Reliable training of Raman spectra-based tumor classifiers relies on a substantial sample pool. This study explores the impact of cryofixation (CF) and formalin fixation (FF) on Raman spectra using samples from surgery sites and a tumor bank. A robotic Raman spectrometer scans samples prior to the neuropathological analysis. CF samples showed no significant spectral deviations, appearance, or disappearance of peaks, but an intensity reduction during freezing and subsequent recovery during the thawing process. In contrast, FF induces sustained spectral alterations depending on molecular composition, albeit with good signal-to-noise ratio preservation. These observations are also reflected in the varying dual-class classifier performance, initially trained on native, unfixed samples: The Matthews correlation coefficient is 81.0% for CF and 58.6% for FF meningioma and dura mater. Training on spectral differences between original FF and pure formalin spectra substantially improves FF samples' classifier performance (74.2%). CF is suitable for training global multiclass classifiers due to its consistent spectrum shape despite intensity reduction. FF introduces changes in peak relationships while preserving the signal-to-noise ratio, making it more suitable for dual-class classification, such as distinguishing between healthy and malignant tissues. Pure formalin spectrum subtraction represents a possible method for mathematical elimination of the FF influence. These findings enable retrospective analysis of processed samples, enhancing pathological work and expanding machine learning techniques.

High subzero cryofixation: A technique for observing ice within tissues

Previously, we showed that cryo fixation of adult mouse brain tissue gave a truer representation of brain ultrastructure in comparison with a standard chemical fixation method (Korogod et al., 2015). Extracellular space matched physiological measurements, there were larger numbers of docked vesicles and less glial coverage of synapses and blood capillaries. Here, using the same preservation approaches, we compared the morphology of dendritic spines. We show that the length of the spine and the volume of its head is unchanged; however, the spine neck width is thinner by more than 30% after cryo fixation. In addition, the weak correlation between spine neck width and head volume seen after chemical fixation was not present in cryo-fixed spines. Our data suggest that spine neck geometry is independent of the spine head volume, with cryo fixation showing enhanced spine head compartmentalization and a higher predicted electrical resistance between spine head and parent dendrite.

Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.DOI:http://dx.doi.org/10.7554/eLife.05793.001

Cellular uptake of clustered α2β1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication.

Peyronie’s disease, a connective tissue disorder of penile tunica albuginea (TA) associated with penile deformity, curvature, pain and erectile dysfunction, is best managed surgically, but suitable graft biomaterials are not available. To establish whether cryopreservation affects human TA in view of its use in allotransplants. The effects on TA samples of the two most widely used tissue cryopreservation methods were investigated using an ad hoc panel of histochemical, immunohistochemical and ultrastructural tests. Apoptotic cells were evaluated using the TUNEL assay. Assessment of tissue integrity and arrangement of collagen and elastic fibers in thawed TA. Both cryofixation methods provided TA tissue suitable for use as graft material. Significant ultrastructural changes, namely a greater diameter of collagen fibrils, were detected in sections preserved in liquid nitrogen; nonetheless, such increase never exceeded the normal range. The comprehensive panel of assays used proved suitable to characterize the thawed tissue. Human TA is suitable for cryopreservation; freezing at -80 °C provides better results than preservation in liquid nitrogen.

X-ray microanalysis is a useful technique to qualify and quantify basic elements in biological specimens. This article reviews the principles and techniques for applying intermediate high voltage electron microscopy to X-ray microanalysis of various elements in biological specimens developed in our laboratory since the late 20th century. We first quantified the endproducts of histochemical reactions such as Ag in radioautographs, Ce in acid phosphatase reaction and Au in colloidal gold immunostaining using semi-thin sections by high voltage electron microscopy at 300-400 kV. We then analyzed various trace elements such as Zn, Ca, and S, which originally existed in the cytoplasmic matrix or cell organelles of various cells in different organs, and some absorbed elements introduced by experimental administration into cells and tissues such as Al, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning, freeze-drying, or freeze-substitution. As a result, we showed that the peak to background (P/B) ratios of all the elements analyzed had high P/B ratios at 300-400 kV. It was concluded that X-ray microanalysis using semi-thin sections by high voltage electron microscopy is of great utility for quantifying trace elements in biological specimens.

Abstract Cryo techniques of specimen preparation have become the standard for cytological studies of biological specimens. From the pioneering work of Feder and Sidman, and Zalokar, the now ubiquitous application of freeze substitution for transmission electron microscope studies of fungi was a direct result of work reported in 1979. Since then, cryopreparative methods have also become the standard for SEM studies and may, for certain purposes, replace conventional methods of fixation for light microscopy as well. Undoubtedly, there are instances where non-cryo methods might be preferred, or where comparisons of results using cryo vs. chemical fixations can provide unique information. On the whole, however, the advantages offered to mycologists and plant pathologists by cryo fixation over any chemical methods are many, and include (a) the opportunity to preserve specimens in a non-aqueous environment, (b) preservation of labile structures such as certain organelles, extracellular matrices, or various cellular content such as ions or that of vacuoles, (c) preservation of cells in a more life-like state,

Common methods for preparing samples for immunoelectron microscopy involve glutaraldehyde fixation (GA) followed by chemical dehydration (CD) or cryofixation (CF) succeeded by physical dehydration, i.e., freeze drying (FD) or freeze substitution (FS). The effects of these techniques have been evaluated with regard to the sizes of epoxy resin embedded rat somatotrophic secretory granules as well as the immunolabeling densities over these granules. The measurements were performed by computerized image analysis using electron microscopy in transmission (TEM) and scanning transmission (STEM) modes, which allowed us to define the immunolabeling in detail. The embedded secretory granules showed the same diameters after GA (2 hr) with CD and GA (15 min) with CF and FS, but were smaller after CF-FS, and smallest after GA (15 min) with CF and FD. The highest labeling density appeared after GA (15 min) and physical dehydration, in particular after freeze substitution. Based on our STEM pictures a new factor for evaluating and interpreting immunolabeling of granules is introduced; the "accessible immunogold labeling surface." It defines the fraction of the epoxy resin surface that is labeled and varies with the preparation methods. By using this factor, an order of labeling densities/micron 2 over the accessible areas could be established for the different techniques: GA-CF-FS > CF-FS > GA-CF-FD > GA-CD. The high labeling after GA-CF-FS may be due to the combination of a large accessible area and accurate preservation of the antigenicity of the hormones in the granules.