Electron Microscope + Hemocytometer - (Dec/06/2013 )
I would like to seek for your advice. I noticed that the chamber lines of hemocytometer are not showed clearly under electron microscope. May I know is it only can be view under inverted microscope?
Electron microscope? Why would you use that on a haemocytometer? Most people would use them with a conventional upright or an inverted microscope - both light microscopes.
The grids are just etched into the glass, so they are just grooves in the glass surface. You should be able to see this by SEM I would have thought.
Why do you want to use the haemocytometer in the EM? just wondering...
as bob1 said, if you use SEM the "lines" on the glass should be really easy to see
There are 2 reasons that I am using EM,
first, is because currently only EM is available in my lab,
secondly, is because of the protocol that showed in this link http://www.youtube.com/watch?v=pP0xERLUhyc
Using EM to count cells!? Wow
By the way, SEM? You only don't see the haemocytometer lines or you don't see the cells either?
Leaving aside the suitability of using the EM for counting cells... of it is SEM, do you sputter-coat your sample/haemocytometer? A lack of a conductive layer/compounds makes the imaging of any EM rather difficult. It also depends a lot on the type of EM you have
I can see the haemocytometer line clearly without loading any sample in. However, after I load the sample (even just pure distiller water) it also will make the line become invisible.
That's normal due the light refraction..
Wait, you don't place water in the EM, do you!?!?
Place water in the EM? Nope. I didn't do it.
So what vehicle will you use to install cells?
Also, please recall hemocytometers are validated with water as carrier and examination via light microscopy. You can't assume that a major change in protocol will maintain that validity so you'll need to validate your protocol.
Could you post what do you exactly do since you load the sample onto the haemocytometer until you place it in the EM?
Didn't mention before but haemocytometer counting is only valid when you use the coverslip provided, as it creates a layer of liquid of known depth that plus the known area of the squares on the glass you can calculate the concentration of cells. So, it can only be effectively used with liquids... and you cannot place them in the EM chamber unless it is a special EM such environmental EM or low vacuum models. In any case, cells are not electron -dense material nor conductive so you will have problems to look at them... and you won't be able to sputter-coating as they will be inside the haemocytometer