Conventional wide-field fluorescence microscopy imaging was carried out on an AxioObserver inverted microscope (Carl Zeiss, Germany), equipped with 100×/1.45 oil Plan Apo Lambda phase objective, Orca FLash4 V2 CMOS camera (Hamamatsu) and a Colibri 7 LED light source (Carl Zeiss, Germany). Acquisition was performed using ZEN Blue 3.2 (Carl Zeiss, Germany) with different filters (Carl Zeiss, Germany) and parameters, depending on the fluorescent reporter or chemical dye (table S5).
Orca flash 4v2 cmos camera
Manufactured by Hamamatsu Photonics
Sourced in Germany, Japan
The Orca Flash 4v2 CMOS camera is a high-performance imaging device manufactured by Hamamatsu Photonics. It features a 4.2-megapixel CMOS sensor with a maximum frame rate of 100 frames per second. The camera is capable of capturing high-quality images and videos with low noise and high sensitivity.
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4 protocols using orca flash 4v2 cmos camera
1
Fluorescence Microscopy Imaging Protocol
2
Microscopic Imaging with Nikon Eclipse Ti
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The Micro-Manager program (Edelstein et al., 2014) was used to control the microscope (Nikon Eclipse Ti) which is equipped with perfect focus (Nikon), a 100x VC DIC lens (Nikon), a fluorescent filter for YFP (Semrock), an X-cite fluorescent light source (Excelitas) and an Orca Flash 4v2 CMOS camera (Hamamatsu). For step input a pump rate of 0.1 ml/minute was used.
3
Rapid Automated Modular Microscopy Imaging
4
Quantifying Hog1 Nuclear Localization in Yeast
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For the quantification of nuclear enrichment of Hog1 terminal kinase in individual yeast cells as an indicator of pathway activation, we employed a strain in which a yellow-fluorescent protein (YFP) was integrated at the C-terminus of the endogenous Hog1 protein in Saccharomyces cerevisiae BY4741 yeast, using homologous DNA recombination.
Our imaging setup consisted of an inverted microscope, specifically the Nikon Eclipse Ti, which was equipped with several components, including perfect focus (Nikon), a 100× VC DIC lens (Nikon), a YFP fluorescent filter from Semrock, an X-cite fluorescent light source (Excelitas), and an Orca Flash 4v2 CMOS camera from Hamamatsu. The entire system was controlled via the Micro-Manager program.
To capture images, we conducted the following steps:
Utilizing the microscope’s xy-plane movement, we selected a field of view that exhibited an optimal density of yeast cells within a single z-plane. The z-focus was carefully adjusted to visualize the boundary of most cells, appearing as a white ring. Time-lapse images were recorded, encompassing both bright-field images, taken at intervals of every 10 seconds, and fluorescent channel images, acquired at intervals of every minute.
Our imaging setup consisted of an inverted microscope, specifically the Nikon Eclipse Ti, which was equipped with several components, including perfect focus (Nikon), a 100× VC DIC lens (Nikon), a YFP fluorescent filter from Semrock, an X-cite fluorescent light source (Excelitas), and an Orca Flash 4v2 CMOS camera from Hamamatsu. The entire system was controlled via the Micro-Manager program.
To capture images, we conducted the following steps:
Utilizing the microscope’s xy-plane movement, we selected a field of view that exhibited an optimal density of yeast cells within a single z-plane. The z-focus was carefully adjusted to visualize the boundary of most cells, appearing as a white ring. Time-lapse images were recorded, encompassing both bright-field images, taken at intervals of every 10 seconds, and fluorescent channel images, acquired at intervals of every minute.
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