For imaging, a Nikon TI2 Eclipse epifluorescence microscope was used. Fluorescence was measured using appropriate band-pass filter sets (DAPI, FITC, TRITC, Cy5, all Semrock) and a SOLA LED light source (Lumencor). The sample was enclosed in a darkened cage incubator (Okolab) and imaged at 37°C and ambient oxygen concentrations. Images were acquired using a 10× objective (NA 0.45) and camera settings (Orca Fusion or R2, Hamamatsu) specific for each channel. An array of 12 × 12 images was acquired for every time point, where all channels were measured for one image, and then the stage moved to the next image. The samples were measured every 30 min for 4 h. Generated images were saved as.nd2 files and exported to a separate computer for data analysis.
Fluorescein isothiocyanate (fitc)
Manufactured by IDEX Corporation
FITC is a fluorescent dye commonly used in laboratory applications. It absorbs blue light and emits green fluorescence, which can be detected and measured using specialized equipment.
Automatically generated - may contain errors
2 protocols using fluorescein isothiocyanate (fitc)
1
Fluorescence Microscopy Imaging Protocol
2
Multi-color Structured Illumination Imaging
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The slides as prepared above were imaged
on an Olympus BX51 microscope
connected to a Hamatsu ORCA-ER detector and illuminated with a Prior
Lumen 200 source with a Hg lamp (Prior; cat LM200B1-A). Excitation
lines and bandpass emission filter (BPF) pairs are as follows—350
nm/DAPI (Semrock; cat: FF02-447/60-25); 405 nm/FITC (Semrock; cat:
FF01-524/24-25); 488 nm/TRITC (Semrock; cat: FF01-593/40-25); 568
nm/Cy5 (Semrock; cat: FF01-692/40-25); 350 nm/TRITC—and were
used to capture PL from DAPI, AlexaFluor488, AlexaFluor568, AlexaFluor647,
and CdSe/CdS QD, respectively. The emission was collected through
an infinity-corrected 20x UPlanApo 0.70 NA objective (Olympus). The
emission is then passed through an OptiGrid structured illumination
element to form a grid confocal image on the detector. For each sample,
a z-stack was captured at interval steps of 1 μm and compressed
into the extended focus view presented in the representative images
used in this manuscript. The exposure time for each channel was optimized
and kept the same between each sample.
on an Olympus BX51 microscope
connected to a Hamatsu ORCA-ER detector and illuminated with a Prior
Lumen 200 source with a Hg lamp (Prior; cat LM200B1-A). Excitation
lines and bandpass emission filter (BPF) pairs are as follows—350
nm/DAPI (Semrock; cat: FF02-447/60-25); 405 nm/FITC (Semrock; cat:
FF01-524/24-25); 488 nm/TRITC (Semrock; cat: FF01-593/40-25); 568
nm/Cy5 (Semrock; cat: FF01-692/40-25); 350 nm/TRITC—and were
used to capture PL from DAPI, AlexaFluor488, AlexaFluor568, AlexaFluor647,
and CdSe/CdS QD, respectively. The emission was collected through
an infinity-corrected 20x UPlanApo 0.70 NA objective (Olympus). The
emission is then passed through an OptiGrid structured illumination
element to form a grid confocal image on the detector. For each sample,
a z-stack was captured at interval steps of 1 μm and compressed
into the extended focus view presented in the representative images
used in this manuscript. The exposure time for each channel was optimized
and kept the same between each sample.
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