Figure 1.

Schematic diagram of multicolor Lightsheet based OVSS fluorescence system. Two distinct wavelength of light sources (${\lambda }_1=473$ nm and ${\lambda }_2=532$ nm) are used to excite spectrally-distinct fluorophores. A unit magnifier (with lens separated by a distance $>(f_1+f_2)$) is placed in one of the illumination arms to correct for the focal-shift ($\mathrm{\Delta }Z$) due to chromatic aberration induced by 10$\times$ objective lens. Beams are combined by Dichroic mirror (DM1), expanded and passed through cylindrical lens (CL, $f=150$ mm) to generate the light sheet. Additionally, we placed an objective lens (Olympus, 10$\times$, 0.3 NA) at the focus of cylindrical lens to generate diffraction-limited multicolor lightsheet (MLS). The specimen (encaged in a capillary tube) placed on the automated rotating-stage is illuminated by MLS. To observe the fluorescence, an orthogonal detection system is employed that use a separate detection objective lens (Olympus, 4X, 0.1 NA/Olympus, 10$\times$, 0.25 NA) to collect the image data. Iris (IR) and Notch filters (473 NF and 532 NF) are used to cut-off the nonparallel and scattered light respectively. Bandpass fluorescent filters (range $500\pm 12$ nm) along with the dichroic mirror DM2 (with cut-off, ${\lambda }_c=505$ nm), additional filters (F1 and F2), mirror (M2) and tube-lens (TL) are used to divert, filter and focus beam to the respective cameras ports (CCD1 and CCD2) for data recording.