Hi Michael, 

I did some experiments with hydrogels in AFM before. However, I guess your collagen hydrogels have a lower elastic modulus than the ones I measure. The reference of my paper is the following: Flores-Merino et al. (2010) Soft Matter 6:4466. In the methodology you can find your answer, but basically the hydrogels I used (PVP hydrogels) were immobilized onto a glass slide using a water-insoluble adhesive. On the other hand, we also bonded polyacrylamide hydrogels by aminosilane chemistry to cover slides. Hope this helps.

As far I know, only some (equiped with force measurement device - extensometer)  AFM systems may measure mechanical properties of sample. In most cases using modelled of model to designate spring constant and apply it into another math model is not measuremnt, but model :) Anyway, most suitable device is PIUMA (http://optics11.com/products/piuma) dedicated to measure mechanical properties of biological (soft) materials.

Thank you!! This paper looks helpful... How much of a concern is achieving a flat surface? Obviously, this is relative to the level of resolution we are looking at with the AFM tip, and also the fact that hydrogels of a fibrous nature like collagen will have inherent topography at the sub-cellular/sub-micron scale, especially when hydrated.... As a bit of background on our problem: We have a brand new Bruker FastScan Bio AFM coupled to a Renishaw InVia Raman spectrometer/microscope, which provides the level of resolution we are trying to achieve. We are interested in studying the composition and stiffness/modulus of the pericellular matrix of tumor cells as the progress at the scale the cells 'see and feel' the surrounding matrix environment... The gels are about the diameter of the well of a 48 well plate and ~2-3mm in height, so they are relatively small; about the size of a punch biopsy. The other challenge is moving the gels from the wells in which the cells were cultured on the collagen substrates to the slides for AFM sample prep after fixation, as the hydrogels fold over on themselves when pulled out of the media/PBS. The gels expand back to their regular shape once immersed back into liquid, so this adds and additional challenge when trying to set the gels into some type of adhesive, so as to maintain the surface for analysis... I am wondering if using aminosilane (e.g., APTES or MPTMS) or other similar bioconjugation chemistry would allow me to adhere the collagen hydrogels to the surfaces of the slides without changing the chemistry of the surface of the gel we wish to evaluate with AFM and Raman. We have been trying to use a 50:50 mixture of Vaseline and parrafin to provide a water insoluble adhesive with a reasonable viscosity that allows for the gels to be pressed down into it and remain stationary. I figure attempting to match the viscosity of the adhesive to the gel would be best. This works for our stiffer cross-linked and compressed collagen hydrogels, but our regular 3mg/ml collagen hydrogels have a very low modulus, so it becomes difficult to embed them into such a mixture. We have also tried Superglue (cyanoacrylate glue), but this is difficult to do while keeping the gels hydrated and without introducing glue throughout the entire collagen matrix. I was thinking that fibrin glue may be something we could try?... Any other thoughts or suggestions would be greatly appreciated!!