live imaging on slice tissue - (Apr/23/2007 )

Hi, has anyone tried imaging (confocal laser scanning) live tissue in slices? I've tried everything to stop the slice from moving (x, y and z directions) but still have no success...Please help!!!

---

Hi,
are u using inverted or upright mic?
slices on... (millipore inserts, home made nets, coverslips?)
perfusion?
are you using any dyes?

I have other problems, but when I got good slices I can look at them without problems.
if you are more specific we can share experience.

by,

---

Hi,
Thanks for the reply. I have since then sorted out most problems. I'm using an inverted microscope with slices on milipore inserts, perfused at 0.5ml/min. Lateral movement is somewhat solved by a slice anchor. Culturing the slices help with z-movement. But if the whole microscope is not temeperature controlled, there will always be a bit of movement in z. I've overcome that by scanning stacks each time. Hope your experiments go well!

---

there are perfusion chambers to fix a tissue with a transparent filter;

other otion is to embed in collagen, matrigel etc but reduces signal; so 2-photon may help in this case...

---