Hello all, This I suppose this is probably 80% of an optics/ microscopy question, maybe only 20% of a biology question, but I thought that people in this section probably have the most experience with this sort of thing. I am using a setup than involves an objective lens suspended over a microfluidic chip. A camera is positioned behind the the lens (well more precisely, the image actually reflects off a beam splitter into a camera, the beam splitter for the purpose of allowing optical tweezers to be used on the sample while simultaneously imaging on the camera). The student who worked on this before me was able to see the erythrocytes just sitting there on a slide (and trap them with the laser), but in the chip they were nowhere to be found! I attempted the same and so far have also failed. What I probably want to do is maximize scattering/ minimize light hitting the camera that is from anything other than the cells (I suppose they call this dark field imaging?). I have not been successful in seeing anything after several days of messing around with the lighting (the light source is free to move basically anywhere). Some of my co-workers suggested lighting from above, or from the side, but still the only time I can even see the channel walls decently is when lighting from below ("below" meaning pointing at the front of the objective, through the chip). I have "frosted" the surface of the transparent fixture plastic that the chip sits on, again to maximize scattering. This improved the contrast of the chip walls a little bit, but I still see no erythrocytes. I have been able to see 6 micron plastic beads (which were supposed to represent the erythrocytes) but when I put the real thing in I see nothing. So now that I've ranted sufficiently, does anyone have any tips for lighting techniques for this sort of application?