HAL Id: jpa-00223769
https://hal.archives-ouvertes.fr/jpa-00223769
Submitted on 1 Jan 1984
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
MICROANALYSIS ON BIOLOGICAL MATERIAL - THE ROLE OF PREPARATION METHODS AT LOW
TEMPERATURES AND A POSSIBLE LINK WITH (IMMUNO)CYTOCHEMISTRY
P. Frederik, P. Bomans, W. Busing, R. Odselius
To cite this version:
P. Frederik, P. Bomans, W. Busing, R. Odselius. MICROANALYSIS ON BIOLOGICAL MATERIAL
- THE ROLE OF PREPARATION METHODS AT LOW TEMPERATURES AND A POSSIBLE
LINK WITH (IMMUNO)CYTOCHEMISTRY. Journal de Physique Colloques, 1984, 45 (C2), pp.C2-
451-C2-455. �10.1051/jphyscol:19842102�. �jpa-00223769�
JOURNAL DE PHYSIQUE
Colloque C2, supplément au n°2, Tome 45, février 1984 page C2-451
MICROANALYSIS ON BIOLOGICAL MATERIAL - THE ROLE OF PREPARATION METHODS AT LOW TEMPERATURES AND A POSSIBLE LINK WITH (iMMUNO)CYTOCHEMISTRY
P.M. F r e d e r i k , P.H.H. Bomans, W.M. Busing and R. O d s e l i u s *
Department of Pathology, University of Limburg, P.O. Box 616, Maastricht, The Netherlands
*Department of Pathology, University of Lund, Malmo General Hospital, Malmo, Sweden
Résumé - Les coupes à congélation ultrafines sont idéales pour l'analyse et la localisation des éléments dans leur matrice physiologique . Le système de cryotransfert entre microtome et microscope électronique offre la possibilité d'examiner des coupes à l'état hydraté et congelé et d'observer ensuite le processus de sublimation jusqu'au séchage complet. En sublimant les coupes avant l'observation au microscope, on prend un risque de réhydratation avec les artefacts caractéristiques que celle-ci implique. Les artefacts de réhy- dratation peuvent être prévenus en exposant les coupes lyophilisées à des vapeurs fixatrices (formaidéhyde, le tétroxyde d'osmium par exemple). Ce procédé de fixation sèche par la vapeur a l'avantage de faciliter la conser- vation des échantillons en vue d'une microanalyse ultérieure et, de plus, il offre une possibilité d'application des techniques immunocytochimiques. Nous présenterons en illustration un exemple d'étude immunocytochimique et micro- analytique des pancréas de rat.
Abstract- Frozen thin sections are considered the objects of choice for the study and localization of elements in their physiological matrix. A cryo- transfer system between microtome and microscope offers the possibility of studying fully hydrated sections in the frozen state and subsequently ob- serve the sublimation process up to complete dryness. By freeze-drying sections before microscopic observation one takes the risk of rehydration with its accompanying typical artefacts. Rehydration artefacts are preven- ted when freeze-dried sections are exposed to vapour fixatives (e.g. formal- dehyde, osmium tetroxide). A dry vapour fixation enables the safe conserva- tion of samples for microanalysis and, in addition, offers the possibility for conducting immunocytochemical reactions on serial sections. This will be illustrated by an immunocytochemical and microanalyttcal study on the pan- creas of the rat.
The localisation of elements in their biological matrix is a relatively new and rapidly expanding field in cell biology (1). A general acceptation of the micro- analytical approach to biological problems is not to be expected until discussions about the relative merits of preparative methods have come to a conclusion. First of all this discussion concerns the fixation, chemical as well as physical by ra- pid freezing. Chemical fixation requires fast and efficient diffusion of chemical agents across membrane barriers, modification and extraction of cell and tissue components is inherent to this treatment. Microanalysis of at least diffusible ions is thus incompatible with chemical fixation. A conventional preparation gives only relevant microanalytical results in a few well defined cases in which ele- ments are securely anchored in a biological structure.
It is an accepted opinion that physical fixation by rapid freezing results in an instateneous preservation of the local biochemical, physiological and ultrastruc- tural state. The extend to which this objective can be attained is limited by heat diffusion. Only small samples or the surface layers of larger samples can be optimally fixed (2). Starting with frozen material a bewildering range of cryopre-
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842102
C2-452 JOURNAL
DE
PHYSIQUEparative methods is offered (fig.
1).A selection of the proper method for a spe- cified biological problem is difficult because objective criteria derived from comparative microanalytical studies are lacking.
Freezing of fresh tissue
I I I
cryoultramicrotomy fracturing
freeze 0 0
freeze freeze lo 0 0
substitute dry dry
I I 1 drying
(fixation) vapour vapour
I fixation I fixation
embedding embedding
I I bulk
i' sectioning analysis
v
Electron microscope and microanalysis Fig. 1
Not surprisingly from an educational point of view people tend to jncline to em- bedding methods. For the evaluation of the proper method one is confined at pre- sent to morphological criteria.
1.
The final section should reflect the ultrastructure of the frozen tissue.
2.
The ultrastructure of tissue gives a record of the thermal history of the water (crystal 1 ization, recrystal
1ization, drying, rehydration, etc
.).Freeze substitution.
(3),freeze drying followed by embedding as wellas cryoultrami- crotomy
( 4 ) ,all result in a good structural preservation, inclusive the freezing damage, according to the first criterion.
Although one can have serious doubts about the preservation at the molecular level with any of these cryomethods it is still clear that each of these approaches can result in a specimen with enough ultrastructural detail for the spatial resolution of the currently available microanalytical methods (EDX, WDX, LAMiYA, etc.).
Furthermore it has to be noted that plastic embedded material from a freeze drying or freeze substitution procedure can be thin sectioned without the interference of any liquid by using a cryoultramicrotome (5). The only drawback of an embedding procedure in this context is the possible extraction or translocation of tissue components in substitution fluids respectively embedding agents.
By far the most direct way to introduce a frozen specimen into the microscope is
by cryoultramicrotomy for high spatial resolution or fracturing followed by bulk
analysis for a lower spatial resolution. For the bulk analysis of fractured bio-
logical material in a cryo-SEM it has been reported recently
(P.Echlin personal
communication) that considerable progress has been made in the quantitation of
microanalytical results. A renewed interest in frozen-thin sections.stems from the
work of Barnard
(6).Recent developments in the instrumentation, microtomes as well
as cryotransfer systems, enable an old dream to come true: the direct observation
of fully hydrated cryosections in an electronmicrosco e, with the potential of
observing life at the molecular level. Not surprisingPy the realisation of this
objective is not as straight foreward as was dreamed. First of all the effects of freezing have to be taken into account as with any other cryotechnique. Subsequent- ly a range of phenomena may induce artefacts and thus interfere with our inter- pretation of the final image. These phenomena will be discussed in more detail.
Melting during sectioning
For the cutting of thick cryosections melting at tile knife edge has been postulated and used to explain the yield of smooth sections (7, seealso
8).Experimental evidence obtained from thin cryosections (50 - 200 nm) cut at low temperatures
(-80
to -140°C) indicates that through section melting
(9)or appreciable sur- face melting are not involved in cryoultramicrotony
( 10). A number of sectioning artefacts known in the sectioning of metals as well, are occurring in this range of temperatures and detract from the quality of the images. Extreme low tempera- tures (below -140°C) during sect~oning and favorable freezing conditions have been reported to improve the quality of the sections and their microscopic images (11). The mechanisms of (cryo)sectioning are still not fully understood and it might well be a good lead to consider sectioning as a flow process with some pa- rallels with metal cutting (12). In conclusion it can be stated that if melting during sectioning occurs,it has escaped from detection and it will certainly not
interfere with the spatial resolution of available methods for microanalysis.
Freeze drying
When a cryotransfer system for the introduction of frozen hydrated sections frorn tne microtome into the microscope is not available, freeze drying outside the micros- cope is often employed for conserving the ultrastructure. Freeze drying inside the cold and dry tmosphere of a cryochamber of an ultramlcrotome will proceed at a rate 5 x 10-3 slower than the maximum rate of evaporation in vacuum at the same temperature. For practical work this figure iillplies that within fifteen mi- nutes a thin section stored in a chamber at -80°C will be dry. Furthermore for t~re observation of fully hydrated material a sectioning temperature must be selec- ted below a temperature of -120°C. Having a freeze dried section at hand and sto- ring this in normal room conditions carries the serious risk of rehydration with its typical artefacts (4, indistinguishable from melting artefacts, see also 10).
Table:
Immunocytochemical demonstration of amylase in thin cryosections from exocrine pancreas (rat) using anti a-amylase and protein A-gold. Results from freeze-dried cryosections from fresh-frozen tissue are compared with results from prefixed tissue.
Density of gold label is expressed as number of gold particles per square micron of the organelles + SEM. The number of areas counted is indicated bewteenbrackets The density of labFl found in control experiments (normal rabbit serum instead of immune serum, and/or protein A-gold solution alone) did not exceed
10particles per square micron.
zymogen mitochondria RER + Golgi
Vapour fixation dry-formaldehyde
Aqueous prefixation
0.2
%glutaraldehyde
2.0
%formaldehyde in
phosphate buffer pH
7.3C2-454 JOURNAL DE PHYSIQUE
A fixation in the vapour phase with osmium tetroxide or formaldehyde protects the ultrastructure from the harmful effects of rehydration. Recently we have investi- gated the possibility to use dry-sections in fixed formaldehyde vapourfor immuno- cytochemistry. This treatment gave comparable results, expressed as the density of immunogold labelling for amylase over exocrine pancreas, as cryosections from tissue that had been chemically fixed before freezing (see Table). In addition it was shown that X-ray microanalysis on vapour fixed material was feasible (in press
13)).
With this approach vapour fixation opens exciting new avenues, serial sections can be used to localize molecular species with immunocytochemistry whereas the dry counterparts can be used for (X-ray) microanalysis.
Cryotransfer
Cryotransfer to the cool specimen holder of the electron microscope is the youngest branch on the tree of cryomethods (14, 15). When the transfer system is used for the observation of fully hydrated cryosections the result is often disappointing
(16,see however II), there is hardly any contrast. Only upon freeze-drying the contrast increases and the outline of organelles can be discerned. It might be that only very thin sections of optimally frozen materialdisplayp contrast in the fully hydrated state
(11).We found that size changes during freeze drying inside the microscope are confined to the final drying stages (see fig. 2). Furthermore the influence of the electron bombardement on the frozen hydrated material is still puzzling. Ionisation and heat effects by the electron beam both exert their effects on the cool specimen and to untangle th? contribution of each of these will be a task for future research.
meanwhilea lot can be learned from studying phasechanges of ice (e.g. recrystallization, crystal growth, freeze drying).
exp.
time
Figure 2: Cryotransfer and freeze drying of cryosections.
2a: transmission of the cryosection is given as exposure time at fixed beam con- ditions. Exposure time indicated in seconds
( - )for consecutive pictures
(X-axis). The distance between two measuring points
(0)is indicated and shrinkage is only observed when most of the water is already sublimed.
2b: Freeze drying of an ice crystal overlying a cryosection. The length to width
ratio is presented (hexagonal)
( % )on consecutive pictures.
Sublimation of hexagonal crystals was found to be related to the orientation of the crystal (fig. 2). Heat contact between frozen material and the cold support is essential in freeze-drying. Even when part of a section is dry according to visual criteria we found a beam induced effect, strongly resembling melting, upon focussing at a small area. An isolated area can dry at lower rate when not exposed to the electron beam. If the electron beam is focussed on an isolated area explo-
sive effects are to be expected.
The field of cryomethods is rapidly evolving. Concerted efforts towards an impro- ved quality of cryosections in conjunction with observation at low temperatures are soon expected to become
fruitful.In relation to this development image for- mation of unstained biological material will be further explored (e.g. by using EELS). There will be a spin-off towards higher spatial resolution in microanalysis (EDX) and detection limits are to be pushed foreward (EDX, EELS, mass spectrometer based methods). At this moment there is already offered an approach to combine results from X-ray microanalysis (localising atomic species) with the results from immunocytochemical incubations (localising molecular species) on serial sections from the same material. Vapour fixation can be the link between two active fields
in
cryosectioning,thoseconcernedwith immunocytochemistry and those merely interested in microanalysis.
References
ROOMANS G. SEM 1981/II, 345.
VAN VENROOIJ GEPM. et al. (AMHJ Aertsen, WMA Hax, PHJT Ververgaert
&HA van der Vorst) Cryo-biology 12 (1975) 46.
DEMPSEY GP.
&BULLIVANT S. J. MErosc. 106 (1976) 261.
FREDERIK PM.
&BUSING WM. J. Microsc. I T - (1981) 191.
FREDERIK PM. et al. (HJ van der M o l e n , T I<lepper
&H. Galjaard) J. Cell Sci. 26 (1971) 339.
BERNHARD W. &-NANCY MT. J. Microscopie
3(1964) 579.
THORNBURG W.
&MENGERS PE. J. HistochemTCytochem.
-5 (1956)
47.SAUBERMANN AJ. SEM 19791111, 261.
FREDERIK PM.
&BUSING WM. J. Microsc. 122 (1981) 217.
FREDERIK PM., BUSING WM.
&PERSSON A. JTFlicrosc. 125 (1981)
191.MCDOWELL AW. et al. (J.-J. Chang. R. Freeman. J. L F u l t , CA. Walter
&J.
