I have been preparing the sample for the cryo-TEM of the fibroblast cells, ribosomes and liposomes but for each sample i have to look for the blot force, blot time, drain time, etc. So I think is that the blot force is the main part to look for and it depends upon the thickness of the specimen you want to blot but I am quite confuse how to determine the blot force for samples.
Article Cryo electron tomography of vitrified fibroblasts: Microtubu...
In my experience what is most critical about blotting single particles (ribosomes) and liposomal samples is how hydrophilic the surface of the electron microscopy grid is (actually, the carbon support layer). And we use glow-discharging to achieve sufficient spreading of the water on the grid. This results in a nicely spread water layer. The blot time, force etc I find of less importance and values between 1 and 2 seconds seem to get me good results for all samples. I never looked at the blot force though with cells you do not want to blot too hard since this might result in total drying of your cells and loss of structure and breaking of the support layer.
I agree with Roman's comments - I've never really worried too much about blot force. For single particles we generally use ~1-4s blot time - a second or so for drain time depending on the ice thickness we want (and whether the vitrobot's humidty control is working that day!). In my experience its actually quite difficult to blot cells dry, but its quite easy to blot cells off your grid when blotting from both sides as with a vitrobot. Sometimes one sided blotting (from the reverse side!) is useful. Something like a Leica EM GP does this, or a manual blotting device.
hello Roman and Neil.. I am glad to read your comment, but i guess still my question is remained unanswered. I have blotted two specimen like liposome and ribosome and with various study found that the blot force required is -2 and 5 sec. and there occurred no damage to the polymer layer and grid and cells. So i am pretty sure that blot force plays the good role with sample thickness. since the liposome were of size below 20 nm and ribosome were of size between 15 - 25 nm range. I am now confused what blot force to use for the dermal fibroblast cell..
Start with the same conditions, which should give useful results. You might adapt the conditions if necessary when the cells do not appear to be good.
You should always start with 0 blot force when you have a new sample. Setting the Vitrobot blot force to 0 is not always "0 blot force" , it dipends on the Vitrobot (for most Vitrobots this is around blot force -10). Next information : 100% humidity, temperature 4 degrees, real blot force 0.
Drain time and wait time, set in to zero. At 100%humidity this does not play any role at all. The blot time, I normaly started with 5 seconds, but sometimes I need only 2, sometimes more.Only if changing the blot time does not work you should change the temperature,humidity or blot force. Grid surface hydrophilic.
Edyta Reichman i am very much thanks for the answer so you mean to say that for every sample the blot force is need to be adjusted with hit and trial method. and drain time and wait time do not play any major role for vitrification process.?
There are a couple of caveats to what Edyta says. Firstly, without wanting to be sued by FEI, it is not my experience that humidity ever (or often) goes to 100%, despite what it says. Even at 100% humidity leaving the grid before blotting does tend to lead to thinner ice in the holes as the meniscus in each hole thins over time, although this is a relative minor effect. As Roman said, if you have working blotting conditions for a single particle specimen, I would start there and see what happens.
But the basic statement Bhanu makes is correct. The vitrobot increases reproducibility a lot but it is by no means perfect. Our basic procedure is to start with a standard condition and optimise by trial and error. We have found that the blot force is a) a minor component that we leave alone, and b) that it is anyway dependent on alignment of the arms and therefore not very constant over time between services! Blot time is the major variable in all our cell and single particle work.
Just my two cents worth.
Thanks Neil Ranson for explanation. So i can finally say that-
1. drain time increases, vitrified ice formation increases, as it expose the grid in humidity, so it should be reduced.?
2. wait time - the time between the sample loading and blotting is to be reduced. as it expose the grids more in humidity.
so if they are all of no use then why the company people are providing that option?
one more thing i seem confused that if blotting is required to remove the excess media then what is the sense to provide the drain time, which unnecessary will lead to exposure of the grids to the humidity.
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