Lipi-Blue (Dojindo Molecular Technologies, Rockville, MD, USA) and HCS LipidTOX Red neutral lipid stain (Invitrogen) were used for LD staining. LD measurements were performed as previously described (Li et al, 2019 (link)). LD numbers / cell were manually quantified in the absence of oleic acid treatment using Nikon NIS Elements AR software. LD diameter was measured after treatment with 100 μM oleic acid using Nikon NIS Elements AR software. The cells or images for LD analysis were chosen randomly.
Nis elements ar software
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Germany, Azerbaijan, Canada, Austria
NIS-Elements AR software is a comprehensive imaging and analysis platform designed for Nikon microscopes. It provides tools for image acquisition, processing, and analysis to support a wide range of microscopy techniques.
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Lab products found in correlation
Eclipse ti e, by Nikon (18 mentions)
Eclipse ti, by Nikon (15 mentions)
Eclipse ti inverted microscope, by Nikon (14 mentions)
Hoechst 33342, by Thermo Fisher Scientific (14 mentions)
A1r confocal microscope, by Nikon (11 mentions)
Eclipse ti e inverted microscope, by Nikon (9 mentions)
Perfect focus system, by Nikon (9 mentions)
Eclipse 90i microscope, by Nikon (8 mentions)
Nis elements software, by Nikon (7 mentions)
Eclipse ti2, by Nikon (7 mentions)
Fura 2 am, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Ti e inverted microscope, by Nikon (6 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (6 mentions)
Eclipse 90i, by Nikon (6 mentions)
Optoscan ele 450, by Cairn Research (6 mentions)
A1 confocal microscope, by Nikon (6 mentions)
Eclipse ti microscope, by Nikon (6 mentions)
Zyla 4, by Oxford Instruments (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
427 protocols using nis elements ar software
1
Lipid Droplet Quantification Protocol
2
Long-term Imaging of HCT-116 Cells
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Long-term time-lapse imaging was performed on an Eclipse Ti-E inverted microscope (Nikon Instruments, Amsterdam, The Netherlands) using a temperature- and humidity-controlled chamber (Okolabs, Pozzuoli, Italy) with 5% CO2 at 37 °C. HCT-116 cells were seeded on 35-mm dishes with low density and phase contrast images were captured every 10 minutes. Images and videos were analysed by NIS elements AR software (Nikon Instruments).
Fluorescence and confocal microscopy were performed as previously described63 (link),64 (link). Briefly, cells were grown on cover slip and fixed with 2% paraformaldehyde (Sigma-Aldrich). Actin cytoskeletons and nuclei were stained with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific) and DAPI (Sigma-Aldrich), respectively. Fluorescence microscopy was performed on an Eclipse 90i microscope (Nikon Instruments) equipped with a 12-bit, black and white CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Images were analysed by NIS elements AR software (Nikon Instruments). Cell counting was performed in at least five fields of triplicate cover slips. Confocal microscopy was performed on a D-Eclipse C1 confocal microscope (Nikon Instruments). Images were processed by EZ-C1 Freeviewer software (Nikon Instruments).
Fluorescence and confocal microscopy were performed as previously described63 (link),64 (link). Briefly, cells were grown on cover slip and fixed with 2% paraformaldehyde (Sigma-Aldrich). Actin cytoskeletons and nuclei were stained with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific) and DAPI (Sigma-Aldrich), respectively. Fluorescence microscopy was performed on an Eclipse 90i microscope (Nikon Instruments) equipped with a 12-bit, black and white CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Images were analysed by NIS elements AR software (Nikon Instruments). Cell counting was performed in at least five fields of triplicate cover slips. Confocal microscopy was performed on a D-Eclipse C1 confocal microscope (Nikon Instruments). Images were processed by EZ-C1 Freeviewer software (Nikon Instruments).
3
Microscopic Analysis of Pneumococcal Cell Morphology
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For static images of cells analyzed in S2 and S3 Figs, cells were grown in C+Y medium to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide. A polylysin-coated coverslip was placed on top to fix the cells in position. Images were captured and processed using the Nis-Elements AR software (Nikon). Analysis of cell dimensions was carried out using the MATLAB-based open source software MicrobeTracker [46 (link)]. Cell contours were obtained using the alg4 spneumoniae3 algorithm implemented in MicrobeTracker, a derivative of alg4 ecoli2 with parameters spliltTreshold, joindist and joinangle refined to fit the shape of S. pneumoniae.
Time-lapse microscopy was carried out as previously described [47 ], with modifications. Briefly, pneumococcal precultures were grown in liquid CAT medium at 37°C to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide containing a slab of 1.2% CAT agarose before imaging. Images were captured at 5 min intervals for 6 hours and processed using the Nis-Elements AR software (Nikon).
Time-lapse microscopy was carried out as previously described [47 ], with modifications. Briefly, pneumococcal precultures were grown in liquid CAT medium at 37°C to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide containing a slab of 1.2% CAT agarose before imaging. Images were captured at 5 min intervals for 6 hours and processed using the Nis-Elements AR software (Nikon).
4
Fluorescence Spectral Imaging of ViBac Probes
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To measure the fluorescence spectra of ViBac1 and ViBac2 in living cells, cells expressing ViBac1 or ViBac2 were placed on an M9 gel pad and observed using a laser scanning confocal microscope (Nikon A1R si+, Japan) equipped with a 100×/1.4 NA oil immersion objective lens. Fluorescence images were obtained using a laser scanning confocal microscope (Nikon A1R si+, Nikon Instruments, Japan) equipped with an APO 40×1.25 NA water immersion objective. The fluorescent spectra of ViBac1 (under 488 nm excitation) and ViBac2 (under 488 nm and 561 nm excitation) were obtained using a 32-PMT spectral detector and then used for spectral unmixing. Spectral images were unmixed using NIS-elements AR software (Nikon, Japan) to obtain images of ViBac1 and ViBac2. All images were captured and analyzed using NIS Elements AR software (Nikon, Japan).
5
Fungal Fate Analysis via Time-Lapse Imaging
6
Time-lapse Imaging of Fungal Fate
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Time-lapse images for fungal fate analysis were captured on a TE2000 (Nikon) with Digital Sight DS-Qi1MC camera (Nikon), × 60 objective (DIC PLAN APO), using NIS elements AR software (Nikon). Transmitted light images were captured every 2 min and fluorescence images were captured every 10 min for 12 h. Two hundred and eighty-eight and 177 cells for AIg54 and AIg56, respectively, were analysed over four separate experiments. Time-lapse images for fungal fate analysis in the presence of apocynin were captured on a Ti (Nikon) with Neo (Andor), × 60 objective (DIC PLAN APO), using NIS elements AR software (Nikon). Transmitted light images were captured every 2 min and fluorescence images were captured every 10 min for 12 h. After completing the infection protocol, macrophages were treated with either 0.5 mM apocynin or 0.5% dimethyl sulphoxide (control). For strain AIg54 191 control and 129 apocynin-treated macrophages, and for strain AIg56 164 control and 148 apocynin-treated macrophages were analysed over three separate experiments. Three-dimensional imaging of mitochondrial tubularization was acquired with Nikon A1R confocal microscope (Nikon) in resonant scanning mode and with a piezo stage (MadCity Labs) for fast z-sectioning. C. gattii AIg54 was imaged in PBS containing 0.7 mM H2O2 to induce tubularization. Seventy-four z-sections were captured every 60 s for 40 min.
7
SARS-CoV-2 Spike Protein Syncytia Formation
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Using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA), U2OS cells were co-transfected with pmCherry and pCAGGS vector encoding SARS-CoV-2 Spike protein. For a negative control, U2OS cells were transfected with pmCherry only. 24-hours post-transfection, U2OS cells were trypsinized, mixed with Calu-3 cells on 96-well μ-plate (Ibidi, Germany), and incubated for 24 h in the presence (40 µM; final concentration) or absence of HNP1 or RC-101, with (10%; final concentration) or without FBS. Following the incubation, the co-cultures were counterstained with 1 μg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) and imaged using Nikon inverted microscope Eclipse Ti-E (Nikon Instruments, Melville, NY). Nikon NIS Elements AR software was used to quantify fusion indices calculated as number of nuclei per individual mCherry-positive syncytium.
8
Eukitt-Mounted Microscopy Imaging
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Slides were mounted using Eukitt quick-hardening mounting medium (Sigma-Aldrich) and visualized at -0.4°C on a Nikon TiE 3000 inverted microscope (Nikon) equipped with a digital camera (DS-U2/L2-Ri1 digital microscope camera (Nikon) for light microscopy or DXM-1200C coded digital camera (Nikon) for fluorescent microscopy) and Nikon NIS Elements AR software (Nikon). Exposure times were consistent for each staining type. Image levels were equally adjusted using Adobe Photoshop CS6 software (Adobe) to remove nonspecific background staining. Necropsy images were captured on a handheld digital camera and stored as high-resolution JPEG files. Margins of cropped images are indicated by a solid black or white border.
9
Fluorescent Microscopy Imaging Protocol
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Slides were mounted using Eukitt Quick-hardening mounting medium (Sigma-Aldrich) and visualized at − 0.4 °C on a Nikon TiE 3000 inverted microscope (Nikon) equipped with a digital camera (DS-U2/L2-Ri1 digital microscope camera (Nikon) for light microscopy or DXM-1200C coded digital camera (Nikon) for fluorescent microscopy), and Nikon NIS Elements AR software (Nikon). Exposure times were consistent for each staining type. Image levels were equally adjusted using Adobe Photoshop CS6 software (Adobe) to remove nonspecific background staining.
10
Mitochondrial activity assessment in cells
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After 24 h exposure to emodin and vinblastine, cells (1 × 105/mL) were fixed in 3.7% paraformaldehyde and then incubated for 30 min with rhodamine 123 (Sigma Aldrich, St. Louis, MO, USA) at 5 µg/mL ethanol, a fluorochrome that binds to metabolically active mitochondria. Cells were then washed with PBS and analyzed under a Nikon A1R confocal microscope based on a Nikon Eclipse Ti inverted microscope (Nikon Instruments Inc., Melville, NY, USA) and equipped with Nikon Nis Elements AR software (Nikon Instruments Inc., Melville, NY, USA). The experiment was repeated 3 times.
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