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Freeze-drying with a modified Glick-Malmström apparatus
Experimental
228
FREEZE-DRYING
WITH
G. MOBERGER, The
Wallenberg
A MODIFIED APPARATUS
B. LINDSTROM
Laboratory
Cell Research,
October
(1954)
GLICK-MALMSTROM and
L. ANDERSSON
at the Institute for Cell Research, Karolinska Institutet, Stockholm Received
6, 228-237
Medical
Nobel
Institute,
30, 1953
THE
freeze-drying procedure is a valuable method to preserve biological material for histo- and cytochemical investigations. By means of initial rapid freezing of the tissue samples, followed by dehydration in the frozen state and subsequent embedding, thin sections of tissues can be prepared with a minimum of structural and chemical changes (2, 22). Various techniques regarding the different steps in the freeze-drying procedure have been reviewed by several authors (i.a. 2, 6, 7, 10, 12, 16, 20). Highly efficient dehydration has been obtained in equipment with a water condenser at the temperature of liquid nitrogen close to the specimens combined with a high vacuum (9, 14, 21). Based upon theoretical considerations (13) Glick and Malmstriim (8) have devised an efficient freeze-drying apparatus of this type with remarkably short dehydration time. In the present paper the freeze-drying procedure with a modified GlickMalmstram apparatus and a new technique for the embedding in zjaczzo will be communicated. DESCRIPTION
OF
APPARATUS
AND
TECHNIQUE
In the following the three main steps of the freeze-drying procedure, the initial freezing, the dehydration in uacuo, and the embedding technique, will be considered separately. The main features of importance for the choice of apparatus and technique will be briefly discussed. Initial
freezing.
In order to avoid postsampling artefacts such as ion diffusion and enzymatic decomposition (l), as far as possible, it is necessary to freeze the tissue samples instantaneously after their removal from the organism. Ice crystal formation within the specimens is the main cause of structural changes occurring during the freezing procedure. A high rate of ice crystal nucleation, and a low rate of growth of the ice crystal nuclei, give the smallest crystals possible, and thus minimal structural changes. As the rate of forExperimental
Cell Research
6
Freeze-drying
229
Fig.
2.
Fig. 1. Propane container for the initial freezing of the tissue samples. Fig. 2. Tissue sample holders. Fig.
1.
mation of new ice crystal nuclei is increased by a factor of lox8 between -33” C and -43” C (4), the tissue samples should be frozen as rapidly as possible, well below this range of temperature. A freezing medium with a high conductivity and a very low temperature is therefore necessary. A medium such as liquid air (5) has a sufficiently low temperature, but a layer of vaporized air forms an envelope around the specimens, resulting in poor conductivity (17). Isopentane, cooled by liquid nitrogen, has a high conductivity (11) but solidifies at about -150” C. Pure propane has a still higher conductivity (3) and remains fluid at -185” C, and is therefore very useful for the initial freezing. In order to obtain rapid initial freezing of the inner parts of the specimens, these must be small. When too large pieces of tissue are frozen, formation of ice crystals of considerable size will occur which causes structural changes in the inner layers of the tissues (19). The transport of the frozen specimens from the cooling medium to the dehydration chamber must be quick enough to prevent an increase of the temperature to the range where the growth of the ice crystals is accelerated (at about -40” C). Liquid propane was used for the initial freezing of the tissue samples. A metal container with an external loop for the condensation of the propane Experimental
Cell Research
6
230
G. Moberger,
B. Lindstrbm
and L. Andersson
was devised (Fig. 1). It was placed in a Dewar flask filled with liquid nitrogen. When the container had obtained adequately low temperature, propane gas was let through the loop, where it condensed and then dropped into the container. Immediately after the removal from the organism small pieces of tissue (with an extent of less than 2 mm in one dimension) were plunged in the fluid propane. The tissue pieces were allowed to sink free to the bottom of the container, thus continuously being surrounded by fresh volumes of the cooling medium. The use of forceps or of holders for the individual specimens was avoided because of the risk in delaying the freezing process. A small metal basket placed in the bottom of the container served to make it easier to collect the samples for further transport. The tissue sample holders were made of solid iron in which grooves for the individual specimens were drilled (Fig. 2). The grooves were tilled with paraffin, degassed at 1OW’ mm Hg for 30 minutes in a separate simple vacuum unit. Subsequently the tissue holders were placed in the sample tube (see below) which was cooled by a carbon dioxide - acetone mixture in a Dewar flask. The frozen tissue pieces were transferred very rapidly from the propane container to the cooled holders in the sample tube by means of thin forceps, cooled in the liquid propane before touching the specimens. Dehydration. The temperature of the specimens and of the water condenser, the distance between the samples and the cold trap, and the magnitude of the vacuum (pumping speed), arc the important factors to be considered in a freeze-drying equipment with a water condenser at low temperature. Different temperatures of the samples during the dehydration procedure have been suggested (5, 12, 15, 17, 18, 21). As insufficient physical data are available, this temperature cannot be selected theoretically (2). Above a sample temperature of -40” C, morphological changes probably due to recrystallization, are demonstrable in the tissues (19). Theoretically the dehydration temperature should be kept below the eutectic point for the salt systems present in the tissues (about -55” C). Otherwise ion diffusion may occur. As low a temperature as -60” C would be safe also for highly diffusible ions (8). If the time for dehydration at such a low temperature is to be reasonably short, a water condenser with very low temperature, placed close to the specimens, and a high vacuum in the system are required. Liquid nitrogen provides sufficiently low water vapor tension in the trap (1O-24 mm Hg). Experimental
Cell Research
6
Freeze-drying
231
Fig. 3. View of the freeze-drying apparatus. A. Sample tube with the close cold trap. i3. Distant cold trap. C. Dewar flask for-carbon dioxide - acetone mixture. D. Dewar flask for liquid nitrogen. E. Oil diffusion pump. F. Pirani vacuum gauge. G. Vacuumeter. H. Oscillator.
A short distance (less than 4 cm) between the samples and the trap accelerates the removal of water from the specimens (8). Further, a high vacuum (of the magnitude of 1O-5 mm Hg) increases the mean free path of water molecules, and therefore shortens the time for dehydration (13). According to these principles Glick and Malmstriim (8) have devised an efficient freeze-drying apparatus consisting of a Pyrex glass unit with a close and a distant cold trap and a high vacuum system. However, the use of liquid nitrogen in the traps in combination with the high vacuum was considered to be hazardous because of the fragility and risk of explosion of the rather complex glass unit. Therefore, a modified dehydration apparatus was built in metal with a minimum of glass parts (Fig. 3). The vacuum system consists of a three stage oil diffusion pump (Distillation Products Type MC 275) with a capacity of 275 l/set. at 10m4 mm Hg, backed by a two stage rotary mechanical fore-pump (Pfeiffer ivo. 3006) with a capacity of 5 m3/hour. A detailed diagram of a slightly changed model is shown in Fig. 4. The apparatus is built in metal except for the cooled parts, consisting of the tubes A, B, and G, which are made of Pyrex glass. The metal parts are connected either by solders or by means of O-rings (G. Angus and Co. Ltd., Newcastle upon Tyne) into a vacuum-tight unit. The glass tubes A, B, and G and the stainless steel tube F are attached to the metal blocks C resp. E. These connections are made vacuum-tight by means of a system of metal discs Experimental
Cell Research
6
232
G. Moberger,
B. LindstrGm
and L. Andersson
and O-rings, as shown in the figure. The system permits an adjustment in height of the close cold trap tube B as well as of the steel tube F. The two blocks C and E are mounted in a stable stage. The tombac tube H and the adjustability in height of the tube F make small adjustments in the distance between the two blocks possible. All vacuum leads are wide enough to permit a high pumping speed.
Fig. 4. Diagram of the freezedrying unit. A. Sample tube (Pyrex glass). B. Close cold trap tube (Pyrex glass). Metal block to connect tubes A and B. Holder for the Pirani vacuum gauge. Central metal block in the distant cold trap. Central tube of stainless steel. Distant cold trap tube (Pyrex glass). Tombac tube. Tombac tube connection to the oil diffusion pump.
A complete view of the freeze-drying apparatus is shown in Fig. 5. In this model the vacuum pumps consist of a two stage oil diffusion pump (Kylteknik, Stockholm, No. 2560) with a capacity of 500 l/set. at 1O-4 mm Hg, and a two stage rotary mechanical fore-pump (Kylteknik, Stockholm, No. 2500) with a capacity of 5 m3/hour. Before the dehydration was started the distant cold trap tube was surrounded by liquid nitrogen in a Dewar flask. Surrounded by the carbon dioxide - acetone mixture in a Dewar flask, the sample tube with the tissue holders carrying the specimens was attached to the apparatus. The close cold trap was filled with liquid nitrogen. Refilling was required every 4 hours. High vacuum (about 1O-5 mm Hg) was obtained in 15 minutes with the vacuum pumps used. Initial dehydration with the sample tube cooled below -60” C was performed for 6-12 hours, depending upon the size of the tissue pieces and the nature of the material. Subsequently the Dewar flask Experimenlal
Cell Research
6
Freeze-drying
233
with the carbon dioxide - acetone mixture was removed, and the sample tube was allowed to reach room temperature. Simultaneously ihe liquid nitrogen in the close cold trap evaporated. In general the dehydration was now continued for 8-12 hours (over night) with the samples at room temperature and the distant cold trap surrounded by liquid nitrogen (refilling required every 12 hours). The moisture initially condensed on the walls of the close cold trap was then rapidly transferred to the distant cold trap. Subsequently, addition of liquid nitrogen to the close cold trap did not result in condensation of visible moisture if the dehydration was complete. Various tissues required different times for the initial dehydration at a temperature below 410” C. For pieces with a maximal thickness of 2 mm the following approximate times have been found sufficient:
Fig. 5. View of the ing apparatus (later tion). A-H. The same as J. Copper wire loop inductance of the
freezc-drymodificain Fig. 3. (the outer oscillator). Experimenlnl
Cell Research
6
234
G. Moberger.
B. Lindstr6m
and L. Andersson
Tissues with a moderate water and lipid content (e.g. epithelial tissues, liver, pancreas) . . . . . . . . . . . . . . . . Tissues rich in water (e.g. intestinal mucosa, testis) . . . . Tissues with a high lipid content (e.g. adrenals, renal parenchyma, cerebral cortex) . . . . . . . . . . . . . . .
Fig. 6. Wiring T,. Transformer, T,. -
6 hours 8 hours 12 hours
diagram of the oscillator circuit. 220/500-1000 volts. 220/s volts. 220/6.3 volts. N 100 pH (air-winded coil.) (Coil, diam. 00 mm. Copper 5 x 3 mm, 6-8 turns.) (Coil, diam. 55 mm. Copper 3.5 x 2 mm, 11 turns.) 2.5 mH (choke coil.) 4.3 mH (choke coil.)
wire, wire,
Dehydration at temperatures close to -40” C is less time-consuming but has not given reliable results. Initial dehydration for a limited time (minimum 6 hours) with a temperature of less than -60” C followed by drying at higher temperatures (-30” C to -40” C) gave no demonstrable tissue changes. The total time for complete dehydration could thus be shortened considerably. Embedding
in paraffin
in uacuo.
The embedding procedure in uacuo is preferred in order not to expose the dried specimens to the moisture of the atmosphere. The infiltration of paraffin is also facilitated. For this purpose a new technique for the heating of the tissue holders within the sample tube was developed. This was Experimental
Cell Research
6
Freeze-drying
235
achieved by means of an oscillator. The wiring diagram of the oscillator circuit is shown in Fig. 6. The primary side of the transformer T, has six steps by which the secondary voltage can be varied in 100 volts steps between 500 and 1000 volts at a constant input of 220 volts A.C. Two selfexcited oscillator triodes (RCA 1623) are used in Hartley coupling with a common oscillator circuit. The secondary voltage from the transformer (TX) and therefore the tubes function is used as anode voltage in the triodes, alternately during the positive half periods. A high frequency A.C. of about 1.5 MC is obtained in the common oscillator circuit (the inductance L, and its self-capacitance). Via the inductance (L,) the high-frequency polver is induced in the outer inductance (La), consisting of a removable copper wire loop outside the main oscillator (as shown in Fig. 5, J). The magnitude of the capacitance (C,) is essential for obtaining maximal high-frequency power in the external loop (L3). During the embedding procedure the loop is placed around the sample tube containing the tissue holders of iron. Heating of the tissue holders is caused by the energy of the electro-magnetic field in the centre of the loop (L3). The tissue holders were warmed to the melting point of the paraffin in 3-13 minutes, depending upon the secondary voltage chosen. \Vhen the paraffin started to melt at the edges of the individual grooves, the oscillator was put off in order to avoid overheating of the paraffin. The great mass of the metal holders secured complete melting of the paraffin blocks. The specimens were rapidly infiltrated with the melted paraffin and sank to the bottom of the grooves. After 10 minutes the paraffin was allowed to solidify. During the whole embedding procedure high vacuum was maintained. SOME
APPLICATIONS ON
DIFFERENT
OF THE
FREEZE-DRYING
BIOLOGICAL
PROCEDURE
MATERIALS
Fig. 7 serves to illustrate the results obtained when applying the freezedrying procedure described in this paper. Because of the minute size of the ice crystals in well preserved frozendried structures, phase-contrast or dark field illumination microscopy are the most reliable methods to estimate the morphological results of the freezedrying procedure. A, II, and C show mouse ascites tumor cells (Krebs II) in the living state (A) and after freeze-drying (R and C). D and E illustrate certain structural elements in frozen-dried specimens after removal of the paraffin from the sections. The tonofibrils in the human epidermis are well preserrcd (II). In the aortic wall (E) the elastic fibrils can be seen to consist 14 - 633iO5
Experimental
Cell Research
6
236
G. Moberger,
R. Lindsfriim
and L. Anderson
Freeze-drying of dense bundles of finer threads. F illustrates the preservation of the different cell layers in a tubule in the testis of the Louse. Tissues with a high lipid content are known to be difficult to freeze-dry. G, H, and J show a fe\v examples of frozen-dried tissues with varying lipid contents. SUMMARY
A freeze-drying apparatus built in metal with a minimum of glass parts and a new technique for the embedding in paraffin in UCIL’IZOis reported. Tl~e freeze-drying procedure with the apparatus is described and some applications on different biological material are given. REFERENCES ~ARTEIAIISZ, G. \I’., A nat. Rec., 77, 509 (1940). rwL, L. G. E., ~nternofl. Heu. C~tol~yy, 1, 35 (1952). I:MMEL, 1’. hf., Anal. Rec., 95, I59 (1946). IQsntxr, J. C., HOLLOMAN, J. I-I., and ‘YURSHULL, L)., Science, 109, 168 (1949). GE~SII, I., Anat. Rec., 53, 309 (1932). ~-Uull. Intern. Ass. Med. Museums, 28, 179 (1948). GLICK, I>., Techniques of Histoand (Zytochemistry, Interscience I’ublishers Inc., New York, pp. 3-5, 1949. GLICK, D., and MALMSTtliiM, 13. G., EX[lt[. Cell Rrseurch, 3, 125 (1952). GCSTAFSSON, B., Communication at Symposium on Freezing and Drying, Stocl;holm, 1952. I~AIlRIS, 13. J. C., i%‘crttZre, 168, 851 (1951). Hor
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. IX. 19. 20. 21. 22.
-
.--
I:ig. 7 (opposite). n. Mouse ascites tumor cell (Krcbs II) in the living state. I’hase contrast, x 8iO. I<. Mouse ascites tumor cell (Krebs II) after freeze-drying of a fresh squash preparation, in anhydrous glycerin. Phase contrast, x 870. CJ. Same as B. D. Hutnan epidermis. Frozen-dried, 3 p paraffin-section, deparnffinized in xylol Mounted in nonylic alcohol. Phase contrast, x 900. E. r\orta from rat. I>rozctt-dried, mounted in nonylic alcohol. Phase contrast, ’ 900. (B-J, 3p paraffitt-sections.) F. ‘restis from mouse. I:rozctt-dried. Gallocyanin, x 500. G. Pancreas from rat. I’rozctt-dried for 6 hours at -70” C. Gallocvanin. ): 1 050. H. Cerebral ganglion cells from rat. I;rozett-dried for 8 hours at --70” C. Nissl-blue. x 710. J. Medullsr adrenal cells frotn rat. l:rozen-dried for 12 hours at -70” C. Ilhrlich hetnatoxylinx 1 050. eosin. 14*
-
.533iO,5
Experimenial
Cell Research
6
228
FREEZE-DRYING
WITH
G. MOBERGER, The
Wallenberg
A MODIFIED APPARATUS
B. LINDSTROM
Laboratory
Cell Research,
October
(1954)
GLICK-MALMSTROM and
L. ANDERSSON
at the Institute for Cell Research, Karolinska Institutet, Stockholm Received
6, 228-237
Medical
Nobel
Institute,
30, 1953
THE
freeze-drying procedure is a valuable method to preserve biological material for histo- and cytochemical investigations. By means of initial rapid freezing of the tissue samples, followed by dehydration in the frozen state and subsequent embedding, thin sections of tissues can be prepared with a minimum of structural and chemical changes (2, 22). Various techniques regarding the different steps in the freeze-drying procedure have been reviewed by several authors (i.a. 2, 6, 7, 10, 12, 16, 20). Highly efficient dehydration has been obtained in equipment with a water condenser at the temperature of liquid nitrogen close to the specimens combined with a high vacuum (9, 14, 21). Based upon theoretical considerations (13) Glick and Malmstriim (8) have devised an efficient freeze-drying apparatus of this type with remarkably short dehydration time. In the present paper the freeze-drying procedure with a modified GlickMalmstram apparatus and a new technique for the embedding in zjaczzo will be communicated. DESCRIPTION
OF
APPARATUS
AND
TECHNIQUE
In the following the three main steps of the freeze-drying procedure, the initial freezing, the dehydration in uacuo, and the embedding technique, will be considered separately. The main features of importance for the choice of apparatus and technique will be briefly discussed. Initial
freezing.
In order to avoid postsampling artefacts such as ion diffusion and enzymatic decomposition (l), as far as possible, it is necessary to freeze the tissue samples instantaneously after their removal from the organism. Ice crystal formation within the specimens is the main cause of structural changes occurring during the freezing procedure. A high rate of ice crystal nucleation, and a low rate of growth of the ice crystal nuclei, give the smallest crystals possible, and thus minimal structural changes. As the rate of forExperimental
Cell Research
6
Freeze-drying
229
Fig.
2.
Fig. 1. Propane container for the initial freezing of the tissue samples. Fig. 2. Tissue sample holders. Fig.
1.
mation of new ice crystal nuclei is increased by a factor of lox8 between -33” C and -43” C (4), the tissue samples should be frozen as rapidly as possible, well below this range of temperature. A freezing medium with a high conductivity and a very low temperature is therefore necessary. A medium such as liquid air (5) has a sufficiently low temperature, but a layer of vaporized air forms an envelope around the specimens, resulting in poor conductivity (17). Isopentane, cooled by liquid nitrogen, has a high conductivity (11) but solidifies at about -150” C. Pure propane has a still higher conductivity (3) and remains fluid at -185” C, and is therefore very useful for the initial freezing. In order to obtain rapid initial freezing of the inner parts of the specimens, these must be small. When too large pieces of tissue are frozen, formation of ice crystals of considerable size will occur which causes structural changes in the inner layers of the tissues (19). The transport of the frozen specimens from the cooling medium to the dehydration chamber must be quick enough to prevent an increase of the temperature to the range where the growth of the ice crystals is accelerated (at about -40” C). Liquid propane was used for the initial freezing of the tissue samples. A metal container with an external loop for the condensation of the propane Experimental
Cell Research
6
230
G. Moberger,
B. Lindstrbm
and L. Andersson
was devised (Fig. 1). It was placed in a Dewar flask filled with liquid nitrogen. When the container had obtained adequately low temperature, propane gas was let through the loop, where it condensed and then dropped into the container. Immediately after the removal from the organism small pieces of tissue (with an extent of less than 2 mm in one dimension) were plunged in the fluid propane. The tissue pieces were allowed to sink free to the bottom of the container, thus continuously being surrounded by fresh volumes of the cooling medium. The use of forceps or of holders for the individual specimens was avoided because of the risk in delaying the freezing process. A small metal basket placed in the bottom of the container served to make it easier to collect the samples for further transport. The tissue sample holders were made of solid iron in which grooves for the individual specimens were drilled (Fig. 2). The grooves were tilled with paraffin, degassed at 1OW’ mm Hg for 30 minutes in a separate simple vacuum unit. Subsequently the tissue holders were placed in the sample tube (see below) which was cooled by a carbon dioxide - acetone mixture in a Dewar flask. The frozen tissue pieces were transferred very rapidly from the propane container to the cooled holders in the sample tube by means of thin forceps, cooled in the liquid propane before touching the specimens. Dehydration. The temperature of the specimens and of the water condenser, the distance between the samples and the cold trap, and the magnitude of the vacuum (pumping speed), arc the important factors to be considered in a freeze-drying equipment with a water condenser at low temperature. Different temperatures of the samples during the dehydration procedure have been suggested (5, 12, 15, 17, 18, 21). As insufficient physical data are available, this temperature cannot be selected theoretically (2). Above a sample temperature of -40” C, morphological changes probably due to recrystallization, are demonstrable in the tissues (19). Theoretically the dehydration temperature should be kept below the eutectic point for the salt systems present in the tissues (about -55” C). Otherwise ion diffusion may occur. As low a temperature as -60” C would be safe also for highly diffusible ions (8). If the time for dehydration at such a low temperature is to be reasonably short, a water condenser with very low temperature, placed close to the specimens, and a high vacuum in the system are required. Liquid nitrogen provides sufficiently low water vapor tension in the trap (1O-24 mm Hg). Experimental
Cell Research
6
Freeze-drying
231
Fig. 3. View of the freeze-drying apparatus. A. Sample tube with the close cold trap. i3. Distant cold trap. C. Dewar flask for-carbon dioxide - acetone mixture. D. Dewar flask for liquid nitrogen. E. Oil diffusion pump. F. Pirani vacuum gauge. G. Vacuumeter. H. Oscillator.
A short distance (less than 4 cm) between the samples and the trap accelerates the removal of water from the specimens (8). Further, a high vacuum (of the magnitude of 1O-5 mm Hg) increases the mean free path of water molecules, and therefore shortens the time for dehydration (13). According to these principles Glick and Malmstriim (8) have devised an efficient freeze-drying apparatus consisting of a Pyrex glass unit with a close and a distant cold trap and a high vacuum system. However, the use of liquid nitrogen in the traps in combination with the high vacuum was considered to be hazardous because of the fragility and risk of explosion of the rather complex glass unit. Therefore, a modified dehydration apparatus was built in metal with a minimum of glass parts (Fig. 3). The vacuum system consists of a three stage oil diffusion pump (Distillation Products Type MC 275) with a capacity of 275 l/set. at 10m4 mm Hg, backed by a two stage rotary mechanical fore-pump (Pfeiffer ivo. 3006) with a capacity of 5 m3/hour. A detailed diagram of a slightly changed model is shown in Fig. 4. The apparatus is built in metal except for the cooled parts, consisting of the tubes A, B, and G, which are made of Pyrex glass. The metal parts are connected either by solders or by means of O-rings (G. Angus and Co. Ltd., Newcastle upon Tyne) into a vacuum-tight unit. The glass tubes A, B, and G and the stainless steel tube F are attached to the metal blocks C resp. E. These connections are made vacuum-tight by means of a system of metal discs Experimental
Cell Research
6
232
G. Moberger,
B. LindstrGm
and L. Andersson
and O-rings, as shown in the figure. The system permits an adjustment in height of the close cold trap tube B as well as of the steel tube F. The two blocks C and E are mounted in a stable stage. The tombac tube H and the adjustability in height of the tube F make small adjustments in the distance between the two blocks possible. All vacuum leads are wide enough to permit a high pumping speed.
Fig. 4. Diagram of the freezedrying unit. A. Sample tube (Pyrex glass). B. Close cold trap tube (Pyrex glass). Metal block to connect tubes A and B. Holder for the Pirani vacuum gauge. Central metal block in the distant cold trap. Central tube of stainless steel. Distant cold trap tube (Pyrex glass). Tombac tube. Tombac tube connection to the oil diffusion pump.
A complete view of the freeze-drying apparatus is shown in Fig. 5. In this model the vacuum pumps consist of a two stage oil diffusion pump (Kylteknik, Stockholm, No. 2560) with a capacity of 500 l/set. at 1O-4 mm Hg, and a two stage rotary mechanical fore-pump (Kylteknik, Stockholm, No. 2500) with a capacity of 5 m3/hour. Before the dehydration was started the distant cold trap tube was surrounded by liquid nitrogen in a Dewar flask. Surrounded by the carbon dioxide - acetone mixture in a Dewar flask, the sample tube with the tissue holders carrying the specimens was attached to the apparatus. The close cold trap was filled with liquid nitrogen. Refilling was required every 4 hours. High vacuum (about 1O-5 mm Hg) was obtained in 15 minutes with the vacuum pumps used. Initial dehydration with the sample tube cooled below -60” C was performed for 6-12 hours, depending upon the size of the tissue pieces and the nature of the material. Subsequently the Dewar flask Experimenlal
Cell Research
6
Freeze-drying
233
with the carbon dioxide - acetone mixture was removed, and the sample tube was allowed to reach room temperature. Simultaneously ihe liquid nitrogen in the close cold trap evaporated. In general the dehydration was now continued for 8-12 hours (over night) with the samples at room temperature and the distant cold trap surrounded by liquid nitrogen (refilling required every 12 hours). The moisture initially condensed on the walls of the close cold trap was then rapidly transferred to the distant cold trap. Subsequently, addition of liquid nitrogen to the close cold trap did not result in condensation of visible moisture if the dehydration was complete. Various tissues required different times for the initial dehydration at a temperature below 410” C. For pieces with a maximal thickness of 2 mm the following approximate times have been found sufficient:
Fig. 5. View of the ing apparatus (later tion). A-H. The same as J. Copper wire loop inductance of the
freezc-drymodificain Fig. 3. (the outer oscillator). Experimenlnl
Cell Research
6
234
G. Moberger.
B. Lindstr6m
and L. Andersson
Tissues with a moderate water and lipid content (e.g. epithelial tissues, liver, pancreas) . . . . . . . . . . . . . . . . Tissues rich in water (e.g. intestinal mucosa, testis) . . . . Tissues with a high lipid content (e.g. adrenals, renal parenchyma, cerebral cortex) . . . . . . . . . . . . . . .
Fig. 6. Wiring T,. Transformer, T,. -
6 hours 8 hours 12 hours
diagram of the oscillator circuit. 220/500-1000 volts. 220/s volts. 220/6.3 volts. N 100 pH (air-winded coil.) (Coil, diam. 00 mm. Copper 5 x 3 mm, 6-8 turns.) (Coil, diam. 55 mm. Copper 3.5 x 2 mm, 11 turns.) 2.5 mH (choke coil.) 4.3 mH (choke coil.)
wire, wire,
Dehydration at temperatures close to -40” C is less time-consuming but has not given reliable results. Initial dehydration for a limited time (minimum 6 hours) with a temperature of less than -60” C followed by drying at higher temperatures (-30” C to -40” C) gave no demonstrable tissue changes. The total time for complete dehydration could thus be shortened considerably. Embedding
in paraffin
in uacuo.
The embedding procedure in uacuo is preferred in order not to expose the dried specimens to the moisture of the atmosphere. The infiltration of paraffin is also facilitated. For this purpose a new technique for the heating of the tissue holders within the sample tube was developed. This was Experimental
Cell Research
6
Freeze-drying
235
achieved by means of an oscillator. The wiring diagram of the oscillator circuit is shown in Fig. 6. The primary side of the transformer T, has six steps by which the secondary voltage can be varied in 100 volts steps between 500 and 1000 volts at a constant input of 220 volts A.C. Two selfexcited oscillator triodes (RCA 1623) are used in Hartley coupling with a common oscillator circuit. The secondary voltage from the transformer (TX) and therefore the tubes function is used as anode voltage in the triodes, alternately during the positive half periods. A high frequency A.C. of about 1.5 MC is obtained in the common oscillator circuit (the inductance L, and its self-capacitance). Via the inductance (L,) the high-frequency polver is induced in the outer inductance (La), consisting of a removable copper wire loop outside the main oscillator (as shown in Fig. 5, J). The magnitude of the capacitance (C,) is essential for obtaining maximal high-frequency power in the external loop (L3). During the embedding procedure the loop is placed around the sample tube containing the tissue holders of iron. Heating of the tissue holders is caused by the energy of the electro-magnetic field in the centre of the loop (L3). The tissue holders were warmed to the melting point of the paraffin in 3-13 minutes, depending upon the secondary voltage chosen. \Vhen the paraffin started to melt at the edges of the individual grooves, the oscillator was put off in order to avoid overheating of the paraffin. The great mass of the metal holders secured complete melting of the paraffin blocks. The specimens were rapidly infiltrated with the melted paraffin and sank to the bottom of the grooves. After 10 minutes the paraffin was allowed to solidify. During the whole embedding procedure high vacuum was maintained. SOME
APPLICATIONS ON
DIFFERENT
OF THE
FREEZE-DRYING
BIOLOGICAL
PROCEDURE
MATERIALS
Fig. 7 serves to illustrate the results obtained when applying the freezedrying procedure described in this paper. Because of the minute size of the ice crystals in well preserved frozendried structures, phase-contrast or dark field illumination microscopy are the most reliable methods to estimate the morphological results of the freezedrying procedure. A, II, and C show mouse ascites tumor cells (Krebs II) in the living state (A) and after freeze-drying (R and C). D and E illustrate certain structural elements in frozen-dried specimens after removal of the paraffin from the sections. The tonofibrils in the human epidermis are well preserrcd (II). In the aortic wall (E) the elastic fibrils can be seen to consist 14 - 633iO5
Experimental
Cell Research
6
236
G. Moberger,
R. Lindsfriim
and L. Anderson
Freeze-drying of dense bundles of finer threads. F illustrates the preservation of the different cell layers in a tubule in the testis of the Louse. Tissues with a high lipid content are known to be difficult to freeze-dry. G, H, and J show a fe\v examples of frozen-dried tissues with varying lipid contents. SUMMARY
A freeze-drying apparatus built in metal with a minimum of glass parts and a new technique for the embedding in paraffin in UCIL’IZOis reported. Tl~e freeze-drying procedure with the apparatus is described and some applications on different biological material are given. REFERENCES ~ARTEIAIISZ, G. \I’., A nat. Rec., 77, 509 (1940). rwL, L. G. E., ~nternofl. Heu. C~tol~yy, 1, 35 (1952). I:MMEL, 1’. hf., Anal. Rec., 95, I59 (1946). IQsntxr, J. C., HOLLOMAN, J. I-I., and ‘YURSHULL, L)., Science, 109, 168 (1949). GE~SII, I., Anat. Rec., 53, 309 (1932). ~-Uull. Intern. Ass. Med. Museums, 28, 179 (1948). GLICK, I>., Techniques of Histoand (Zytochemistry, Interscience I’ublishers Inc., New York, pp. 3-5, 1949. GLICK, D., and MALMSTtliiM, 13. G., EX[lt[. Cell Rrseurch, 3, 125 (1952). GCSTAFSSON, B., Communication at Symposium on Freezing and Drying, Stocl;holm, 1952. I~AIlRIS, 13. J. C., i%‘crttZre, 168, 851 (1951). Hor
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. IX. 19. 20. 21. 22.
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I:ig. 7 (opposite). n. Mouse ascites tumor cell (Krcbs II) in the living state. I’hase contrast, x 8iO. I<. Mouse ascites tumor cell (Krebs II) after freeze-drying of a fresh squash preparation, in anhydrous glycerin. Phase contrast, x 870. CJ. Same as B. D. Hutnan epidermis. Frozen-dried, 3 p paraffin-section, deparnffinized in xylol Mounted in nonylic alcohol. Phase contrast, x 900. E. r\orta from rat. I>rozctt-dried, mounted in nonylic alcohol. Phase contrast, ’ 900. (B-J, 3p paraffitt-sections.) F. ‘restis from mouse. I:rozctt-dried. Gallocyanin, x 500. G. Pancreas from rat. I’rozctt-dried for 6 hours at -70” C. Gallocvanin. ): 1 050. H. Cerebral ganglion cells from rat. I;rozett-dried for 8 hours at --70” C. Nissl-blue. x 710. J. Medullsr adrenal cells frotn rat. l:rozen-dried for 12 hours at -70” C. Ilhrlich hetnatoxylinx 1 050. eosin. 14*
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Experimenial
Cell Research
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