Age dependence of T-lymphocyte apoptosis induced by high-energy proton exposure

Peripheral blood samples from three donors of different ages were exposed to 300 kVp x-rays or 138 MeV protons (0, 2, and 9 Gy dose). After 48 h incubation, CD4 and CD8 T-lymphocytes were labelled with specific monoclonal antibodies and cellular DNA was stained by propidium iodine. Radiation-induced apoptosis was followed by flow cytometry and the data were processed by LYSIS II software. The data analysis revealed an age-dependent sensitivity to radiation-induced apoptosis by 300 kVp x-rays and 138 MeV protons, for both CD4 and CD8 T-lymphocytes. Radiation-induced apoptosis was about 4 times greater in CD4 lymphocytes from the youngest donor than the oldest donor and was about 2 times greater in CD8 T-lymphocytes, both after x-ray and proton exposures. RBE values for CD4 T-lymphocytes ranged from 0.9 to 1.4 and for CD8-positive cells from 0.7 to 0.9. It is concluded that radiation-induced apoptosis of CD4 and CD8 T-lymphocytes, which is already exploited to predict patient response in conventional radiotherapy, may also be used to predict response in proton treatment planning. Received: 23 June 2000 / Accepted: 29 January 2001     

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

Scanning tunneling microscopy with applications to biological surfaces

Each major advance in the field of microscopy has eventually been translated into major advances in the biological and medical sciences. The scanning tunneling microscope (STM) offers exciting new ways of imaging biological surfaces with resolution to the sub-molecular scale. Rigid, conductive surfaces can readily be imaged with the STM with atomic resolution. Unfortunately, few biological surfaces are sufficiently conductive or rigid enough to be examined directly with the STM. At present, non-conductive surfaces can be examined in two ways: 1) Sufficiently thin molecular layers attached to conductive substrates so that tunneling can occur through the molecules; or 2) coating or replicating non-conductive surfaces with metal layers so as to make them conductive, then imaging with the STM. We present images of biological and organic molecules obtained with these techniques that demonstrate the possibilities and limitations of each. Future advances leading to atomic resolution STM of biological surfaces depend on significant progress in the art and science of making biomaterials compatible with the restrictions of the instrument.     

Statistical evaluation of confocal microscopy images

BACKGROUND: The coefficient of variation (CV) is defined as the standard deviation (sigma) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean (mu) intensity (CV = sigma/mu). The field of flow cytometry has used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In a similar manner, the analysis of CV has been applied to the confocal laser scanning microscope (CLSM) to determine machine performance and sensitivity. METHODS: Instead of measuring 10,000 beads using a flow cytometer and determining the CV of this distribution of intensities, thousands of pixels are measured from within one homogeneous Spherotech 10-microm bead. Similar to a typical flow cytometry population that consists of 10,000 beads, a CLSM scanned image consists of a distribution of pixel intensities representing a population of approximately 100,000 pixels. In order to perform this test properly, it is important to have a population of homogeneous particles. A biological particle usually has heterogeneous pixel intensities that correspond to the details in the biological image and thus shows more variability as a test particle. RESULTS: The bead CV consisting of a population of pixel intensities is dependent on a number of machine variables that include frame averaging, photomultiplier tube (PMT) voltage, PMT noise, and laser power. The relationship among these variables suggests that the machine should be operated with lower PMT values in order to generate superior image quality. If this cannot be achieved, frame averaging will be necessary to reduce the CV and improve image quality. There is more image noise at higher PMT settings, making it is necessary to average more frames to reduce the CV values and improve image quality. The sensitivity of a system is related to system noise, laser light efficiency, and proper system alignment. It is possible to compare different systems for system performance and sensitivity if the laser power is maintained at a constant value. Using this bead CV test, 1 mW of 488 nm laser light measured on the scan head yielded a CV value of 4% with a Leica TCS-SP1 (75-mW argon-krypton laser) and a CV value of 1.3% with a Zeiss 510 (25-mW argon laser). A biological particle shows the same relationship between laser power, averaging, PMT voltage, and CV as do the beads. However, because the biological particle has heterogeneous pixel intensities, there is more particle variability, which does not make as useful as a test particle. CONCLUSIONS: This CV analysis of a 10-microm Spherotech fluorescent bead can help determine the sensitivity in a confocal microscope and the system performance. The relationship among the factors that influence image quality is explained from a statistical endpoint. The data obtained from this test provides a systematic method of reducing noise and increasing image clarity. Many components of a CLSM, including laser power, laser stability, PMT functionality, and alignment, influence the CV and determine if the equipment is performing properly. Preliminary results have shown that the bead CV can be used to compare different confocal microscopy systems with regard to performance and sensitivity. The test appears to be analogous to CV tests made on the flow cytometer to assess instrument performance and sensitivity. Published 2001 Wiley-Liss, Inc.     

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.     

Multiphoton microscopy in biological research

From its conception a decade ago, multiphoton microscopy has evolved from a photonic novelty to an indispensable tool for gleaning information from subcellular events within organized tissue environments. Its relatively deep optical penetration has recently been exploited for subcellularly resolved investigations of disease models in living transgenic mice. Its enhanced spectral accessibility enables aberration-free imaging of fluorescent molecules absorbing in deep-UV energy regimes with simultaneous imaging of species having extremely diverse emission spectra. Although excited fluorescence is the primary signal for multiphoton microscopy, harmonic generation by multiphoton scattering processes are also valuable for imaging species with large anharmonic modes, such as collagen structures and membrane potential sensing dyes.     

Concepts in imaging and microscopy. Exploring biological structure and function with confocal microscopy

Confocal microscopy is providing new and exciting opportunities for imaging cell structure and physiology in thick biological specimens, in three dimensions, and in time. The utility of confocal microscopy relies on its fundamental capacity to reject out-of-focus light, thus providing sharp, high-contrast images of cells and subcellular structures within thick samples. Computer controlled focusing and image-capturing features allow for the collection of through-focus series of optical sections that may be used to reconstruct a volume of tissue, yielding information on the 3-D structure and relationships of cells. Tissues and cells may also be imaged in two or three spatial dimensions over time. The resultant digital data, which encode the image, are highly amenable to processing, manipulation and quantitative analyses. In conjunction with a growing variety of vital fluorescent probes, confocal microscopy is yielding new information about the spatiotemporal dynamics of cell morphology and physiology in living tissues and organisms. Here we use mammalian brain tissue to illustrate some of the ways in which multidimensional confocal fluorescence imaging can enhance studies of biological structure and function.     

Grayscale representation of infrared microscopy images by extended multiplicative signal correction for registration with histological images

Fourier‐transform infrared (FTIR) microspectroscopy is rounding the corner to become a label‐free routine method for cancer diagnosis. In order to build infrared‐spectral based classifiers, infrared images need to be registered with Hematoxylin and Eosin (H&E) stained histological images. While FTIR images have a deep spectral domain with thousands of channels carrying chemical and scatter information, the H&E images have only three color channels for each pixel and carry mainly morphological information. Therefore, image representations of infrared images are needed that match the morphological information in H&E images. In this paper, we propose a novel approach for representation of FTIR images based on extended multiplicative signal correction highlighting morphological features that showed to correlate well with morphological information in H&E images. Based on the obtained representations, we developed a strategy for global‐to‐local image registration for FTIR images and H&E stained histological images of parallel tissue sections.     

Edge detection in microscopy images using curvelets

Background  

Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories.     

Cryo-electron microscopy of artificial biological membranes

Vitrified synthetic phosphatidycholine liposome suspensions were studied by cryo-electron microscopy. The bilayer structure is resolved on vitrified liposome images. The packing of the aliphatic chains of the lipid within vitrified liposomes can be determined by the analysis of electron diffraction patterns. Images and electron diffraction patterns show that the structure of vitrified liposomes is related to the structure that liposomes have before vitrification. In fact, vitrified liposomes have a different structure, depending whether they are maintained before cooling at a temperature higher or lower than that corresponding to the ‘melting’ of the hydrocarbon chain of the lipids. Below the melting temperature, liposomes are formed by small domains.     

Quantification and calibration of images in fluorescence microscopy

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue “phantoms” that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (?40 and ?4 °C).     

Feature extraction from three-dimensional images in quantitative microscopy

Different methods are investigated in selecting and generating the appropriate microscope images for analysis of three-dimensional objects in quantitative microscopy. Traditionally, the ‘best’ focused image from a set is used for quantitative analysis. Such an objectively determined image is optimal for the extraction of some features, but may not be the best image for the extraction of all features. Various methods using multiple images are here developed to obtain a tighter distribution for all features.Three different approaches for analysis of images of stained cervical cells were analyzed. In the first approach, features are extracted from each image in the set. The feature values are then averaged to give the final result. In the second approach, a set of varying focused images are reconstructed to obtain a set of in-focus images. Features are then extracted from this set and averaged. In the third approach, a set of images in the three-dimensional scene is compressed into a single two-dimensional image. Four different compression methods are used. Features are then extracted from the resulting two-dimensional image. The third approach is employed on both the raw and transformed images.Each approach has its advantages and disadvantages. The first approach is fast and produces reasonable results. The second approach is more computationally expensive but produces the best results. The last approach overcomes the memory storage problem of the first two approaches since the set of images is compressed into one. The method of compression using the highest gradient pixel produces better results overall than other data reduction techniques and produces results comparable to the first approach.     

Measuring the size of biological nanostructures with spatially modulated illumination microscopy

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.     

NanoSIMS chemical imaging combined with correlative microscopy for biological sample analysis

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