A mercury arc lamp (50 W) was used for excitation, in combination with an FT510 dichroic mirror, 450 490 nm band-pass excitation filter, and emission filters (570DF20, 560DF40, BP515 565 nm, BA590, or BP530 550 nm; Omega Optical, Brattleboro, VT).
Therefore, many readers for UCP assays are complex, using laser diodes for excitation, in combination with other hardware such as lenses, filters, photomultiplier tubes, preamplifiers, and stepper motors.
We introduce a more selective strategy, involving dye laser excitation and background subtraction by means of off-resonant excitation in combination with a bootstrap evaluation of sets of individual events.
In fluorescence microscopy, image contrast is created by spectral separation of the reflected illumination light and the imaging light by application of an excitation and an emission filter in combination with a dichroic mirror.
Fast switching (<50 ms) excitation and emission filter wheels used in combination with a multiple band dichroic mirror (filter set 86006, Chroma, Bellows Falls, VT) allows for rapid switching between channels.
The entire well was imaged in four fields at 25-ms exposure time using excitation filter D360/40 and emissions filter HQ460/40 in combination with a 10X/0.45 NA objective.
Excitation at 488 nm with an argon laser in combination with a 505 530 nm bandpass emission filter were used for imaging of GFP-expressing cells.
All fluorescence measurements were performed with a spectrofluorometer (Horiba, Fluorolog3) at 300 nm excitation wavelength (power: 0.9 µW, front face geometry) in combination with a home build stirring system, as described previously [ 47].
A 150 W halogen lamp (Schott 1500, Schott, Mainz, Germany) in combination with a fluorescence filter block (excitation: 450 490 nm, emission >520 nm) was used to visualise FITC-Dextran.
Finally, we show how using these techniques in combination with a two-color two-photon excitation could permit to further improve the resolution to 60 nm.
