I think usng AFM for topographic images entails using a substrate i.e. wafers. From the wafers, you should be able to isolate the cells. Aside that, depend on the type of nanoscope, ther is always a software to visualized the morphologies such as Nanoscope IIIa and Nanoscope 7.2 software (Veeco Instruments Inc.) and JPK Nano wizard AFM. For now I have both. But I don't know if mechanical properties and adhension force can be measured, rather size distribution of the cell, cell height and others can be assessed.

Can you give a little more detail about the parameters you are using. And also the details of the substrate and the tips.

The substrates are SiO2 wafers that would allow you ti immobilized your samlple using molecular combing method (Bensimon et al., (1995) Phys.Rev. Lett 74, 4745 or Science, 1994, 265, 2096. The parameter of interest to are height/size or diameter and topographies of my nanowire in real time and in 2D.  

Thanks Zubairu and Loveleen..!!

@Zubairu, I have attached cells on a glass substrate. And not interested on cell height etc, want to measure membrane properties.

@Loveleen: I have tried folloving parameters:

Mode: contact:

k= 0.18 N/m

set point: 3 nN- 400 nN

p-gain: 600

i gain:  600

Thanks

@chiththaka:

mechanical information about the cells you will be able to get with force-distance curves. have you tried that? What is the medium you are using? Since cell properties will change with medium.

As i understand from your query you are trying to push/move the cells with the cantilever to understand the adhesive property b/w the cell and the substrate. Even for these experiments medium will be very crucial for deciding the final outcome.

@Loveleen: Yes exactly, I am tring to get delocalise cells in PBS pH 7.0 buffer. I could not deattach them, which is the problem. Have tried changing the parameters. But no through understanding of each parameter which SW shows!

Your medium is very important. as sited by Brar I think you can work with less polar medium then isolation will be very easy. i.e. glycerol. Aq. medium is unfriendly.

@chiththaka:

Mode: contact: k= 0.18 N/m:  this is a good k for contact mode imaging

set point: 3 nN- 400 nN: This is your most crucial parameter since it will be a deciding factor in how much you are in contact with the surface when you start pushing.

p-gain: 600 i gain: 600: these are P and I gains for the feed back. any way during pushing i think the feed back should not be working (check)

What is missing from here is the speed at which you are trying to move the cell. I would suggest you use slow speed and high set point. 

The high setpoint limit will be decided by height of your cell and also the type and the number of points of adhesion.

eg. most demonstration of the push mode consist of pushing the latex or silica nanospheres around. Since in that case there is only one point of contact. your cell on the other hand is going to be flattened and attached to the surface at multiple points. 

I feel @Zubairu has given a good suggestion that you should first try to decrease the cell surface interaction by changing the pH. try and move that. then come back to pH 7 samples and go ahead from there.

Also i will suggest you try a slightly higher k cantilever (more force). but be careful while imaging in contact mode. Are your cantilevers rectangular or triangular?

@Zubairu:

Thank you for your comments and sugessions. Really appritiate them. I have used pH 7 as it is the best pH where cells can be attached. This is the pH I have used for earlier studies. So canging this value result in challenging the pervious work I have done. The pH 7 is the value our study is based on.

@Loveleen:

400nN is the max I can set as the set point. I have started from 3nN and increased to 400nN.

Actually what does p and i-gain mean?? I have no cluue!! Once these are incresed, I do hear a sound when scanning.

I have used rectangular cantilver.

I will try again with slowing down the scanning speed. If not the next option would be using higher force cantilever. Thanks.

Dear Chiththaka,

Working on bacterial cells with an AFM and keeping the whole system alive is not a simple task. I believe that you need to separate your work in 3 different approaches as each of them will require a different set of parameters:

For imaging : use a soft lever and PBS medium for start (you can add charges to the buffer if needed too). You will quickly find out that contact mode in liquid with live bacteria is tricky as the bacteria move ! so you may need to functionalise your surface using poly-dopamine for example to immobilise your cell. Next is the question of the shear force done by contact mode imaging : these are usually quite high and induce morphological change in the cell.  Thus tapping may be the preferred option , yet tapping in liquid is also very challenging. My advice ?  if you want to get some nice image of bacterium cell : fix them histologically using 4% PFA for 10 min and then you can do contact in air at your will. You will conserve an adequate cell morphology even though your sample is effectively bow dry.

For indentation and adhesion: it gets a lot trickier. I have a PhD student working on this and would be happy to share some expertise offline. We have a paper in preparation about this exact topic related to Gram+  and Gram-  as well as a review coming up early next year in Nanotechnology.  Force distance curves are the way to go but needs to be performed very carefully and with the right probe. There is quite a bit of literature available on the topic but yet we have not found a standard protocol that we could duplicate. Each bacterial species react differently. So from my comments, you will see that it is more about knowing how to prepare the sample than running the AFM itself! This had to be one of the more challenging experiments we have run for a while but with dedication it does work.

Finally, I would not use "said parameter value" on the AFM without understanding their impact on the running of the instrument. There is no default values to be inserted to get a decent image as it is highly dependent on the surface/sample you are trying to image. If you want to start reading about AFM, i would highly recommend an invaluable book from my good friend Patrick Gunning "Atomic Force Microscopy for Biologists". It is essential to actually read the first 2 chapters of that book before switching on an AFM.

Best of Luck

Laurent

Dear Laurant,

Thank you for a detailed answer for my issue. I have spent lot of time in AFM now. Your insights are really important. I would eagerly wait for your paper for Gram+/- as I wanted to know membrane properties of both species as well.

So far I have not experienced the moving of the bacterial cell for some reason. But I always doubt that the contact mode might damage the carbohydrate layer of the cell!

I want to know the adhesion properties od the cell to the different surfaces I have made. So immobilisation is not required! I would be glad if you can share your expertise with me regarding AFM with cells.

Do you have the same instrument?

Thanks for the recommondation, I will find it and read during these holidays.

Thanks.