Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures.

The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging Property (SIP) charts (Brakenhoff et al., 2005). It is shown that the integrated intensity of a z-stack from a biological sample, imaged under identical conditions as the layer, can be calibrated in terms of fluorescence layer units of the calibration layer. The imaging after such calibration becomes, as a first approximation, independent of the microscope system and imaging conditions. This is demonstrated on axially integrated images of standard fluorescent beads and standard BPAE Fluorocells. Corrections on the microscope imaging conditions include shading effects, imaging with different magnifications and objectives, and using different microscope systems. It is also shown that with the present approach the actual underlying three-dimensional (3D) fluorescence data set itself can be corrected for variations in point spread function (PSF) imaging efficiency over the imaging data cube. Realizing such calibration between imaging conditions or systems requires basically only the 2D fluorescer molecule density of the reference layers and the section distances with which the layer data are collected.

Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts.

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.

The effects of selection of pigs on growth rate vs leanness on histochemical characteristics of different muscles.

The microstructure of two type of muscles was studied in a selection experiment conducted with Dutch Large White pigs (boars and gilts) selected for either low backfat thickness (L-line) or fast growth (F-line). Second- and fourth-generation pigs were used to determine effects of selection on fiber type composition, fiber area, and capillary density in the longissimus lumborum (LL) and biceps femoris (BF) muscles. Immediately after slaughter samples were taken from the LL and BF muscles. The latter was divided into an inside (BFi) and outside (BFo) portion, which refer to the red and white portions of the biceps femoris. Serial sections were stained for ATPase (pH 4.60), succinate dehydrogenase (SDH), and alpha-amylase-periodic acid shiff (PAS) to determine fiber type and capillary density. The LL and BFo muscles had predominantly type IIBw fibers, whereas the BFi muscle had a 2 to 4 times higher amount of type I fibers. In most muscles there were more type I and fewer type IIBw fibers in F- than in L-line pigs (P < .05), except in the LL muscle of second-generation pigs and in the red part of the BF muscle of fourth-generation pigs. In both selection lines lower type I and higher type IIBw percentages were found in muscles from gilts than in those from boars (P < .05). Capillary density and fiber area of L- and F-lines showed minor differences, which could be explained by differences in weight and age of the pigs of both lines. The results suggest that selection for low backfat thickness in pig breeding compared with increased growth rate resulted in fewer oxidative and more glycolytic muscle fibers. The magnitude of the effect depended on muscle type and duration of the selection period.

Muscle fibre type and meat quality.

Muscle morphology and fibre type composition are briefly reviewed in relation to colour stability and tenderness in beef, and water holding capacity, colour and eating quality in pork. A large inter-muscle and inter-animal variation exists in meat quality, which is often related to metabolic and contractile properties as determined by their muscle fibre type distribution. Characteristics of different muscles may be modified in living animals by environmental conditions and genetic selection. Selection experiments based on biochemical and histochemical characteristics determined in biopsies or otherwise, and study of correlated selection responses, may lead to the development and applications of (new) muscle traits in future breeding programmes.

Histochemical characteristics in relation to meat quality properties in the Longissimus Lumborum of fast and lean growing lines of Large White pigs.

A selection experiment was carried out to study genetic and physiological factors influencing meat quality in lines of Large White pigs selected for lean (L) or fast (F) growth. Second and fourth generation pigs were used to determine effects on fibre type composition, fibre diameters and capillary density in the Longissimus Lumborum (LL). Significant differences in histochemical properties were found in the LL between L- and F-pigs, but only in the 4th generation. L-pigs had significantly less type I and more type IIB myofibres compared to F-pigs in both sexes. In both lines lower type I and higher type IIB percentages were determined in gilts compared with boars. Significant differences in meat quality properties between L- and F-pigs were found in the LL, but only in the 4th generation. The a* values were significantly higher in F- versus L-pigs and in boars versus gilts. L* values were not significantly different between lines. However, L* values were significantly lower in 4th versus 2nd generation pigs and in boars versus gilts. In conclusion, genetic selection on lean versus fast growth induces differences in fibre type composition of the LL. These differences become visible in the 4th generation, when a certain selection effect is achieved. Fibre type composition was influenced by the gender of the animal. Meat quality showed significant differences between lines in meat colour (a* value), but only in the 4th generation. Capillary density and fibre area between L- and F-lines showed minor differences, which could be explained by the differences in weight and age of the pigs of both lines.

Effects of early immobilization on the functional capacity of dystrophic (ReJ 129 dy/dy) mouse leg muscles.

Hindleg muscles of dystrophic (ReJ 129 dy/dy) mice were immobilized during the second post-natal week. Two months after remobilization histopathological features and isometric force characteristics of the m. extensor digitorum longus (EDL) and the m. tibialis anterior (TA) were studied. As a result of early transient immobilization significant differences were observed in muscle morphology and isometric force compared with untreated dystrophic muscles. Restriction of dynamic use of the muscles during this second post-natal week largely prevents the muscles of dystrophic mice from becoming affected by the disease process. Even after two months of remobilization pathology appears to be reduced.

Myosin isoforms in hindlimb muscles of normal and dystrophic (ReJ129 dy/dy) mice.

Myosin isoform expression was studied in hindlimb muscles of control (Dy/Dy) and dystrophic (dy/dy) mice of the ReJ129 strain during postnatal development. Three myosin heavy chain isoforms (fast II-B MHC, neonatal MHC, and slow or I MHC) were identified using monoclonal antibodies. Only original fibers, i.e., fibers formed during fetal life, were studied. Necrotic and regenerating fibers were excluded. The disappearance of neonatal MHC was found to be delayed in all muscles of dystrophic mice, except the soleus. The fraction of fibers containing I MHC was similar in control and dystrophic animals at all ages, except during the third postnatal week. The developmental increase in the fraction of fibers expressing II-B MHC was interrupted in dystrophic mice by two marked declines. The first occurred during the second postnatal week at the beginning of the main wave of fiber necrosis, and the second occurred at between 30 and 90 postnatal days.

Vimentin and desmin expression in degenerating and regenerating dystrophic murine muscles.

The distribution of the intermediate filament proteins (IFP) desmin and vimentin was studied in gastrocnemius, plantaris and soleus muscles of the dystrophic mouse strain ReJ 129 during postnatal development. Special attention was paid to the overall morphological changes in the distribution of these cytoskeletal constituents in degenerating and regenerating muscle fibres. In contrast to their normal counterparts, the dystrophic mice (ReJ 129 dy/dy) appeared to develop four types of distinct muscle fibres with immunohistochemically detectable aberrant IFP patterns. The distribution of desmin IFP differed in the dystrophic muscle fibres as compared to the normal fibres in that juxtanuclear aggregates of IFP were frequently seen. In contrast to the recent literature we conclude that these cells are regenerated myofibres exhibiting defective nuclear migration.