between the RI of the immersion medium and the RI of the sample). SA due to RI mismatches has many deleterious effects on imaging. Perhaps most important for 3D imaging is that the distance the image plane moves in a sample is not equivalent to the distance travelled by an objective (or stage) during Z-stack acquisition. This non-uniform translation along the Z-axis gives rise to artifactually elongated images (if the objective is immersed in a higher RI medium than the sample) or compressed images (if the objective is immersed in a lower RI medium than the sample) and alters the optimal axial sampling rate. In this Tutorial, we describe why this distortion occurs, how it impacts quantitative measurements and axial resolution and what can be done to avoid SA and thereby prevent distorted images. In addition, this tutorial aims to better inform researchers of how to correct RI mismatch-induced axial distortions and provides a practical ImageJ/Fiji-based tool to reduce the prevalence of volumetric measurement errors and lost axial resolution.