Franck–Condon-like Progressions in Infrared Spectra of Biological Molecules

High resolution dark field microscopy is becoming an important tool for the investigation of unstained and specifically stained biological molecules. Of primary consideration to the microscopist is the interpretation of image Intensities and the effects of radiation damage to the specimen. Ignoring inelastic scattering, the image intensity is directly related to the collected elastic scattering cross section, σɳ, which is the product of the total elastic cross section, σ and the eficiency of the microscope system at imaging these electrons, η. The number of potentially bond damaging events resulting from the beam exposure required to reduce the effect of quantum noise in the image to a given level is proportional to 1/η. We wish to compare η in three dark field systems.

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3-D reconstruction of molecular shape from microcrystal thin sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077311 ◽

1983 ◽

Vol 41 ◽

pp. 748-749

Author(s):

S. Cusack ◽

J.-C. Jésior

Keyword(s):

Three Dimensional ◽

Molecular Shape ◽

Biological Molecules ◽

Fourier Space ◽

Single Particles ◽

Thin Sections ◽

Standard Methods ◽

X Ray ◽

X Ray Crystallography ◽

Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

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Direct visualization of phase-separated domains in lipid membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082315 ◽

1978 ◽

Vol 36 (2) ◽

pp. 290-291

Author(s):

S. W. Hui ◽

T. P. Stewart

Keyword(s):

Lipid Membranes ◽

Structural Information ◽

Freeze Fracture ◽

Biological Molecules ◽

Vacuum Drying ◽

Direct Visualization ◽

X Ray Diffraction ◽

Electron Microscopic ◽

X Ray ◽

Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

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Correction of the contrast transfer function to determine the radial density distribution of frozen-hydrated tobacco mosaic virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123088 ◽

1992 ◽

Vol 50 (1) ◽

pp. 536-537

Author(s):

Michael F. Smith ◽

John P. Langmore

Keyword(s):

Transfer Function ◽

Phase Contrast ◽

Heavy Atom ◽

Biological Molecules ◽

Frequency Compensation ◽

Contrast Transfer Function ◽

High Background ◽

Low Contrast ◽

Radial Density Distribution ◽

Excellent Preservation

The purpose of image reconstruction is to determine the mass densities within molecules by analysis of the intensities within images. Cryo-EM offers this possibility by virtue of the excellent preservation of internal structure without heavy atom staining. Cryo-EM images, however, have low contrast because of the similarity between the density of biological material and the density of vitreous ice. The images also contain a high background of inelastic scattering. To overcome the low signal and high background, cryo-images are typically recorded 1-3 μm underfocus to maximize phase contrast. Under those conditions the image intensities bear little resemblance to the object, due to the dependence of the contrast transfer function (CTF) upon spatial frequency. Compensation (i.e., correction) for the CTF is theoretically possible, but implementation has been rare. Despite numerous studies of molecules in ice, there has never been a quantitative evaluation of compensated images of biological molecules of known structure.

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Methylamine vanadate (nanovan) negative stain

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168396 ◽

1994 ◽

Vol 52 ◽

pp. 132-133

Author(s):

James F. Hainfeld ◽

Daniel Safer ◽

Joseph S. Wall ◽

Martha Simon ◽

Beth Lin ◽

...

Keyword(s):

Electron Beam ◽

Low Dose ◽

Biological Molecules ◽

Positive Staining ◽

Signal To Noise ◽

Negative Stain ◽

Beam Attenuation ◽

Coarse Grains ◽

Lower Contrast

Uranyl and tungstate compounds have found favor as negative stains because of their high scattering power relative to biological molecules. However, other properties, such as specimen preservation, resistance to alterations or crystallization in the electron beam, and signal to noise (S/N) ratio, are also important. It may be that lower density materials may have advantages in these areas. A new negative stain, methylamine vanadate, CH3 NH2.VO3 ("NanoVan"), offers a near physiological pH of 8, similar to phosphotungstate (pH 7) with much smoother background. It is also very stable in the electron beam with minimal granulation at a dose of l04 el / nm2 . The resolution obtainable with vanadate appears to be comparable to uranyl at low dose, but superior at higher dose where uranyl forms coarse grains (see Fig. 1). Problems with uranyl such as unwanted positive staining and need for pH below 4 can be avoided. The lower contrast permits use of thicker stain embedment for better preservation and less flattening without excessive beam attenuation.

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