Figure 2

Evaluation of the optical features of the MIM relative to commercial microscopes. (a–d) Ultrathin 90 nm (a,b) and semi-thin 350 nm (c,d) Lowicryl sections from a high-pressure-frozen 3-day old embryo expressing Tg(kdrl:Hsa.Hras-mCherry) in blood vessels. Fluorescence images from a Leica SP5 confocal microscope (a,c, merge of 81 images of the same field of view; 63x, NA 1.2 objective with 3x zoom) and MIM (b,d, 40x, NA 0.6 objective) were shown in a color-code to visualize fluorescence signal intensity. For comparison with MIM, we optimized carefully the parameters of confocal imaging to have the very best image quality by using a high precision HCX PL APO CS 63.0 × 1.20 WATER UV objective: laser intensity (561 nm at AOTF 20%), PMT gain (869 V), emission detection range (583–662 nm), pixel dwell time (2.5 µsec), pinhole size (1 airy unit), averaging (2), and pixel size (80 nm). Sections recorded with MIM and confocal microscope represent images of adjacent parasagittal sections cut from the same animal. Scale bar: 10 µm. (e–l) Lateral resolution comparison between confocal and MIM. Magnified views of a-d for caudal vein wall are shown in (e–h), respectively. Levels of fluorescence intensities were normalized to facilitate comparison. Scale bar: 10 µm. Fluorescence profiles (arbitrary unit [AU]) along the line drawn perpendicular to a vein endothelium wall (distance = 0) toward both directions (+/−1.6 µm) were plotted for each condition (n = 5 juxtapositions, blue lines in e-h). Full width at half maximum (FWHM, red line) of vein endothelium wall for each condition was measured. (m) Exploded view of the light transmission measurement set-up. The transmitted light intensity through inner light pathway lenses and prisms of the optical tubes (OT) was assessed with the same objectives (OBJ) and light source (LS) for the MIM and two commercial epifluorescence compound microscopes. The camera was replaced by a light intensity sensor (LIS) on the tested instruments. (n) The transmitted light intensity is represented for MIM and the two tested epifluorescence microscopes (Leica DMR, Leica DMLB). The MIM performs better in light detection than the two tested commercial epifluorescence microscopes.