Fluorescence confocal microscopy was performed on a Leica TCS SP8 with a Hamamatsu Orca Flash 4.0 cMOS camera and an AOBS laser system (HyD Detector; Wetzlar, Germany). To observe sequestration (Fig. 1 ), samples were incubated in a glass bottom 384-well plate (MatriPlate 384Well Plate, Brooks Life Science Systems, Poway, CA). Since aggregation occurs over long time scales in these plates, to monitor changes over time (Fig. 3E and F ) we incubated samples in a plastic bottom 384-well plate (MICROPLAE 384Well, μClear, Non Binding, Greiner Bio-One, Kremsmünster Austria). Images were recorded at λem = 520 nm after excitation at λex = 500 nm for Atto-488 and at λem = 485 nm after excitation at λex = 405 nm for ThT.
Tcs sp8
Manufactured by Hamamatsu Photonics
Sourced in Germany
The TCS SP8 is a laser scanning confocal microscope system manufactured by Hamamatsu Photonics. It is designed for high-resolution imaging of biological and materials samples. The system features a scanning unit, detection unit, and control software, enabling comprehensive analysis of specimen characteristics.
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Lab products found in correlation
2 protocols using tcs sp8
1
Fluorescence Confocal Microscopy of Protein Aggregation
2
Quantifying Protein Droplet Volume Fraction
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The volume fraction of the dense phase was estimated from z-stacks of protein droplets acquired using confocal microscopy. Samples were prepared in 384-well plates with glass bottom (Matriplate, Brooks) using protein labelled with atto488 at a final concentration of 30 µM. A total of four z-stacks per condition were acquired using a Leica TCS SP8 confocal microscopy with a Hamamatsu Orca Flash 4.0 cMOS camera and an AOBS laser system (HyD Detector). Each z-stack covered a surface of 8,545.15 µm2, and the distance between images in the vertical direction was 0.1 µm. Image acquisition was controlled using the Leica LAS X SP8 software (version 1.0). Data were analysed with a customized Python script by counting the number of pixels with intensity above an arbitrarily defined threshold for each image. From the volume of a voxel (0.00324 µm3), the area of the well (10.9 mm2), the sample volume (20 µl), and assuming that protein droplets are homogeneously distributed on the well area, we estimated the total volume fraction of the dense phase from each z-stack.
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