NanoSIMS imaging of Bacillus spores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top‐sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift‐out. The lift‐out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top‐cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ～300 nm for top‐cut sections versus ～100 nm for electron‐transparent sections.     

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0·3–1·0 μm). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than ～10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the ‘restored’ images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.     

A comparison of subcellular element concentrations in frozen-dried, plastic-embedded, dry-cut sections and frozen-dried cryosections

Biological X-ray microanalysis of diffusible elements within cellular and subcellular compartments requires preparation methods to retain electrolytes in the compartments they occupied in vivo. X-ray microanalysis of frozen-dried, plastic-embedded samples has been used to quantitate electrolytes at the cellular level. We have compared the subcellular elemental distribution in dry cut sections from such samples with that in ultrathin frozen-dried cryosections. Rat pancreases were quench-frozen onto a helium-vapor-cooled copper block. Cryosections were cut at 130-150 K, transferred using a Gatan cold stage, frozen-dried in the column and analysed at 190 K. Tissue fragments were frozen-dried at 190 K, and cut on a dry knife at 293 K. Both samples provided images permitting unambiguous identification of all major compartments except the Golgi complex. Intracellular potassium-to-sodium ratios obtained on frozen-dried plastic-embedded sections were lower than for cryosections (e.g. 1.77 in basal cytoplasm in plastic sections as compared to 4.34 for cryosections) and varied with the pre-embedding procedure (e.g. 1.77 in formaldehyde-fixed as compared to 2.87 in osmium-fixed plastic sections). Potassium gradients between adjacent organelles were large in cryosections and insignificant in plastic-embedded material. Higher cytoplasmic phosphorus, potassium and sulfur concentrations were observed in cryosections. Therefore, a redistribution of electrolytes and covalently bound elements occurred subcellularly in the plastic sections. Calcium was quantifiable in most organelles in cryosections but the plastic lowered sensitivity too much to permit routine calcium quantification. We conclude that in our hands frozen-dried, plastic-embedded samples were compromised and provided lower sensitivity than cryosections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specimen thickness dependence of hydrogen evolution during cryo‐transmission electron microscopy of hydrated soft materials

The evolution of hydrogen from many hydrated cryo‐preserved soft materials under electron irradiation in the transmission electron microscope can be observed at doses of the order of 1000 e nm^?2 and above. Such hydrogen causes artefacts in conventional transmission electron microscope or scanning transmission electron microscopy (STEM) imaging as well as in analyses by electron energy‐loss spectroscopy. Here we show that the evolution of hydrogen depends on specimen thickness. Using wedge‐shaped specimens of frozen‐hydrated Nafion, a perfluorinated ionomer, saturated with the organic solvent DMMP together with both thin and thick sections of frozen‐hydrated porcine skin, we show that there is a thickness below which hydrogen evolution is not detected either by bubble observation in transmission electron microscope image mode or by spectroscopic analysis in STEM electron energy‐loss spectroscopy mode. We suggest that this effect is due to the diffusion of hydrogen, whose diffusivity remains significant even at liquid nitrogen temperature over the length scales and time scales relevant to transmission electron microscopy analysis of thin specimens. In short, we speculate that sufficient hydrogen can diffuse to the specimen surface in thin sections so that concentrations are too low for bubbling or for spectroscopic detection. Significantly, this finding indicates that higher electron doses can be used during the imaging of radiation‐sensitive hydrated soft materials and, consequently, higher spatial resolution can be achieved, if sufficiently thin specimens are used in order to avoid the evolution of hydrogen‐based artefacts.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Note: direct measurement of the point-to-point resolution for microns-thick specimens in the ultrahigh-voltage electron microscope

We report on a direct measurement method and results of the point-to-point resolution for microns-thick amorphous specimens in the ultrahigh-voltage electron microscope (ultra-HVEM). We first obtain the ultra-HVEM images of nanometer gold particles with different sizes on the top surfaces of the thick epoxy-resin specimens. Based on the Rayleigh criterion, the point-to-point resolution is then determined as the minimum distance between centers of two resolvable tangent gold particles. Some values of resolution are accordingly acquired for the specimens with different thicknesses at the accelerating voltage of 2 MV, for example, 18.5 nm and 28.4 nm for the 5 μm and 8 μm thick epoxy-resin specimens, respectively. The presented method and results provide a reliable and useful approach to quantifying and comparing the achievable spatial resolution for the thick specimens imaged in the mode of transmission electron including the scanning transmission electron microscope.     

A new method to correlate acoustic spectroscopic microscopy (30 MHz) and light microscopy

A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 μm and 10 μm thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2·5 × 2·5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 μm. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 × 250 μm. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.     

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.     

The preparation,examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and X-ray microanalysis

A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X-ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen-hydrated. Criteria for assessing whether or not the tissue remains frozen-hydrated are reported. After quench freezing, 1–2 μm thick sections of mouse liver were cut at 193°K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143°K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X-ray analyser. Concentration of intra-nuclear and intracytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.     

Soft X-ray contact microscopy with nanosecond exposure times

High resolution (better than 20 nm) contact micrographs have been produced with exposure times of about a nanosecond. The illuminating source was a short-lived carbon plasma produced by focusing a single short (～1 ns) 100 J pulse from the Vulcan laser at the Rutherford Appleton Laboratory (RAL) to a 300 μm spot on a graphite target. This plasma emits strongly in the soft X-ray region, particularly at the CVI (3.37 nm) and CV (4.03 nm) lines. The specimens were behind a 100 nm thick Si₃N₄ window, at atmospheric pressure in an environmental cell. The images of diatoms recorded on X-ray resist showed features down to the limit of resolution of the SEM used to view the developed resist, which was about 20 nm.     

Estimation of organelle water fractions from frozen-dried cryosections

Local dry mass or water fractions can be measured on frozen-dried cryosections assuming constant section thickness in the hydrated state and no net water movements and no differential shrinkage during freezing and drying. These assumptions have been tested on a model consisting of isolated rat liver mitochondria in an albumin matrix with a concentration similar to the dry mass concentration of the cytoplasm. The dry mass concentrations of mitochondria before freezing as measured by interference microscopy and after freezing and freeze-drying of the sections as measured by X-ray microanalysis and scanning microdensitometry are shown to be equal as long as the ice crystals in the medium are smaller than about 100 nm. It is concluded, therefore, that the above-mentioned assumptions could also hold for the cryopreparation of cells and tissues.     

Sectioning and imaging hard mineral fibres in biological tissues

A technique is described which overcomes the problems associated with sectioning biological tissue containing hard mineral fibres. 0·2–0·5 μm thick sections were cut with a diamond knife, placed in a folding grid, conventionally stained with uranyl acetate and lead citrate and viewed at an accelerating voltage of 200 kV in the scanning transmission mode.     

Properties of frozen sections relevant to quantitative microanalysis

Difficulties in the quantitative X-ray microanalysis of frozen sections may conceivably arise from ice-crystal damage and from electron-beam damage. X-ray peak-to-continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice-crystal formation existing prior to sublimation. The experiments in these reports are re-interpreted here; it is argued that peak intensities may be affected by ice-crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak-to-continuum method is affected by beam-induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold-stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen-hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen-hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X-ray analysis at very high resolution.     

Increasing membrane contrast by means of imidazole-osmium post-fixation as exemplified by skeletal muscle fiber

Until now, the interpretation of findings derived from investigations on membrane structures (T tubules, sarcoplasmic reticulum, the Golgi apparatus) in thick sections of mammalian muscle tissue has been limited in TEM due to the lack of sharp resolution of the membrane contours. This article shows how the imidazol-osmium post-fixation of tissue blocks can be used to achieve well-contrasted, sharply defined membrane contours. Therefore, unstained sections from imidazol-osmium post-fixed tissue can be examined immediately. But protein structures (e.g., ribosomes) remain uncontrasted with this technique. If needed, it is possible to visualize the protein structures by conventional section staining with uranyl acetate and lead citrate. This method is suitable for both ultrathin and thick sections (>150 nm).     

Investigation of dyed human hair fibres using apertureless near-field scanning optical microscopy

We present the first studies of dyed human hair fibres performed with an apertureless scanning near‐field optical microscope. Samples consisted of 5‐µm‐thick cross‐sections, the hair fibres being bleached and then dyed before being cut. Hair dyed with two molecular probes diffusing deep inside the fibre or mainly spreading at its periphery were investigated at a wavelength of 655 nm. An optical resolution of about 50 nm was achieved, well below the diffraction limit; the images exhibited different optical contrasts in the cuticle region, depending on the nature of the dye. Our results suggest that the dye that remains confined at the hair periphery is mainly located at its surface and in the endocuticle.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

Silver impregnation applied to striated muscle: The sarcoplasmic reticulum

The classical black reaction developed by Camillo Golgi is shown to impregnate the tubules and fenestrations of the sarcoplasmic reticulum (SR) in striated muscle. This is a double impregnation of chromate and silver, which usually fills extracellular spaces. The method is difficult insofar as long incubation times are required, and location of the successfully “stained” SR in plastic-embedded tissue blocks is unpredictable. The light microscope is absolutely necessary to find the good regions which can then be cut from the blocks in 1-μm-thick sections and examined in the electron microscope. Stereo pairs give the best results since these resolve overlap problems common to thick sections. A variety of artifacts are illustrated which can help avoid erroneous interpretations. The Golgi-“stained” SR shows this elusive network with unsurpassed contrast and should benefit the morphological studies of muscle-membrane enthusiasts.