Biomedical subjects

C A Putman

Publications and source records attributed to C A Putman.

5 recordsLinked to original sources

PubMed1994

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy.

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm).

Animals↗

PubMed1993

Detection of in situ hybridization to human chromosomes with the atomic force microscope.

Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1-79 results in clear marking of the telomere region 1p36. The diameter of the probe p1-79 linked reaction product was 75-100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material.

Chromosomes, Human↗

PubMed1993

White blood cell differentiation using a solid state flow cytometer.

A flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set-up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche photodiode, which was used to detect the weak scattered light in the orthogonal direction. The new flow cytometer set-up is very small, relatively cheap and yields similar results as a standard flow cytometer set-up using a helium-neon laser and photomultipliers.

Cell Differentiation↗

PubMed1992

High-resolution imaging of chromosome-related structures by atomic force microscopy.

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein.

Animals↗

PubMedDate not supplied

Analysis of immunolabeled cells by atomic force microscopy, optical microscopy, and flow cytometry.

In this study we investigated the applicability of the (silver-enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied on these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a direct correlation was made between the force and the fluorescent images. Additionally, we performed flow cytometric analysis. From the results we conclude that immunogold labeling using small labels (1 nm) in combination with silver enhancement (30 min) proves to be a reliable method for high-resolution cell surface antigen detection in atomic force microscopy.

Animals↗