Bioimaging最新文献

We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP).

Imaging performance in a confocal fluorescent microscope can be improved by using multiple detectors. The use of a large detector radius in a confocal fluorescent microscope, while giving a strong signal for lower spatial frequencies, results in negative components at some higher frequencies. Using this fact, effects of a simple detector array consisting of the difference signal between small and large detectors are considered. The effects on the transverse and axial variation of the three-dimensional optical transfer functions (OTFs) of the confocal fluorescent microscope are considered. The results show that the resulting OTFs are boosted, resulting in a better resolution.

Knowledge of flow in porous media can be improved by analyzing three-dimensional (3-D) images of the water distribution in a soil. We report on such a study using computed tomography (CT) scanning of a clay soil sample first in dry conditions. Then, after an infiltration experiment, by scanning the sample again in wet conditions. We used test phantoms to determine the optimal scanning parameters to obtain images with a bimodal gray value distribution with high contrast and small standard deviation. We also determined the effect of slice thickness and reconstruction algorithm on the restoration and segmentation of the images. For a good 3-D representation of the pore space we scanned the slices adjacently at an axial resolution of 1 mm and a pixel size of 0.27 × 0.27 mm². The high contrast between air in the pores and the clay background allowed a global thresholding for the segmentation whereby the connectivity and topology of the pore networks is conserved. From the difference between the images of the dry sample and the wet sample we determined the water content distribution. We used the image data for measurements of the water distribution to study the relation between the structure of the pore networks and water flow.

The results of a Monte Carlo computer simulation of photon transport through biological tissue are presented. A comparison is made of the performance, in terms of resolution for a given signal-to-noise ratio, of spatial filtering, time-of-flight imaging and a combination of these approaches. The results show that a careful choice of time gate and aperture dimensions can significantly improve the performance of time-gated optical imaging systems.

Currently, two methods of detection and identification of single molecules are widely used: fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC). We present a thorough theoretical analysis of the error rates for identifying single molecules according to their diffusion coefficients (using FCS), and to their fluorescence lifetimes (using TCSPC). In most cases, the error rate using TCSPC is much lower. TCSPC is thus proven to be more versatile for analyzing single molecule events. The study is significant for a broad range of ultra-sensitive fluorescence detection applications.

Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. Thus, this protein appears particularly appropriate for studying the microheterogeneity of the macromolecule GFP on a single molecule level.

In ultrathin sections for electron microscopy the evaluation of images generated with immunocytochemical or in situ hybridization techniques can become difficult if the colloidal gold label is very small and the underlying structure shows a strong heavy metal contrast caused by conventional uranium/lead staining. Normally, the problem is overcome by analyzing specimens at higher magnifications or by enlarging the gold grains in a secondary process called silver enhancement. We present a method to visualize the small gold particles with electron spectroscopic imaging (ESI). Three energy-filtered images acquired at energy losses of 0, 45 and 120 eV are analyzed by digital image treatment so as to allow the optical separation of the Au-markers from the image background. This protocol can be adapted for applications related to the automated counting of the label.

The utility of autofluorescence imaging for early lung cancer detection has been previously demonstrated. The aim of this work is to extend the use of real-time autofluorescence imaging to the early cancer detection in the esophagus, stomach, and colon. A prototype fluorescence imaging system for the gastrointestinal (GI) tract was developed which produces real-time video images of tissue autofluorescence. It consists of a filtered mercury arc lamp light source, two intensified charge coupled device (ICCD) cameras, a fiber optic endoscope, and a computer-based control console. The system is capable of working with three different imaging modalities: (1) conventional white light imaging mode; (2) light induced fluorescence (LIF) imaging mode based on the fluorescence imaging of two wavelength bands (green and red); and (3) light induced fluorescence and reflectance (LIFR) imaging mode based on the combination of a green band fluorescence image and a red–near-IR reflectance image. The imaging wavelength bands were selected based on in vivo fluorescence spectroscopic studies. The fluorescence images (both LIF image and LIFR image) clearly delineate the abnormal tissue areas for biopsy. Early cancer sites are better visualized under fluorescence imaging than under conventional white light examination. Initial clinical tests demonstrated the usefulness of the imaging prototype system for early cancer detection in the GI tract.

A method is described that demonstrates a new technique for rapid and high-throughput single molecule sequencing. This sequencing technique is based on the successive enzymatic degradation of fluorescently labeled single DNA molecules, and the detection and identification of the released monomer molecules according to their sequential order in a microstructured channel.

The detection technique is evolved from confocal fluorescence microscopy, with two different laser sources to excite the individual mononucleotides that are either labeled with tetramethylrhodamine (TMR) or Cyanine5 (Cy5). The handling of DNA which is immobilized on carrier beads, and the detection of the cleaved monomers is performed in optically transparent and biochemically inert microstructures (glass or PMMA) with detection channels of 7 μ × 10 μm.

The projected rate of sequencing is ≈100 bases min⁻¹, dependent solely on the rate of the enzymatic DNA cleavage.