ultrastructural morphometric analysis by transmission...

Ultrastructural morphometric analysis by transmission electron microscopy associated with stereology methods

N. Nathaly Rigoglio*1, M. V. Mendes Silva1, V. Pavanelo Junior1, S. A. Ferreira Lima1, J. Luiz Nogueira1, R. Avancini Fernandes1 and M. A. Miglino1

1Surgery Department , Faculty of Veterinary Medicine and Animal Science (FMVZ-USP), São Paulo University (USP), Cidade Universitária, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo SP 05508270, Brazil. *Corresponding author: [email protected]

The stereology is a method used to quantify uniform, systematic and random samples. Usually use the method a two-dimensional (2D), with limitations. For extrapolated from planar measurements performed on 2D slices images, to three-dimensional (3D), the stereology use a test-system usually composed by test-points, test-lines over a known frame (or test-area). All indices are estimated by the application of specific formulas. However, the accuracy of calculations is based on the statistics principles (sample size, randomization, and isotropy). A new tool that can complement a stereological analysis is transmission electron microscopy (TEM) to obtain ultrastructural information. This technique can be used, for example to quantify morphological changes of mitochondrial component, such as relate it to the ATP synthesis. Furthermore, estimate the mitochondrial volume density (Vvm), numerical density (Nvm), absolute volume (Vm) and numerical density per area (Nam) for each cellular type, to verify if the cells have some differences. This study aimed to describe the importance of the stereology as a method to quantify organelles and other structures.

Keywords: stereology; ultrastructural; mitochondria; transmission electron microscopy (TEM)

1. Introduction

To understand physiological adaptations that occur on the structure of the organs is essential to be able to characterize and quantify the underlying structural changes in the tissues. A common approach is to examine some microscopic sections taken from the organ of interest and to qualitatively describe the structures observed. But the 2D methods have some limitations when are used to quantify biological structures. These can be avoided by stereology, where the stereological methods offer practical and scientifically valid approaches for obtaining accurate, precise quantitative estimates of subtle structural changes in tissues from histological sections [1]. The three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology, as well as, to obtain quantitative insights into the subcellular localization, geometric arrangement and distribution of cellular components [2]. Stereology is the first choice whenever 3D structural quantities need to be extrapoled from planar measurements performed on 2D slice images [3]. To obtain ultrastrustural information some studies have used the Transmission Electron Microscopy (TEM) [4]. As an alternative to TEM, scanning electron microscopy methods have been developed [2]. The first application of confocal microscopy for stereological measurements was presented by Howard et al. [5] in their concept of unbiased sampling brick. The aim of the sampling design for stereology is to obtain the maximal amount of quantitative structural information at a given total cost or effort [6]. The stereology use a test-system, for example test-points, test-lines over a know frame (test-area). We can estimate densities per volume: volume density (Vv), length density (Lv), surface density (Sv), and numerical density (Nv). And densities per area can also be estimated: area density (Aa) and numerical density per area (Na or Qa) [7,8]. Volume density, Vv, is the volume proportion of one phase within a reference volume. Is also known as volume fraction; Surface density, Sv, is the area of an interface within a unit reference volume; Length density, Lv, is the length of a linear feature within a unit reference volume; for example, the length of fibers per cm3; Numerical density, Nv, is the number of discrete objects in a unit reference volume.

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

© 2012 FORMATEX 309

Table 1 The dimension, units and notation used for some parameters

PARAMETER DIMENSION UNITS TEST SYSTEM

Volume density (Vv) L3/L3= L0 None Points Surface density (Sv) L2/L3= L-1 µm-1 Lines Length density (Lv) L1/L3= L-2 µm-2 Planes

Number density (Nv) L0/L3= L-3 µm-3 Volumes Volume (V) L3 µm3 Surface (S) L2 µm2 Length (L) L1 µm

Number (N) L0 None Connectivity (X) L0 None

Profile density(QA) L/L2= L-2 µm-2 Intersection density (IL) L0/L1= L-1 µm-1

Specific formulas are applied to estimate all indices. However, the accuracy of calculations is based on the statistics principles, such as sample size, randomization and isotropy. The validity and the efficiency (number of samples and counts of interaction with geometric probes) are determined by design-based random sampling. The random samples can be applied at all levels of sampling process, e. g. cut an organ into uniform random slices, select tissues blocks at a set distance, and place a regular pattern of test probes on those sections [3]. Intrinsic distortion of structures can occur due the skrinkage caused by fixation, dehydration and paraffin infiltration, and the compression cause by the knife action in microtomy direction [9], and we can considered this [10].

2. Basic principles

The tissues, structurally, occupy 3D space, associated with length, space and height. But when these structures are sectioned with a microtome, we have a series of 2D planes and the reconstruction of 3D structural information from histological sections is not simple. The position and orientation of the sectioning plane will influence size, shape, and frequency of these 2D profiles. To obtain statistically valid histological sections from an organ to reduce the amount of tissue for analysis without reducing the precision of the estimate are used the statistical sampling principles to guarantee that no bias is introduced in choosing sections or microscopic fields for analysis. According to [11], appropriate geometric probes are superimposed on sections and the number of interactions of the probes with the structural features being estimated is determined. To ensure that probe-structure interactions are random, the position of the relevant probe set is always randomized relative to the structure, that is, the probe sets are randomly positioned onto the sections. There is no direct mathematical relationship between the number of structures profiles counted in a single thin histological section and the number of structures that exist in 3-D tissue space. So, the problem of how to accurately estimate cell number in sections was solved with the development of the disector, a probe based on pairs of thin sections [11]. Conventional transmission electron microscopy (TEM) played an early and essential role in formulating and shaping the concepts of mitochondrial bioenergetics [12]. In many types of cell the mitochondria have different geometric shapes, the predominant being elliptical, spherical and cylindrical. In hepatocytes the mitochondria have cylinder or ellipsoid shape [13], and in lymphocytes it has been treated as spheres [14]. The problem of particle shape heterogeneity is of especial concern to those interested in counting and sizing mitochondria.

3. Material and methods

3.1 Transmission Electron Microscopy

The tissue was cut into a series of slices. A single slice was then divided in blocks, approximately equal in size. After these blocks were rinsed in buffer, and post-fixed in 1% osmium tetroxide in cacodylate buffer for 1 h. Blocks were rinsed, dehydrated in acetone, and embedded in Spurr Resine® (Low Viscosity Embedding Kit, Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were prepared from randomly area. These sections were mounted on 200-mesh grid (G200-Cu, Electron Microscopy Sciences, Hatfield, PA), stained with uranyl acetate and lead citrate and examined in a electron microscope Morgagni 268 microscope (FEI Company, Oregon, USA) through the Mega View – Soft Imaging System.



Microphotographs, generally, were taken at a low (x3600), intermediate (x11000) and high (x22000) magnification. The micrographs with low magnification are taking to show the whole cell and the other two magnifications with focus on mitochondria.

3.2 morphometric analysis

Random test points were assigned on the low power micrographs (x3600), and estimates of volume density (VV) of mitochondria in the cells:

Vvm = Pm\Pt

where Pm is the number of points on mitochondria, and Pt is the number of points in the frame [15]. The absolute volume (Vm) for a cellular component can be calculated for the multiplying volume density by organ volume:

Vm = Vvm x V(organ) The number of mitochondria per unit area (Nam) was obtained using random test-points on the intermediate power micrographs (x11000):

Nam = number of points hitting profile Weibel and Gomez [16] developed a formula for deriving the numerical density of particles from the number of objects per unit area (Nam) and their volume density (Vvm):

Nvm = K x Nam 3\2

β Vvm 1\2

The factor K (>1) is a dimensionless coefficient which depends on the mitochondrial size distribution. The shape of the object examined depends on factor β which is therefore variable and should be taken into account when calculating numerical density. Two values of β have been employed, the one appropriate for spheres (β=1.382) and the other for ellipsoids of axial ratio 4:1 (β=2.25). The absolute number of mitochondria can be estimate if the organ (tissue) volume (Vorgan) is known:

Nm = Nvm x V The surface density (SV) of the mitochondrial outer membrane can be estimate on the high power micrographs (x22000):

Svm = 2 x I\Lt

where I is the number of intersections of the structure boundary with the lines, and Lt is the total length of the lines [6]. The surface area of the structure can be estimate using the formula:

Sm = 2 · I x V(organ)

The most complex problems involved in obtaining stereological parameters is the variant of shapes and sizes of biological objects which difficult the determination of numerical density of the structures.

Table 2 Units used to specific parameters

PARAMETERS UNITS Mitochondrial Volume Density µm3/µm3

Mitochondrial Numerical Density no./µm3

Mean Mitochondrial Volume µm3

Mitochondria per cell no.



3.3 Models of probes

Fig. 2 – Models of probes used to quantify area and volume (A), number estimation (B) and length and surface (C).

BA

C Fig. 1 – Electronmicrographs model for studies related to stereological estimates. A – For volume density (VV) of mitochondria in the cells (x3600); B – For the number of mitochondria per unit area (Nam) (x11000), and C – For the surface density (SV) of the mitochondrial outer membrane (X22000).



Table 3 The conversion units

Length Conversion of mm to m 1mm = 1 x 10-3m Conversion of µm to m 1µm = 1 x 10-6m

Area Conversion of mm2 to m2 1mm2 = (1 x 10-3m)2 = 10-6m2 Conversion of µm2 to m2 1µm2 = (1 x 10-6m)2 = 10-12m2

Volume Conversion of mm3 to m3

1mm2 = (1 x 10-3m)3 = 10-9m3

Conversion of µm3 to m3

1µm = (1 x 10-6m)3 = 10-18m3

3.4 Statistical analysis

Statistical analysis was performed using the independent samples Student’s t test, and means were considered significantly different if p<0.05.

4. Discussion

When the relationships between function and structure will be analyze, the morphometrical measurements of structural components of organs, tissues, cells or organelles are the main prerequisite for quantitative analysis in a number of studies in biological research [17], such as features of development, repair, natural aging, and normal anatomy which could not be detected otherwise. Nowadays, a large number of design-based stereological methods offer an efficient quantitative approach to intriguing questions [17] and their methods provides the tools for obtaining accurate, precise quantitative structural data from tissue sections [1]. Stereology’s fundamental development has been the unbiased estimation of geometric properties, such as length, area, volume, and population size [18], and have scientific advantages over qualitative studies. These methods are numerical and can be reproduced at any time by others laboratories, but various investigators could arrive at startlingly different estimates of these parameters, even when they were outcomes of very similar experimental situations [19]. Addition, these methods are useful to comparing different groups (ages, species, treatments, etc). Due the strict sampling strategy, the laborious work is almost nil [6]. Since the advent of electron microscopy, a clear picture of the organization of eukaryotic cells has evolved, based on descriptive studies, often combined with histochemical methods [20]. The limitation of conventional TEM operating at 100 kV is that it only provides single images from ultrathin (50-100 nm thick) sections. This does not permit adequate sampling from mitochondria with dimensions that are typically 0.2 to 2 μm or resolution of mitochondrial substructures that range from 2-50 nm. According to [21], the increased magnification is better for the identification of nuclear and cytoplasmic characteristics of the various cell types in an attempt to increase accuracy in classifying cytoplasmic cross sections, however, with the disadvantage that less tissue can be examined per photomicrograph. White and Gohari [22] quantified the morphological alterations occurring in the mitochondrial component of hamster cheek pouch epithelium during differentiation. Whereas Campbell et al. [23] used the technique to relate if the luteal mitochondria synthesize ATP a rate twice that of follicular mitochondria in porcine ovarian. There are many studies that realize morphometric analysis of small and large luteal cells in different mammals [15,21,23,24]. Schwerzmann et al. [25] studied the molecular architecture of the inner membrane of mitochondria from rat liver. These studies show us the wide variety of research where the stereological methods can be applied. Computer microscopy was developed to quantify the 3D morphology of structures seen in light microscopy, with the goal to obtain accurate representations of the anatomical structures. The integration of classical stereology and computer microscopy has resulted in a flexible system that permits the combination of rigorous stereological measurements with elective user-controlled morphometric maps and studies. The mapping provides the framework for the combination of stereological and anatomical data and also obtains a better understanding of how variations in estimates can arise [18]. Confocal microscopy allows optical sectioning of relatively thick pieces of tissue and are enables one to obtain perfectly registered stacks. Digital images of such stacks represent suitable data for quantitative measurements as well as for computer 3D reconstructions that can be made without having to solve the tedious problem of the alignment of images of successive sections [26]. It is possible to generate different virtual test probes with arbitrary predefined (e.g., random) position and orientation within the stack of sections and apply them directly to this image data. It should be noted that the geometrical characteristics of 3D objects can also be measured by other than interactive stereological methods, e.g., automatic digital methods [27]. More recently, the development and refinement of 3-D electron imaging using high-voltage electron tomography has provided significant new insights into mitochondrial bioenergetics as well as the ultrastructure and substructure of normal and apoptotic mitochondria [28,29,30].



To obtain introductions to the basic concepts of quantification, probes and sampling, we can use as references the following authors [3,31,32,33], whereas to obtain the application we can use as references [34,35].

5. Conclusions

Stereological methods provide the means of efficiently producing quantitative data on the internal structure of organs, tissues, and cells. These methods can easily be applied to cytological work at the electron microscope level of resolution. The techniques of stereology allow quantification of structural features within three-dimensional organs that relate in a meaningful way to integrated function

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