A tandem-lens epifluorescence macroscope: hundred-fold brightness advantage for wide-field imaging.

The design of a macroscope constructed with photography lenses is described and several applications are demonstrated. The macroscope incorporates epi-illumination, a 0.4 numerical aperture, and a 40 mm working distance for imaging wide fields in the range of 1.5-20 mm in diameter. At magnifications of 1X to 2.5X, fluorescence images acquired with the macroscope were 100-700 times brighter than those obtained with commercial microscope objectives at similar magnifications. In several biological applications, the improved light collection efficiency (20-fold, typical) not only minimized bleaching effects, but, in concert with improved illumination throughput (15-fold, typical), significantly enhanced object visibility as well. Reduced phototoxicity and increased signal-to-noise ratios were observed in the in vivo real-time optical imaging of cortical activity using voltage-sensitive dyes. Furthermore, the macroscope has a depth of field which is 5-10 times thinner than that of a conventional low-power microscope. This shallow depth of field has facilitated the imaging of cortical architecture based on activity-dependent intrinsic cortical signals in the living primate brain. In these reflection measurements large artifacts from the surface blood vessels, which were observed with conventional lenses, were eliminated with the macroscope.