Characterization of apoptosis mainly derives from morphological and ultrastructural observations (1). Intracellular and plasma membrane structural modifications have been widely recognized as crucial factors involved in cell injury and death. Changes in nuclear morphology and in organelle structure as well as specific phenomena at the cell surface level, namely surface smoothing and surface blebbing, are often considered as markers associated with cell pathology (2). In addition, it must be recalled that these structural findings are intimately related to the cascade of biochemical and physiological events leading to changes in cellular homeostasis, to the loss of cell volume regulation, to some modifications of macromolecule synthesis and, finally, to the loss of cell viability.

A single intracellular event can be extensively analyzed by using, in parallel, biochemical, molecular or ultrastructural approaches. The complex sequence of structural modifications ultimately leading to cell death can be recognized by light and electron microscopy techniques. These analyses are mainly qualitative and can indicate: i) the different features of the apoptotic process in terms of appropriate markers, e.g. histotype-associated, and ii) the staging of the process, e.g. early or late phases (also called secondary necrosis). However, quantitative analyses, i.e. cytometric and morphometric, can also be performed by using light microscopy, fluorescence microscopy, confocal microscopy, scanning electron microscopy and, in some conditions, transmission electron microscopy (3-5).

Subcellular effects induced by physical, chemical and biological agents are often similar and provide some clue to the mechanisms underlying the structural modifications leading to cell death. These changes can also be experimentally induced in vitro by using specific "injury-tools" and can be evaluated by immunochemical and ultrastructural analyses. These models, although semplified with respect to the in vivo situations, can provide useful information for the understanding of the apoptotic process. However, several questions remain unanswered regarding i) the possible relationships between cell structure and function; ii) the possibility of detecting target structures or organelles related to precise pro-apoptotic stimuli; iii) structural differences and homologies between various injury models in various cell histotypes; iv) the ability of a cell to recover (from a precise morphological marker of injury come back to a normal morphology) and v) the point of "no return" and the consequent identification of the boundary line between cell injury and cell death.

In this brief review, the main morphological approaches will be described and some aspects of apoptotic cell death will be shown. The figures are proposed as a sequence of pictures to consult, to look at for comparison or as simple reference material: a sort of small atlas.

The techniques herein described are listed as follows:

 

A) Light microscopy

B) In situ immunocytochemistry

C) Scanning electron microscopy

D) Transmission electron microscopy

A) Light microscopy
 
A1. Introduction

In the last years, numerous different techniques have been considered for studying apoptosis: i) light microscopy staining procedures, e.g. MayGrunwald-Giemsa, and ii) fluorescence microscopy techniques, e.g. DNA staining fluorochromes. Some of them are of great use in the laboratory practice while others are specifically employed in certain experimental conditions. We report here the most suitable for studying apoptosis. In fact, some important old and new procedures, such as those using non-specific stainings, or stainings with compounds used with other technical approaches (e.g. annexin V staining usually employed by flow cytometry) are still controversial, at least from a morphological point of view (in static cytometry studies), and will be omitted.

In the following protocols different cell culture conditions will be considered: a) cells growing in suspension; b) detached cells derived from a monolayer; c) cells grown in monolayer and adhering on a coverslip.

 

a) Cells Growing In Suspension

Aa.2.1 Materials

1. Polylysine-coated (0.1 mg/ml dH2O) coverslips (12 mm)

2. Slides (25.4 x 76.2 mm)

3. PBS

4. 3.7% paraformaldehyde in PBS

5. 0.5% Triton X-100 in PBS

6. Hoechst 33258

 

b) Cells growing in monolayer: detached cells (i.e., following apoptotic treatments).

Ab.2.1. Materials

1. Polylysine-coated (0.1 mg/ml dH2O) coverslips (12 mm)

2. Slides (25.4 x 76.2 mm)

3. PBS

4. 3.7% paraformaldehyde in PBS

5. 0.1% Triton X-100 in PBS

6. Hoechst 33258

Ab.2.2. Methodology

c) Cells growing on coverslips

(or remaining adhering to the substrate).

Ac.2.1. Materials

1. Slides (25.4 x 76.2 mm)

2. PBS

3. 3.7% paraformaldehyde in PBS

4. 0.5% Triton X-100 in PBS

5. Hoechst 33258
 

1. Sonicate the coverslips in acetone for 15 min at room temperature.

Samples can also be stained with propidium iodide (fixing cells with ethanol 70% at +4°C for 30 min) or acridine orange 0.1% (fixing the cells with ethanol) in citrate buffer (pH 3).

 
B) In situ immunocytochemistry

 
B1.Introduction

The importance of cell loss through apoptosis or necrosis has been increasingly recognized, and the focus is now on the balance between cell proliferation and cell elimination, and on the search for a simple and efficient way for specifically identify and quantify these two phenomena. Therefore, an increasing number of investigators favor in situ labeling of apoptotic cells versus proliferating cells, which allows high sensitivity and precise identification of the cell population involved in the two phenomena. In fact, specificity is secured by simultaneous evaluation of apoptotic cell morphology. The technique relies on the use of endogenous enzymes that allow incorporation of labeled nucleotides into the 3'-hydroxyl (3'OH) recessed termini of DNA breaks. The added value in the use of in situ immunocychemical techniques resides in the possibility to evaluate both morphological and staining features in the same sample. In this chapter the use of Tunel method implemented with the APAAP and PAP immunocytochemistry methods will be described (6-9).

 In general, both materials and methods are roughly the same for the various tissue or cell samples used. In fact, the pretreatment of biological samples to obtain a suitable access to DNA breaks for enzymatic reactions is highly impaired by fixation procedures, such as cold absolute acetone treatment. Actually, the incubation of samples with detergents, such as Triton X-100 did not show to highly improve Tunel sensitivity. The immunocyto-histochemistry, using either the PAP or APAAP methods, to show Tunel positive cells needs a convertion procedures that is very poorly described.

1. slides or 8chamber-slides

2. Tris buffer solution (TBS) pH 7.6

3. poly-L-lysyn (Sigma)

4. Acetone (100%)

5. Ethanol (80% in DW)

6. Methanol (70% in DW)

7. Tunel mixture (Boehringer Mannheim)

8. Mouse-anti-FITC Ab (DAKO)

9. RAM (DAKO)

10. APAAP (DAKO)

11. DAB+developing solution (DAKO)

12. Fast red+developing solution (DAKO)

13. NBT+developing solution (DAKO)

14. Aquamount (BDH)

15. OCT
 

B2.2.Methodology

*Cytospin preparation:

To obtain the best cell morphology the use of chamber slides is recommended. Of course in this case the amount of Tunel mixture as well as of the other reagents highly exceeded that used for cytospin preparation (roughly 5 folds more for chamber slides). Therefore, we use this technique precisely when we have some problem in identifying some morphological parameters. The method is roughly the same of the cytospin preparation, with the only exception of the number of cells used, the technique to attach non-adherent cells on L-polylysin-covered chamber-slide and the permeabilization procedure (unfortunately acetone cannot be used with plastic materials):

The technique is roughly the same described in (a) with the only exception of the preliminary procedures for collecting, cutting and treating tissue specimens. In particular:

 
C1. Introduction
 

This technical approach allows the analysis of surface of materials. In the case of cells and tissues different technical methods can be used (which can be found in specific SEM textbooks). The use of SEM in the analysis of apoptosis is mainly referred to the study of cell surface alterations such as smoothing, loss of microvillous structures, blebbing, shrinking etc. However, these are important signs of cell injury. They can not be considered as specific markers of apoptosis.

Cell lines which grow in monolayer (or dispersed cells seeded on polylysine-coated coverslips) are fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer. This fixation procedure can induce some changes on the cell surface. To avoid artifacts fixative has to be gently dropped on the samples. After post-fixation in 1% osmium tetroxide, cells are dehydrated through graded ethanols and critical point dried in CO2. The critical point-drying technique provides a much better means of avoiding the damaging effects of surface tension forces than does air drying. The specimen is thus transferred into a closed pressure vessel where it is first infiltrated and surrounded by liquid CO2. The temperature and pressure within the vessel are then raised above the critical values at which point, the critical point, the liquid and gas phases have the same density. After passing the critical point, the pressure is slowly reduced so that the recondensation of the fluid through adiabatic cooling is avoided.

To allow scanning electron microscopy observation the specimens are then normally coated with a conducting layer to inhibit charging. Sputter coating by gold has the advantage of requiring only a low vacuum and has been proved to be satisfactory for observational work.
 

 

a) Cells growing in suspension

Ca.2.1. Materials
 

1. Polylysine-coated (0.1 mg/ml dH20) coverslips (12 mm)

2. 0.1M cacodylate buffer (pH 7.4)

3. 2.5% glutaraldehyde in 0.1M cacodylate buffer

4. 0.2M cacodylate buffer (pH 7.4)

5. 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer

6. Graded ethanols

7. Absolute ethanols

8. Stubs

9. Silver print
 
 

Fixation

b) Cell lines which grow in monolayer

(on the coverslips)

C.b.2.1. Materials

1. coverslips

2. 0.1M cacodylate buffer (pH 7.4)

3. 2.5% glutaraldehyde in 0.1M cacodylate buffer

4. 0.2M cacodylate buffer (pH 7.4)

5. 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer

6. Graded ethanols

7. Stubs

8. Silver print

C.b.2.2. Methodology

 

D) Transmission Electron Microscopy (TEM)

 
D1. Introduction

TEM analysis has been considered a milestone of the research in the field of apoptosis. A pletora of findings contributing to the knowledge of the process, e.g. the role of subcellular structures and organelles, have been widely published. In this section we describe only some basic methods to approach the problem. Several figures are provided to help in the evaluation of cell alterations.

Ultrathin sections. Cell monolayers (and cells recovered from the surnatant and gently centrifuged) can be fixed in glutaraldehyde and post fixed in OsO4. Different protocols can be used depending on the type of sample considered. In particular, cell pellets or monolayer are easily fixed (only vapours are sometime sufficient) while tissues need longer times of exposure to fixative. However, glutaraldehyde is capable of penetrating very poorly inside tissues (no more then 2mm).

Specific beems can be used to collect both these samples. In particular, cell monolayers are embedded directly in the culture flasks after at last three different embedding steps. A first step is performed by using low resin concentrations. Other two or three steps are then performed by using increasing concentrations of embedding resin. Finally, absolute resin is used in two embedding steps of one-two hours.

 

D2.1. Materials

1. 0.1M cacodylate buffer (pH 7.4)

2. 2.5% glutaraldehyde in 0.1M cacodylate buffer

3. 0.2M cacodylate buffer (pH 7.4)

4. 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer

5. Agar 2% in dH20

6. Graded ethanols

7. Absolute ethanol

8. Propylene oxide

9. Agar 100 resin

D2.2. Methodology

Fixation:

*Dispersed cells

 Dehydrate the samples through graded ethanols as follows:

 
 

	Cultures	Monolayers	Tissue
30% ethanol	2 x 10 min	2 x 5 min	2 x 10 min
50% ethanol	2 x 10 min	2 x 5 min	2 x 10 min
70% ethanol	2 x 10 min	2 x 5 min	2 x 10 min

 

 
 

70% ethanol

2 x 10 min

2 x 5 min

2 x 10 min

 

  At this point the dehydration procedure may be stopped. Samples can be kept in 70% ethanol at +4°C.

 

85% ethanol	1 x 10 min	1 x 10 min	1 x 10 min
95% ethanol	1 x 30 min	1 x 10 min	1 x 30 min
Absolute ethanol	3 x 20 min	3 x 10 min	2 x 30 min
Propylene oxide  (or toluene)	1 x 10 min		1 x 20-30 min

 

Inclusion

Include the samples as follows:
 

	dispersed cells	tissues
Propylene oxide/Resin 2:1	1 x 1h	1 x 1h
Propylene oxide/Resin 1:1	1 x 1h	1 x 1h
Propylene oxide/Resin 1:2	1 x 2h	1 x 2h

At this point the inclusion procedure is usually stopped for an overnight. Samples can be kept in Propylene oxide/Resin 1:2 at +4°C.

 

Absolute resin	2 x 1h	1 x 2h
Absolute resin into the beems	1 x 1h	1 x 2h

 
 
 
 

	Monolayer
Absolute ethanol/Resin 2:1	1 x 1h
Absolute ethanol/Resin 1:1	1 x 1h
Absolute ethanol/Resin 1:2	1 x 1h

 

At this point the inclusion procedure may be stopped for an overnight. Samples can be kept in absolute ethanol/Resin 1:2 at +4°C.

 

Absolute resin	2 x 1h
Absolute resin with the beems	1 x 1h

 

Monolayers: fill beems with absolute resin and put them upside down on the cell layer.
 

Polymerization

Monolayers: gently detach the beems. They will detach with small fragments of polymerized resin. A dark surface will indicate the presence of cells.

• Polymerize at 60°C for 48h.
 
3. Commentary

 
3.1. Background information

In order to detect and study apoptotic cells as well as for obtaining extensive data on the morpho-functional state of a cell population, various technical approaches should be utilized. The use in parallel of different methods is crucial for: i) quantify the phenomenon; ii) analyze qualitative differences between experimental conditions; iii) determine the different stages iv) validate observations obtained with other approaches etc. In situ detection of apoptotic cells may be a checkpoint approach for a better and a more complete sight of the problem of apoptosis both in a certain cell population and at tissue level. In general, the light microscopy approach can provide both qualitative and quantitative data, TEM analysis is essentially qualitative, SEM studies can provide information of the cell surface, cell-cell and cell-substrates interactions (but it is very difficult to evaluate apoptotic features by SEM).

The cytochemical and immunocytochemical techniques allow to investigate morpho-functional correlates of the various apoptotic pathways. Double staining procedures can be often used to correlate both apoptotic cells to their phenotype, cytokine production, virus and microbial infections, cytoskeletal organization, transcription factor expression, calcium ions, redox alterations and so on. Moreover, in situ immunocytochemical techniques are useful to investigate the distribution of the various cell proteins (e.g. adhesion molecules) both at the single cell level and at the cell-to-cell contact sites. In other words, the in situ evaluation of apoptosis through immucyto- and histochemistry provides unique information for studies aimed to identify apoptotic mechanisms. Electron microscopy, TEM in particular, should be avoided for routinary studies.

At least three different apoptotic morphologies can be detected depending on: i) the stimulus; ii) the histotype; iii) the stage of apoptosis. For instance: i) four main pro-apoptotic stimuli can exist: a) those receptor-mediated (e.g. by cytokines, protein toxins etc), b) chemical agents, capable of entering the cells (e.g. toxicants, anticancer drugs, etc.), c) physical agents which do not interact "directly" with cells (e.g. by ionizing radiations); d) the lack of molecules of relevance in the cell life, (e.g. withdrawal of growth factors). ii) Different histotype, e.g. epithelial, neuronal etc, undergo apoptosis following specific pathways with precise features which also depend on their relationships with their environmental growth conditions, e.g. the type of interaction with other cells (e.g. desmosome mediated, gap junctions, synaptic connections etc) or with basement membranes. iii) being an asyncronous process, apoptotic process can be detected as a phenomenon with marked structural discrepancies which are associated with earlier or latest phases. All these conditions are intertwined together and share common features. These can provide useful information but also result in artifacts, errors and misleading results.

3.4. Time considerations

As a general rule, light microscopy (2-6 hours) and SEM (24 hours) analyses can be considered as very fast preparation procedures. Both cytospin and chamber-slides preparations need one day, with the only exception of double stainings which need two days. TEM methods are very slow procedures (5-6 days)

The authors are indebted with G. Rainaldi and A.Tinari for their precious help in the choice of some pictures and their interpretation.

 

 

Fig. 1

1A. Hoechst staining. Epithelial cells. Morphological appearance of normal nuclei. Note different chromatin aggregations.
1B. Brightfield. Normal cells (epithelial cells) undergoing retraction
1C. Hoechst staining. Higher magnification of normally stained nuclei, a mitotic nucleus and an apoptotic nucleus.
1D-1F. TUNEL staining. Different features of apoptotic nuclei. Note the "enucleation" in F.

Fig. 2

2A-2D. Hoechst staining. Apoptotic morphology. A) normally shaped nuclei, B) chromatin clumping in apoptotic nuclei; C) Different features of chromatin clumps; D) Chromatin superaggregation. Note the absence of clumping.

Fig. 3

3A-3D. Hoechst staining. Apoptotic stages increasing times of exposure to a proapoptotic stimulus. A) well evident apoptotic figures; B) several different forms of chromatin aggregation; C) late stage of apoptosis; D) Very late apoptosis. Note the presence of several residual bodies.

4a-d. Time course. Lymphocytes. Different aspects of apoptosis. Note: aggregation in (a), kidney shaped nuclei in (b), small nuclear blebs in (c).
 

Fig. 5-14

5-10. Scanning electron microscopy. Epithelial cells. Different stages. Flat cells (Fig. 5) undergo different forms of rounding, surface blebbing and cell retraction (6-9) preceding the typical apoptotic figure shown in (10).

11-14. Scanning Electron microscopy. Different aspects of necrotic cell death. Compare necrotic surface alterations including surface blebbing in (14) with apoptotic ultrastructure (8-10).

Fig. 15-22

15. Transmission Electron Microscopy. Surface blebbing and apoptotic nuclear morphology.

16. Light microscopy. Note the different aspects of apoptotic cells including chromatin marginalization, e.g. compare with Fig. 15.

17. Transmission Electron Microscopy. "In vivo" blebbing. Section from a human tissue. Note a surface bleb between two epithelial cells (a). In (b) is shown a surface blebbing in an apoptotic cell.

18-22. Transmission Electron Microscopy. Different aspects and phases of chromatin aggregation in lymphocytes.
 
 

TEM. Different aspects of apoptosis. Late stages (23,24) show altered cytoplasm with swollen mitochondria (23). Nucleoskeletal structures are also detectable in cells in latest stages (secondary necrosis, 24).

Chromatin aggregation and condensation shows sometimes a paracristalline structure (25). The chromatin "superaggregation" of an apoptotic cells is shown in figure 26. To note the mitochondrial lesions and the lack of nuclear envelop which can be detected at this stage.

Fig. 27 shows a cell with several ultrastructural features typical of apoptotic cell death. Note: "condensed" mitochondria, chromatin marginalization and two nuclear clumps with nuclear pore overlapping.