Light from 405, 488 and 561 nm laser (Melles Griot 405, Coherent Inc., Coherent Inc.) is coupled into a single mode optical fiber and collimated with a 25 mm lens (ACN127-025-A, Thorlabs Inc.). The light is directed via a 405/488/561/640 nm quad band dichroic mirror (ZT405/488/561/640rpc, Chroma Technology Corp.) to the first xy-scanner (GVSM002/M, Thorlabs Inc.) at the telecentric point of the scan lens of effective focal length 70 mm (CLS-SL, Thorlabs Inc.) and directed to a standard Nikon Ti-E body. The objective in use is 100x Oil 1.49NA Apo-TIRF (Nikon). Fluorescence light is, after de-scanning, focused with a 200 mm achromat (AC254-200-A, Thorlabs Inc.) on a 200 μm pinhole (Precision Pinholes, Thorlabs Inc.). Light after the pinhole is collected with a 150 mm achromat (AC254-150-A, Thorlabs Inc.) and re-scanned with a second xyscanner (GVSM002/M, Thorlabs Inc.) and projected with a scan lens of effective focal length 70 mm (CLS-SL, Thorlabs Inc.) on a sCMOS camera (Hamamatsu Flash 4.0) with 2048 × 2048 pixels of 6.5×6.5 μm 2 . Electronic components of the setup are interfaced by a Nidaq pci-6733 card (National Instruments Co.) via a plug-in in NIS Elements (Nikon Instruments). For line-by-line multicolour imaging mode and fast line scanning, electronic components of the setup are interfaced via LabView.
DE Luca G.M.R., Desclos E., Breedijk R.M.P., Dolz-Edo L., Smits G.J., Nahidiazar L., Bielefeld P., Picavet L., Fitzsimons C.P., Hoebe R, & Manders E.M.M. (2017). Configurations of the Re-scan Confocal Microscope (RCM) for biomedical applications. Journal of microscopy, 266(2).
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