Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microscope enables a direct comparison between features observed in the fluorescence microscope and those observed in the images obtained with the AFM, in air or under liquid. The cracks in polymerized Langmuir-Blodgett films of lO,l2-pentacosadiynoic acid as observed in the fluorescence microscope run parallel to one of the lattice directions of the crystal as revealed by molecular resolution images obtained with the AFM. The orientation of these cracks also coincides with the polarization direction of the fluorescent light, indicating that the cracks run along the polymer backbone. Ripple-like corrugations on a submicrometer scale have been observed, which may be due to mechanical stress created during the polymerization process

Atomic Force Microscopy of DNA Electrophoresed onto Silylated Mica

A new technique has been developed for electrophoresing DNA molecules from an agarose gel onto a silylated mica substrate where they can be imaged with an atomic force microscope (AFM). With a simple modification, the technique can also be used for polyacrylamide gels. This method does not require purification of samples from the gels. Using tapping mode AFM, we have observed plasmids after electrophoretic separation into two bands. Differences in conformation were observed between the plasmids in the two bands

Improved Visualization of DNA in Aqueous Buffer with the Atomic Force Microscope

An improved method has been developed for imaging deoxyribonucleic acid (DNA) in aqueous buffer with the atomic force microscope (AFM). DNA on untreated mica can be imaged in aqueous buffer with the AFM if the DNA is deposited onto the mica in a buffer with HEPES and MgCl2, if the sample is rinsed thoroughly with high water pressure, and if the imaging is done with an electron beam-deposited (EBD) tip that has been deposited in the scanning electron microscope (SEM). The water rinse removes DNA that is otherwise easily scraped off the substrate. There is evidence that sharper tips may be more damaging to DNA when imaged in aqueous buffer especially when the DNA is bound tightly to the mica. The ability to image DNA in nearly biological conditions has potential applications for imaging biomolecular processes with the AFM

Changes in the Elastic Properties of Cholinergic Synaptic Vesicles as Measured by Atomic Force Microscopy

Cholinergic synaptic vesicles from Torpedo californica have been probed with the atomic force microscope in aqueous buffers to map and measure their elastic properties. Elastic properties were mapped with a new atomic force microscope technique known as force mapping. Force mapping of vesicles showed that the centers of the vesicles are harder or stiffer than the peripheral areas in the three buffers that were investigated. These were an isoosmotic buffer, a hypoosmotic buffer, and an isoosmotic buffer with 5 mM CaCl(2) added. The hardness of the vesicular centers was quantified by calculation of the elastic modulus. Elastic moduli were in the range of 2-13 × 10(5) Pa. Vesicular centers were hardest in calcium-containing buffer and softest in isoosmotic buffer. Hypotheses are presented for the composition and function of the hard centers

Atomic force microscopy of DNA in aqueous solutions

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers If the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, It shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E.coli RNA polymerase bound to DNA in a propanol water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM