Metamorph

Fluorescence microscopy was performed on an automated Leica DMI6000B fluorescence microscope under Leica MetaMorph control, with a sCMOS camera (pco.edge), an LED illuminator (Lumencor Spectra-X), and a 63× oil immersion objective. Excitation illumination was filtered at the LED source (mCherry imaging λ = 575/25 nm; GFP/AlexaFluor488 imaging or wide-field BcLOV4 stimulation λ = 470/24 nm; miRFP703 imaging λ = 632/22 nm). Fluorescent proteins were imaged with Chroma filters: mCherry (T585lpxr dichroic, ET630/75 nm emission filter, 0.2–0.5 s exposure), GFP (T495lpxr dichroic, ET 525/50 nm emission filter, 0.2 s exposure), miRFP703 (AT655dc dichroic, ET655 nm emission, 0.5 s exposure). Cells were imaged at room temperature in CO2-independent media (phenol-free HBSS supplemented with 1% l-glutamine, 1% penicillin–streptomycin, 2% essential amino acids, 1% nonessential amino acids, 2.5% HEPES pH 7.0, and 10% serum). The spatially patterned illuminator was custom-constructed from a digital light processor (DLP, Digital Light Innovations CEL5500), as previously described.^{[22 (link)]}

Fluorescence microscopy was performed on an automated Leica DMI6000B fluorescence microscope under Leica MetaMorph control, with a sCMOS camera (pco.edge), an LED illuminator (Lumencor Spectra-X), and a 63× oil immersion objective. Excitation illumination was filtered at the LED source (mCherry imaging λ = 575/25 nm; GFP imaging or wide-field BcLOV4 stimulation λ = 470/24 nm; miRFP imaging λ = 632/22 nm). Fluorescent proteins were imaged with Chroma filters: mCherry (T585lpxr dichroic mirror, ET630/75 nm emission filter, 0.2–0.5 s exposure), GFP (T495lpxr dichroic mirror, ET 525/50 nm emission filter, 0.2 s exposure), miRFP703 (AT655dc dichroic mirror, ET655 nm emission filter, 0.5 s exposure). Cells were imaged at room temperature in CO2-independent media (phenol-free HBSS supplemented with 1% l-glutamine, 1% penicillin–streptomycin, 2% essential amino acids, 1% nonessential amino acids, 2.5% HEPES pH 7.0, and 10% serum); LifeAct and lamellipodia imaging were performed in CO₂-independent media without serum. The spatially patterned illuminator was custom-constructed from a digital light processor (DLP, Digital Light Innovations CEL5500), as previously described.⁷