600 677 nm brightline quad band band pass filter

Images were acquired using a modified Olympus IX81 inverted epifluorescence microscope with an oil-immersion objective (UApo N 100x/1.49 NA, Olympus, Vienna, Austria). The sample was positioned with nanometer precision on a XYZ piezo stage (P-733.3DD, Physical Instruments) on top of a mechanical stage with a range of 1 × 1 cm adjusted by precision screws (TAO, JPK Instruments, Berlin, Germany). A tube-lens with an additional magnification of 1.6 was used to achieve a final imaging magnification of 160 (corresponding to a pixel size of 100 nm). Platelets were illuminated with a 642 nm laser light from a diode laser (Omicron-laserage Laserprodukte GmbH, Phoxx 642, Rodgau-Dudenhofen, Germany), a 488 nm laser light from a solid-state laser (diode-pumped, Toptica Photonics, Graefelfing, Germany), and a 405 nm laser light from a diode laser (Insaneware, Gladbeck, Germany). The signal was detected using an Andor iXonEM+ 897 (back-illuminated) EMCCD camera (16 μm pixel size). The following filter sets were used: dichroic filter (ZT405/488/561/640rpc, Chroma, Olching, Germany), emission filter (446/523/600/677 nm BrightLine quad-band band-pass filter, Semrock, Rochester, NY, USA), and an additional emission filter (HQ 700/75 M, NC209774, Chroma Technology GmbH, Olching, Germany).

Fluorescence images were acquired using a modified Olympus IX81. A nanometre precision XYZ piezo stage (Physik Instrumente GmbH, Germany) on top of a mechanical stage with 1 × 1 cm range (JPK Instruments, Germany) was used for sample positioning. The sample was illuminated through a 60× magnification objective lens (60×, 1.42 NA, Olympus, Austria) and an additional 1.6× magnification tube lens with a 642 nm diode laser (Omicron, Germany). A dichroic filter (ZT405/488/561/640rpc, Chroma, Germany), an emission filter (446/523/600/677 nm BrightLine quad-band band-pass filter, Semrock, USA), and an additional emission filter (HQ 700/75 M, NC209774, Chroma, Germany) were used for imaging of the probes of the immobilized fluorophore–biomolecule conjugates (ATTO647N-cNP77 for Cy-LAPAP and ATTO655-SA for Cou-LAPAP and Rho-LAPAP). The fluorescence signal was detected using an Andor iXonEM+ 897 (back illuminated) EMCCD camera (16 μm pixel size) (Andor Technology, United Kingdom).^{93 (link)} 1500 images were recorded with 20 ms illumination time and 4.15 kW cm⁻² excitation intensity at λ_exc = 640 nm.