Plan fluor elwd 20x 0.45 ph1 dm objective

Brightness and illumination flatness in critical and Koehler illumination modes were measured on a custom-built epifluorescence microscope using Nikon Plan Fluor ELWD 20x/0.45 Ph1 DM objective, a Nikon 200 mm tube lens and a Chroma ET-EGFP/mCherry set (Chroma 59022) using a Thorlabs DCU3240 CMOS camera.
All diaphragms and the extra elements to generate conjugate planes were omitted to reduce path length and increase light throughput. LEDs were either collimated and projected directly onto the objective back focal plane (critical illumination) or a 150 mm achromatic lens was used to focus the light onto the objectives back focal plane (Koehler illumination). Lumileds Luxeon Rebel blue (470 nm LED) or Luxeon Rebel PC amber (595 nm LED) were used and driven at 1000 mA. Images were acquired either for 0.2 ms with green Chroma fluorescent plastic slides (470 nm LED) or for 1 ms (595nm LED) with red Chroma fluorescent plastic slides.

LED brightness was measured by coupling the LED illuminator described in this work including a Thorlabs DMLP505R dichroic mirror to combine channels onto the Koehler fluorescence illuminator unit of an Nikon Ti inverted microscope stand equipped with a Nikon Plan Fluor ELWD 20x/0.45 Ph1 DM objective. Chroma red and green fluorescent plastic slides (Chroma 92001) were used as samples. Images were acquired with a Photometrics Coolsnap ES² CCD camera. A Chroma ET-EGFP/mCherry set (Chroma 59022) was used for LED illumination. For Arc-lamp illumination the dual bandpass excitation filter was exchanged with the equivalent single bandpass excitation filters (ET470/40x and ET572/35x). An ND8 filter (12.5% transmittance) was used to attenuate the excitation light and images were taken at 1 ms exposure time. All used LEDs are listed in S4 Table together with their drive currents. An Excelitas X-Cite 120 mercury vapor short arc lamp was used for comparison. The bulb had been used for less than 50 hours with limited on/off cycles. At least 10 images were taken from different sample positions and their mean value was determined in Fiji [3 (link)].