Paired-objectives photon enhancement (POPE) Microscopy

Fluorescence microscopy techniques are widely employed for the visualization, localization, and observation of structures and dynamics in biological samples. In order to obtain optimal image resolution, collecting the maximum number of photons from each fluorophore is essential. Historically, researchers have addressed this need by increasing the excitation energy, engineering higher numerical aperture (NA) objectives, improving the quantum yield of fluorophores, and/or escalating the quantum efficiency of detectors. While these approaches have led to great advances in the field of microscopy, they cannot proceed linearly in a limitless manner. We noticed that more than half of photons are lost as fluorophores emit in a direction other than the objective of either an upright or inverted microscope. To collect these lost photons, we have developed a new system called paired-objectives photon enhancement (POPE) microscopy to increase photon capture of a fluorophore by nearly two-fold. This system captures these lost photons by aligning an 8f optical system and mirror directly opposite the lower objective of an inverted microscope, thereby reflecting more emitted fluorescence back through the lower objective and into the detector. Through the additive effect of the initial fluorescence and the reflected fluorescence combining, the number of photons captured nearly doubles. This improved photon budget allows a microscopy imaging system to have better optical resolution, higher sensitivity, and/or faster detection speed. This new technique has been demonstrated in confocal light microscopy, epifluorescence microscopy, and super-resolution light microscopy and can, in theory, be applied in a modular fashion to any and all other fluorescence microscopy methods.