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Browsing by Author "Nichols, Evan L."
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Item Automatic segmentation of intravital fluorescence microscopy images by K-means clustering of FLIM phasors(OSA, 2019-08) Zhang, Yide; Hato, Takashi; Dagher, Pierre C.; Nichols, Evan L.; Smith, Cody J.; Dunn, Kenneth W.; Howard, Scott S.; Medicine, School of MedicineFluorescence lifetime imaging microscopy (FLIM) provides additional contrast for fluorophores with overlapping emission spectra. The phasor approach to FLIM greatly reduces the complexity of FLIM analysis and enables a useful image segmentation technique by selecting adjacent phasor points and labeling their corresponding pixels with different colors. This phasor labeling process, however, is empirical and could lead to biased results. In this Letter, we present a novel and unbiased approach to automate the phasor labeling process using an unsupervised machine learning technique, i.e., K-means clustering. In addition, we provide an open-source, user-friendly program that enables users to easily employ the proposed approach. We demonstrate successful image segmentation on 2D and 3D FLIM images of fixed cells and living animals acquired with two different FLIM systems. Finally, we evaluate how different parameters affect the segmentation result and provide a guideline for users to achieve optimal performance.Item Generating intravital super-resolution movies with conventional microscopy reveals actin dynamics that construct pioneer axons(The Company of Biologists, 2019-03-08) Zhang, Yide; Nichols, Evan L.; Zellmer, Abigail M.; Guldner, Ian H.; Kankel, Cody; Zhang, Siyuan; Howard, Scott S.; Smith, Cody J.; Medicine, School of MedicineSuper-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a 'top-down' scaffolding event. Further, we identify an F-actin population - stable base clusters - that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals.