Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations.

Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations—causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively—are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

https://www.cell.com/ajhg/pdf/S0002-9297(11)00297-7.pdf

Mutations in DNAJC5, Encoding Cysteine-String Protein Alpha, Cause Autosomal-Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis

Effects of mot gene expression on the structure of the flagellar motor.

Freeze-fracture replication of lamellar granules and intercellular sheets of the horny layer in mouse, chicken, and snake epidermis reveals a pattern of serial fracture faces which is highly suggestive of polar lipids in a bilayer configuration. The occurrence of alternating wide and narrow fracture faces separated by intervening steps supports the view that epidermal barrier bilayers display lipid asymmetry similar to membranes. Within the lamellar granules, bilayers arrange to form disks which in fact are equivalent to flattened unilamellar liposomes. Stacking of the disks in turn gives rise to the lamellar pattern. After exocytosis into the intercellular space, the disks are arranged parallel to the cell membranes. In tangentially fractured specimens, the cleavage plane jumps back and forth from the plasma membrane to a disk-bilayer, thereby giving rise to the known phenomenon of EF-ridges (on the extracellular fracture face) and PF-grooves (in the plasmatic fracture face) which both represent the level of the plasma membrane sur- or subjacent to the aisles between disks. Concomitantly with the upward movement of the keratinocytes, the ridges and grooves become narrower until they fade away by the second or third cell layer of the stratum corneum. This phenomenon is explained by the fusion of adjacent disks at their highly curved brims due to a mechanism similar to the process of membrane fusion which causes the formation of wide, uninterrupted sheets.

Epidermal permeability barrier: Transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freexe-fracture study

Epidermal permeability barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze-fracture study

Background and noise impair image quality by affecting resolution and obscuring image detail in the low intensity range. Because background levels in unprocessed confocal images are frequently at about 30% maximum intensity, colocalization analysis, a typical segmentation process, is limited to high intensity signal and prone to noise-induced, false-positive events. This makes suppression or removal of background crucial for this kind of image analysis. This paper examines the effects of median filtering and deconvolution, two image-processing techniques enhancing the signal-to-noise ratio (SNR), on the results of colocalization analysis in confocal data sets of biological specimens. The data show that median filtering can improve the SNR by a factor of 2. The technique eliminates noise-induced colocalization events successfully. However, because filtering recovers voxel values from the local neighbourhood false-negative ('dissipation' of signal intensity below threshold value) as well as false-positive ('fusion' of noise with low intensity signal resulting in above threshold intensities), results can be generated. In addition, filtering involves the convolution of an image with a kernel, a procedure that inherently impairs resolution. Image restoration by deconvolution avoids both of these disadvantages. Such routines calculate a model of the object considering various parameters that impair image formation and are able to suppress background down to very low levels (< 10% maximum intensity, resulting in a SNR improved by a factor 3 as compared to raw images). This makes additional objects in the low intensity but high frequency range available to analysis. In addition, removal of noise and distortions induced by the optical system results in improved resolution, which is of critical importance in cases involving objects of near resolution size. The technique is, however, sensitive to overestimation of the background level. In conclusion, colocalization analysis will be improved by deconvolution more than by filtering. This applies especially to specimens characterized by small object size and/or low intensities.

Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques

SUMMARY

The geometry of the evaporated metal layer on models of membrane-associated particles has been calculated theoretically and the respective density profiles have been estimated. The models were a hollow cylinder, a hemisphere and a rotary ellipsoid substituting the freeze-fracture appearance of gap junction connexons and globular integral membrane proteins respectively. These calculations may be used as a help for choosing an optimal compromise between shadowing angle and layer thickness. They furthermore provide a basis for assaying the extent of shadowing artefacts such as decoration and lead to a more accurate interpretation of rotary shadowed structures.

Theoretical calculation of layer geometry in rotary shadowed models of membrane-associated particles.

SUMMARY

A fracturing mechanism in the form of a double-wedged cleaver, used previously on other freeze-fracture machines, has been incorporated in the Balzers BAF 400 D apparatus. The design of the stage, with the cleaver, is explained and its performance and advantages are outlined. By minimizing mechanical damage to the exposed surface of the frozen specimen and reducing contamination by effective shrouding, an improvement in the quality of the replicas is achieved. The suitability of this mechanism for the fracturing of tough tissues such as skin is also outlined.

L. Landmann

Papers

Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques

Theoretical calculation of layer geometry in rotary shadowed models of membrane-associated particles.

Design and application of a freeze-fracture stage with a cleaver device for Balzers freeze-etching apparatus