A method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis:A method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

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A series of PPV films with different fluorescent emission colors are prepared using a facile approach. Modified from the conventional Wessling route, the labile sulfonium groups in the polymer precursor (pre‐PPV) are partly substituted to various degrees by relatively stable methoxy groups, followed by a regular thermal elimination step to obtain PPV. Different chemical structures among these films are confirmed by IR and photophysical studies. Two film‐preparative processes are compared and the films are studied by SEM and confocal fluorescent microscopy. From the same polymer precursor, different preparative processes result in PPV films with different morphologies but the same photophysical properties.

NADPH/NADP + redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP + . To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP + sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP + to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose-and glutamine-stimulated NADPH responses by single-and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP + sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.