We used a custom-built STED/confocal setup55 (link) constructed around an inverted microscope body (DMI 6000 CS, Leica Microsystems) which was equipped with a TIRF oil objective (x100, 1.47 NA, HXC APO, Leica Microsystems) and a heating box (Cube and Box, Life Imaging Services) to maintain a stable temperature of 32 °C. A pulsed-laser (PDL 800-D, PicoQuant) was used to deliver excitation pulses at 488 nm and a de-excitation laser (Onefive Katana 06 HP, NKT Photonics) operating at 594 nm was used to generate the STED light pulses. The STED beam was profiled to a donut shape using a spatial light modulator (Easy3D Module, Abberior Instruments). Image acquisition was controlled by the Inspector software (Abberior Instruments). The spatial resolution of the microscope was 175 nm (x-y) and 450 nm (z) in confocal mode and 60 nm (x-y) and 160 nm (z) in STED mode.
Inspector software
Manufactured by Abberior
The Inspector software is an image acquisition and analysis tool developed by Abberior. It is designed to control and operate various microscope systems and acquire high-quality images. The software provides a user-friendly interface for controlling the microscope hardware, configuring imaging parameters, and processing the acquired data. The core function of the Inspector software is to facilitate the efficient capture and management of microscopic images.
Automatically generated - may contain errors
2 protocols using inspector software
1
Super-Resolution Microscopy Setup
2
Multimodal Microscopy for Cellular Imaging
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The images were obtained using a combination of confocal and superresolution microscopy. DAPI and WGA 488 were acquired in confocal mode and images of MPO (STAR RED) and IL-40 (STAR 580) in cell detail were acquired using stimulated emission depletion (STED) microscopy. The imaging was performed on an Abberior Expert Line STED system (Abberior Instruments GmbH) equipped with a Nikon Eclipse Ti-E body (Nikon, Tokyo, Japan), QUAD beam scanner and Nikon CFI Plan Apo 60x/NA 1.40 oil-immersion objective (Nikon). The samples were illuminated with 405 nm, 485 nm, 561 nm and 640 nm lasers. The fluorescence excited with 561 and 640 nm laser was depleted by a pulsed 775 nm STED laser in 2D donut shape. The fluorescence signal passed the pinhole set to 1 AU, was filtered by emission filters (422–467 nm, 506–594 nm, 580–630 nm and 650–720 nm) and the signal was detected with single-photon counting detectors (Excelitas Technologies, Waltham, MA, USA). The images were obtained by the Inspector software (Abberior Instruments GmbH).
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