High Voltage Cryomicroscopy of Human Blood Platelets

O’Toole, Eileen; Wray, G.; Kremer, James R.; McIntosh, J. Richard

doi:10.1006/jsbi.1993.1004

Cited by 43 publications

(13 citation statements)

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“…Near-®eld (Betzig et al, 1991) and related (Zenhausern et al, 1995) microscopes deliver higher resolution images, but only of near-surface features. Using 1 MeV (O'Toole et al, 1993) or energy ®ltered 120 keV (Grimm et al, 1998) electron microscopes, one can study samples at higher resolution in ice as thick as 0´5±1 mm, but this thickness limit is not suf®cient for most intact eukaryotic cells. Cryo X-ray microscopy offers a way to study the 2D and 3D organization of larger scale aggregates in unmodi®ed frozen specimens at a resolution intermediate between the capabilities of optical and electron microscopes.…”

Section: Discussionmentioning

confidence: 99%

Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Mäser

Osanna

Wang

et al. 2000

Journal of Microscopy

138

104

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We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 microm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 1010 Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography (Wang et al. (2000) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc. 197, 80-93) or spectroscopic analysis.

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Section: Discussionmentioning

confidence: 99%

Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Mäser

Osanna

Wang

et al. 2000

Journal of Microscopy

138

104

View full text Add to dashboard Cite

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“…Since sectioning frozen-hydrated samples has been difficult, several scientists have sought cellular samples that can be viewed without it. For example, the thin margin of a cell spread on an electron-transparent substrate has been imaged directly by high voltage electron microscopy using samples maintained at ∼−165°C (O'Toole et al 1993; Fig. 1).…”

Section: Images Of Frozen-hydrated Cells Are Revealing New Aspects Ofmentioning

confidence: 99%

Electron Microscopy of Cells

McIntosh

2001

The Journal of Cell Biology

109

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“…In electron microscopy, stability against radiation damage has long been solved by imaging the specimen at cryogenic temperatures [28–30]. Electron microscopy studies of fixed and dried versus frozen hydrated blood platelets [31] reveal tremendous differences in structural preservation. However, because of the thickness limitations of electron microscopy on frozen hydrated specimens [1–3], to our knowledge only thin, peripheral regions of whole, unsectioned eukaryotic cells have been imaged in a frozen hydrated state using electron microscopy [32,33].…”

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confidence: 99%

Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

et al. 2009

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We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below −170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.X-ray microscopes allow high resolution microscopy of intact, hydrated biological specimens with thicknesses of many micrometers, beyond the limit of biological electron microscopy [1][2][3]. Radiation damage precludes repeated imaging of live specimens [4], but this can be mitigated by working at liquid nitrogen temperature [5,6]. In addition, single view flash imaging of cells using ultrabright sources has been proposed [7,8] as a way of capturing the image before radiolytical and thermal damage become evident.In recent years, there has been much progress in developing zone plate microscopy for 3D imaging of frozen hydrated cells [9][10][11][12][13]. While there are demonstrations of x-ray optics with higher resolution [14][15][16], scientific applications using x-ray microscopes have mainly used Fresnel zone plate optics with 25-40 nm spatial resolution. These optics typically have a focusing efficiency in the 10% range [17] and the modulation transfer function for incoherent bright field imaging decreases the efficiency of utilization of higher spatial frequency information. As a result, while the practical advantages of lens-based microscopes will be the deciding factor for most studies, it is also worthwhile to consider alternative methods for high resolution x-ray imaging.X-ray diffraction microscopy (XDM), also called coherent x-ray diffraction imaging, was proposed by Sayre as an imaging method that dispenses with the technological limits of lens efficiency and resolution [18]. Instead, the far-field diffraction pattern of an isolated object illuminated by a coherent x-ray beam is recorded. If the object is finite, and the diffraction pattern is sampled finely enough, the object can be reconstructed from the measured diffraction intensities alone [19,20]. In this manner one is able to eliminate limitations due to the efficiency and finite numerical aperture of x-ray optics [21]. Following a first demonstration by Miao et *Chris.Jacobsen@stonybrook.edu. An important limitation applies to the demonstrations of x-ray diffraction microscopy of cells, chromosomes, and virions cited above: they have all involved dehydrated specimens at room temperature. Though Nishino et al. [27] have obtained a very exciting 3D XDM image of a dehydrated chromosome, they note significant resolution degradation due to accumulated radiation dose. In electron microscopy, stability a...

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High Voltage Cryomicroscopy of Human Blood Platelets

Cited by 43 publications

References 0 publications

Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy

Electron Microscopy of Cells

Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

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