Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity
MG1655 either unlabeled or expressing YPet. All incubation steps were performed at 37 °C under agitation. Cultures were inoculated in LB broth, supplemented with ampicillin for MG1655-YPet or kanamycin for BL21-EGFP and incubated overnight. Before plating on a microscope slide, cultures were prepared as follows. Cultures of BL21-EGFP were diluted 200-fold in LB supplemented with kanamycin and IPTG , incubated for 2 h and diluted again 200-fold.
Once the liquid was absorbed, the cavity was sealed with a coverslip, and the slide was incubated at 37 °C for several hours before imaging , allowing cells to grow into small microcolonies.The bacterial growth experiments were performed with a Nikon Eclipse Ti microscope controlled by NIS software, whose epifluorescence illuminator was modified to deliver dual visible and NIR illumination .
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