Biocurious is a weblog about biology, quantified.

Hybrid TIRF/AFM Imaging

by Andre on 27 October 2006

One of the things I’ve been working on in the last few months is simultaneous fluorescence and atomic force microscopy (AFM) of a variety of biological samples. I’ve talked about total internal reflection fluorescence microscopy, or TIRF, before and I’ve also shown you some AFM images of leaves and insect wings so I think the time is ripe to show you some work on combining the two. I should point out that all of the samples I’m going to show you were expertly prepared by Vidya Nadar in Peter Baas’s lab at Drexel University College of Medicine. Thanks to Vidya and Peter!

The first image shows the growth cone of an axon. Part (a) is a TIRF image of the stained microtubules that run through the body of the axon and splay into the growth cone. We know that everything in red is a microtubule because the labeling was done with tubulin specific antibodies. That’s the power of fluorescence combined with immunostaining. This technology has become indispensable in cell biology. To complement this information, AFM gives high resolution images with quantitative height information. Part (b) is a TappingMode AFM image of the same region and it is much easier to make out the cell extremities where there are no (or few) microtubules. It also contains information about the microtubule arrangement in the axon that is not available from the TIRF image alone. For example, two microtubule bundles are visible running through the axon in the TIRF image, but in the region of the AFM image highlighted with a white box, their relative position becomes clear: the lower bundle is actually broken and frayed, passing over its neighbor and terminating above the main part of the axon. Without the AFM image one might falsely conclude that the bundles simply merged and continued unobstructed through the rest of the axon. The line profiles in the inset illustrates this more clearly (these just show the intensities measured along the yellow lines in each image).

Here’s another image showing a glial cell from the same sample with the fluorescence overlaid on the AFM height image.

Using AFM for imaging cells is useful and, although you will often get nicer images from electron microscopy, AFM doesn’t require complicated sample preparation and cells can be imaged while they’re alive in ambient conditions. That eliminates some sample preparation artifacts and also makes it possible to make movies of processes occurring of the scale of several minutes.

But that’s not the most exciting thing to do with AFM. It is also possible to interact directly with samples using the AFM tip not for imaging, but for manipulation. Samples can be indented to measure their stiffness and the stiffness of their substrates, something that is increasingly recognized to be important for the regulation of several processes in different cell types (see Dennis’s review here) and most recently to direct stem cell differentiation (see Adam’s Cell paper [pdf]).

That work was done using a standard AFM, but now, using the combined instrument we can simultaneously visualize these manipulations. Here’s an example from a glial cell with labeled microtubules. Look how stretchy the fixed samples are! Sometimes I imagine fixed cells as almost vitrified, but that’s obviously not the case (nor would you expect it to be if you think about it: a crosslinked gel may be stiff, but it’s not solid). This same method was recently used to show that single fibrin fibers (the ones that form the scaffold of blood clots) are remarkably extensible (see also the movies on Martin Guthold’s site). But that’s not all. Since you can also measure the cantilever deflection during the manipulation, mechanical properties are also available from this method. This is a nice idea and you can expect to see a growing number of papers describing this kind of work in the next couple of years (maybe even some from me!).

  1. Nick    2865 days ago    #

    Visualization of the multiple hybrid AFM images in one 3D screen is perhaps one of the most challenging tasks of the computer graphics. ScienceGL Inc. develops an adequate graphics for such data interactive visualization.
    Fluorescence data together with AFM height and some other modes Visualized in 3D can be found here:

    Quoting from ScienceGL web page
    AFM microscopy is getting more and more sophisticated with introduction of the combined AFM instruments that support multiple modes of data acquisition. Modern AFM can produce multiple data sets taken at several modes such as: height, phase, modulated force, optical microscopy, luminescence, Raman spectroscopy, electrical (Kelvin potential), magnetic, etc. The amount of data to be analyzed together might be large so that the traditional 2D and 3D visualization is not adequate any longer for scientist.

    To address the needs of modern AFM microscopy ScienceGL has developed specific 3D graphics for multiple layer data taken at different AFM modes. The software brings together several different data sets into one 3D screen. This approach lets the researcher to reveal the complex trends and relationship between various phenomena under investigation. The software features advanced interactive set of measuring tools that permits fast and accurate data measurement in 3D virtual reality environment. The access to quantitative measurable parameters of data such as intersections, distance, volume, area makes our software the unique analyzing package for science and technology.
    ———end quote———-

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