All imaging was performed on the Bruker Ultima IV multiphoton microscope system described in Dones et al., 2019.26 A Ti:Sapphire pulsed laser (Spectra-Physics Insight DeepSee) was tuned to 820 nm to excite the Cy5 fluorophore of Cy5-CMP, using a 690 (50) nm emission bandpass filter (Chroma Technology Corporation, VT) to isolate the fluorescence emission. SHG of intact collagen was performed on all tissues on the same microscope by tuning the laser wavelength to 890 nm and using a 445 (50) nm bandpass filter (Semrock Inc., Rochester, NY). Samples were imaged using a 10× CFI Super Fluor objective lens (Nikon, NA = 0.50). Images generated were acquired as z-stacks from superficial to deep at 40 micron intervals spanning 800–1200 microns in depth. Slices were summed and stitched to capture the full field of view using FIJI.28 (link) A high pass Gaussian filter was used to correct for gradients in the images in Figures 3 and 4, which were taken with a 4× Super Fluor objective (Nikon, NA = 0.20).
Super fluor objective
Manufactured by Nikon
The 4× Super Fluor objective is a high-performance lens designed for use in microscopy applications. It features a numeric aperture of 0.13 and a working distance of 17 mm. The objective is optimized for fluorescence imaging and provides a wide field of view. Its super fluorite glass elements help to minimize chromatic and spherical aberrations, ensuring high-quality image reproduction.
Automatically generated - may contain errors
Lab products found in correlation
Digital sight ds u3, by Nikon (1 mentions)
Ti e inverted microscope, by Nikon (1 mentions)
Cy5 filter set, by IDEX Corporation (1 mentions)
Nis elements software package, by Nikon (1 mentions)
Imagexpress micro xl, by Molecular Devices (1 mentions)
Spectra x led illumination, by Lumencor (1 mentions)
4 protocols using super fluor objective
1
Multiphoton Imaging of Collagen Structure
2
Wide-field Fluorescence Imaging of Burned Skin
3
Fluorescence Microscopy of Cells
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Fluorescence measurements were performed on a Nikon TiE inverted microscope driven by the NIS-elements software package (Nikon). The microscope was equipped with an Andor Neo 5.5 sCMOS 12 bit camera (Oxford Instruments), a 40 × 0.75 NA Super Fluor objective, a 60 × 1.4 NA oil-immersion apochromatic objective (Nikon), a perfect-focus mechanism (Nikon), and EGFP and Cy3 TE-series optical filter sets (Chroma), as well Cy5 filter set (Semrock).
4
Single Cell Apoptosis Imaging Assay
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Single cell killing assay measurements were run on an ImageXpress Micro XLS screening image analysis system (Molecular Devices). The detection of apoptotic and necrotic target cells with the apoptosis sensor Casper-GR construct was carried out as described before (24 (link)). Briefly, NALM6 pCasper target cells were pulsed with 1 µg/ml SEA in AIM-V medium at 37°C and 5% CO2 for 30 min, washed taken up in 100 µl of phenol-free RPMI-1640/10% FBS and seeded into fibronectin coated wells (coating 30 min, 50 µl of 0.1 mg/ml fibronectin per well) of a 96-well plate (1x104 targets per well). After a short rest (30 min) 2x104 effector cells in 100 µl of phenol-free RPMI-1640/10% FBS were added per well and the measurement was started. Over 8h bright field, GFP- and FRET-signals were acquired with a 20x objective from 1-4 positions per well at a 5 min interval. Objects were excited via Spectra X LED illumination (Lumencor) using LED 470/24 for GFP. The filter sets for FRET were 472/30 nm for excitation and 520/35 nm for emission to measure GFP and 641/75 nm to measure RFP. A Nikon Super Fluor objective (20x/NA 0.75) was used.
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