Mlc monolithic laser combiner

Correlated AFM and fluorescence images were acquired as described previously ^{37 (link)}. Briefly, fluorescence images were acquired with an EMCCD iXon Ultra 888 camera (Andor) mounted on an IX73 inverted optical microscope (Olympus) equipped with a 100X oil immersion objective (UAPON100XOTIRF, Olympus). Illumination was provided by an MLC monolithic laser combiner (Agilent) coupled to an optical fiber. For combined fluorescence imaging of FtsZ-GFP and FM4-64 the excitation filter and dichroic mirror of the EGFP filter cube F36-526 (AHF Analysetechnik, Germany) were used. The emission light was split by an optical system (DV2, Photometrics) equipped with a second dichroic mirror (T565lpxr) placed between the EMCCD camera and the microscope frame to separate the red and green channels each to one half of the EMCCD camera chip. Cleanup filters used were F37-528 (EGFP) for the green channel and F37-624 (TxRed) for the red channel. Additionally, a neutral density filter with OD 0.6 was placed on the red channel. Typically, 5 mW power of 488 nm laser light at the MLC400B output was used for illumination. Images were recorded with EM gain set to 300 and exposure time to 500 milliseconds. The AFM was mounted on top of the inverted microscope (IX73, Olympus) and the AFM laser was switched off before acquiring fluorescence images. Contrast and brightness were adjusted with ImageJ.