Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.

https://web.physics.ucsb.edu/~hhansma/HHansma%20NAR96%20713.pdf

Atomic Force Microscopy of Long and Short Double-Stranded, Single-Stranded and Triple-Stranded Nucleic Acids

One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells. This is correlated with the presence in the membrane of resistant cells of a 150-180-kDa glycoprotein, P-glycoprotein, responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4'-o-tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells. The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe. The data show that the uptake occurs through free permeation of the neutral form of the drug. For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+(7.2) = V+/[D]e.n where V+ and [D]e are the initial rate of uptake and the concentration of drug in the medium at pHe = 7.2 respectively and n is the number of cells. This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer. Efflux coefficients k-(7.2)- at pHi = 7.2 (the intracellular pH value) have also been calculated. In the case of sensitive cells, k+(7.2) and k-(7.2)- are equal. For resistant cells, the efflux coefficient is composed of two terms: (a) (k-)p corresponding to the passive diffusion of the neutral form of the drug and (k-)p = k+; (b) (k-)a corresponding to an active efflux mediated by the P-glycoprotein. Our data suggest that the anthracycline derivatives efflux actively in the neutral form.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb15840.x

Comparison of the membrane transport of anthracycline derivatives in drug‐resistant and drug‐sensitive K562 cells

Anthracyclines in haematology: preclinical studies, toxicity and delivery systems

Contribution of DNA conformation and topology in right-handed DNA wrapping by the Bacillus subtilis LrpC protein.

Among electron beam microanalytical techniques, electron energy loss spectrometry (EELS) offers unique advantages in terms of information content, sensitivity, limits of detection. This paper describes new methods and tools for acquiring families of spectra over many pixels on the specimen, i.e. spectrumimages, and for processing them. Applications in different fields of research, both in materials science and in life sciences, demonstrate the potential impact of the technique for characterizing nano-sized structures.

Electron energy loss spectrometry mapping

/pdf/comparative-observations-of-biological-specimens-especially-2stngojxcs.pdf

Comparative observations of biological specimens, especially DNA and filamentous actin molecules in atomic force, tunnelling and electron microscopes

Relationship between the intracellular level and growth inhibition of a new anthracycline 4′O-tetrahydropyranyl-adriamycin in friend leukemia cell variants

The conditions for the use of Electron Spectroscopic Imaging (ESI) in the observation of DNA molecules adsorbed on carbon film are described. The Zeiss 902 electron microscope with its built-in spectrometer allows original modes of imaging, such as energy-filtered annular dark-field. The six different modes of imaging are illustrated with either positively stained or metallized DNA preparations to show the resulting important differences in contrast. The very strong contrasts obtained in annular or spectroscopic dark-field modes on specimens presenting a very low contrast in traditional bright-field are very useful for the observation of biological macromolecules. The advantages of some observation modes for imaging different types of preparations are discussed.

/pdf/new-possibilities-in-the-observation-of-nucleic-acids-by-1kibxghsn7.pdf

New possibilities in the observation of nucleic acids by electron spectroscopic imaging

Au vu du developpement considerable de l'imagerie des macromolecules biologiques a l'aide des microscopes a champ proche, il est rappele que ce type d'observations d'objets biologiques pose encore de reels problemes, tant au niveau methodologiques pour la preparation des echantillons, qu'au niveau de l'interpretation des images. Une approche interdisciplinaire est recommandable, qui utiliserait conjointement les methodes traditionnelles de la biophysique et de la biochimie, et celles, relativement mieux etablies, de la microscopie electronique. La microscopie en champ proche possede cependant des possibilites uniques d'observation et d'analyse des objets biologiques, qui ne sont pas accessibles en microscopie electronique, et qui conferent a ces nouvelles microscopies une nouvelle dimension a l'imagerie en biologie. L'illustration fournie donne des exemples des possibilites et des limitations de la microscopie electronique, et propose quelques domaines dans lesquels la complementarite des differents moyens d'observation serait la bienvenue

/pdf/are-electron-and-near-field-microscopies-complementary-29n05f5htc.pdf

Alain Fourcade

Papers

Comparative observations of biological specimens, especially DNA and filamentous actin molecules in atomic force, tunnelling and electron microscopes

Relationship between the intracellular level and growth inhibition of a new anthracycline 4′O-tetrahydropyranyl-adriamycin in friend leukemia cell variants

New possibilities in the observation of nucleic acids by electron spectroscopic imaging

Are electron and near-field microscopies complementary?

Cytogenetic modifications of friend leukemia cells resistant to adriamycin