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Biomedical subjects

D Sayre

Publications and source records attributed to D Sayre.

12 recordsLinked to original sources
PubMed2001

An approach to three-dimensional structures of biomolecules by using single-molecule diffraction images.

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-A resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

Computer Simulation↗
PubMed2000

On possible extensions of X-ray crystallography through diffraction-pattern oversampling

It is known that sampling the diffraction pattern of a finite specimen, at a spacing somewhat finer than the Nyquist spacing (the inverse of the size of the diffracting specimen), corresponds to generating a no-density region surrounding the electron density of the specimen. This no-density region can then be used to retrieve the phase information. In earlier papers [Miao, Sayre & Chapman (1998). J. Opt. Soc. Am. A15, 1662-1669; Sayre, Chapman & Miao (1998). Acta Cryst. A54, 232-239], it was demonstrated, in the case of non-crystalline specimens, that this no-density region could be used to retrieve the phase information; here the same is performed for crystalline and near-crystalline specimens. By employment of an iterative algorithm, the phase information could be recovered from computer-generated oversampled diffraction patterns of small specimens that are (a) perfect or imperfect crystals, or (b) have a repeated motif without orientational regularity, or (c) are an unrepeated motif, such as an amorphous glass, a single molecule or a single biological cell. Cases (a) and (b) represent an extension over work recently published [Miao, Charalambous, Kirz & Sayre (1999). Nature (London), 400, 342-344]. Our algorithm requires an approximate envelope for the specimen. It does not require any structural knowledge concerning the specimen and does not require data to atomic resolution (although it can use such data if present). After a few hundred to a few thousand iterations, the correct phase set and image are recovered. The oversampling technique thus greatly extends the specimen range of X-ray crystallography but it imposes a high radiation dose on the specimens compared with the situation in crystallography, in which it is usual for the pattern to be sampled at the (much less fine) Bragg spacing (the inverse of the size of the unit cell). In cases where the specimen is a crystal, there are also possibilities for oversampling relative to Bragg (instead of Nyquist) sampling, thus providing a lesser degree of oversampling and the possibility of lower dosage. Damage of the specimen in consequence of the dose will in many cases seriously affect the quality and resolution of the imaging, but in at least one case [the biological cell in (c) above] the imaging obtainable with the aid of a cryogenic protective technique should surpass any other present method of whole-cell imaging. In addition, with the possible appearance in the future of free electron lasers (>10(12) photons and <200 fs per pulse), it is possible to circumvent the radiation-damage problem by recording diffraction patterns before damage manifests itself.

Journal Article↗
PubMed2000

The oversampling phasing method.

Sampling the diffraction pattern of a finite specimen more finely than the Nyquist frequency (the inverse of the size of the diffracting specimen) corresponds to surrounding the electron density of the specimen with a no-density region. When the no-density region is bigger than the electron-density region, sufficient information is recorded so that the phase information can be retrieved from the oversampled diffraction pattern, at least in principle. By employing an iterative algorithm, the phase information from the oversampled diffraction pattern of a micrometre-sized test specimen has been successfully retrieved. This method is believed to be able to open a door for high-resolution three-dimensional structure determination of complex and non-crystalline biological specimens, i.e. whole cells and sub-micrometre molecular clusters and micrometre-sized protein crystals. With the possible appearance in the future of X-ray free-electron lasers, it may become possible to image single molecules by recording diffraction patterns before radiation damage manifests itself.

Image Processing, Computer-Assisted↗
PubMed1995

X-ray microscopy.

The subject of X-ray microscopy (high-resolution X-ray imaging of general nonperiodic structures), an area in which much progress has been made in recent years, is reviewed. The main techniques are briefly described. Achievable performance levels, which for many years were highly speculative, can now be understood with fair accuracy in terms of basic X-ray and specimen properties, and techniques have progressed to the point where actual results are nearing those levels. In terms of specimen size and imaging resolution, X-ray microscopies lie between electron and light microscopy, and are thus suited to imaging extremely large and complex structures; in addition, they demand little or no specimen preparation, and can be used to observe local composition and chemical state as well as structure. Thus X-rays, which have played the leading role in imaging crystallizable materials, may also prove to be highly valuable in the imaging of very large non-crystalline structures. Throughout the treatment, attention is paid to the relationships connecting the subject with X-ray crystallography.

Crystallography, X-Ray↗
PubMed1986

Contact x-ray microscopy. A new technique for imaging cellular fine structure.

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.

Animals↗
PubMed1985

Direct imaging of live human platelets by flash x-ray microscopy.

A 100-nanosecond pulse of long-wavelength x-rays was used to produce high-resolution stop-motion images of living human platelets. Although some aspects of the structure conform to those seen in dehydrated specimens, novel features are apparent. The technique should permit detailed stop-motion examination of the interaction of platelets with their surrounding medium as well as exploration of the phagocytic and secretory activities of a wide variety of other cells.

Blood Platelets↗
PubMed1981

Improved detail in biological soft X-ray microscopy: study of blood platelets.

Improved image quality in soft x-ray contact microscopy can be obtained by examining the resist with transmission rather than scanning electron microscopy. Application of the new technique to air-dried preparations of human blood platelets reveals structures not visible in the same cells with transmission electron microscopy or when the resist is examined by scanning electron microscopy. As seen by the new technique, platelet pseudopods contain a central structure connected to a network in the platelet and dense bodies exhibit a lamellar structure.

Blood Platelets↗
PubMed1980

Ultrastructural and elemental imaging of biological specimens by soft X-ray contact microscopy.

Soft X-ray contact microscopy offers a means of visualizing unstained as well as stained biological materials at better than 6 nm resolution. Unlike light and transmission electron microscopy, which rely on stains to increase specimen contrast, soft X-ray imaging is dependent upon the differential absorption of incident soft (1-10nm wavelength) X-rays by the endogenous elements within the specimen. The advantages of using soft X-rays for imaging are: 1) reduced specimen damage during exposure; 2) ability to image hydrated specimens at atmospheric pressure; 3) ability to image specimens ranging in thickness from less than 40 nm to as much as 10 microns; and 4) ability to map the elemental composition of the specimen through observation of the differential absorption of properly chosen incident x-ray wave lengths. This paper explains the principles of the image formation and demonstrates the use of soft X-ray contact microscopy with biological samples which could not readily be imaged in their natural form using conventional electron microscopy methods.

Animals↗
PubMed1977

Transmission microscropy of unmodified biological materials: comparative radiation dosages with electrons and ultrasoft x-ray photons.

The minimum radiation dosage in a specimen consistent with transmission microscopy at resolution d and specimen thickness t is calculated for model specimens resembling biological materials in their natural state. The calculations cover 10(4)-10(7) eV electrons and 1.3-90 A photons in a number of microscopy modes. The results indicate that over a considerable part of the (t,d)-plane transmission microscopy on such specimens can be carried out at lower dosage with photons than with electrons. Estimates of the maximum resolutions obtainable with electrons and photons, consistent with structural survival of the specimen, are obtained, as are data on optimal operating conditions for microscopy with the two particles.

Air↗
PubMed1976

X-ray microscopy of biological objects with carbon kappa and with synchrotron radiation.

X-ray micrographs of biological objects have been obtained with a resolution better than 1000 angstroms by using poly(methyl methacrylate) x-ray resist and carbon Kalpha or synchrotron radiation. Synchrotron radiation allows short exposure times; storage rings especially designed as radiation sources and improved x-ray resists would make exposure times under 1 second possible.

Eukaryota↗