Low impact to fixed cell processing aiming transmission electron microscopy

In cell culture, cell structures suffer strong impact due to centrifugation during processing for electron microscope observation. In order to minimise this effect, a new protocol was successfully developed. Using conventional reagents and equipments, it took over one week, but cell compression was reduced to none or the lowest deformation possible.

cell fixation; transmission electron microscopy; low impact; DENV

Authorship

Ortrud Monika Barth ⁺+ Corresponding author: barth@ioc.fiocruz.br

Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Morfologia e Morfogênese Viral, Rio de Janeiro, RJ, BrasilFundação Oswaldo CruzBrasilRio de Janeiro, RJ, BrasilFundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Morfologia e Morfogênese Viral, Rio de Janeiro, RJ, Brasil

Marcos Alexandre Nunes da Silva

Debora Ferreira Barreto-Vieira

+ Corresponding author: barth@ioc.fiocruz.br

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Fig. 1
: protocol of low impact fixation of cells. (1-2) The culture medium was changed by cacodilate buffered 1% glutaraldehyde and aliquots of 1.5 mL of the cell suspension were kept overnight at 8ºC in 2 mL Eppendorf tubes; (3) a portion of the fixative was gently removed and the cell-fixative-suspension was transferred to smaller vials of 500 µL capacity; (4-5) the fixative was changed by 0.2 M cacodilate buffer added to 7% saccharose; (6) 0.2 M cacodilate buffer added to 7% saccharose was changed by 1% aqueous osmium tetroxide; (7) washing with distilled water and 0.2 M cacodilate buffer with 7% saccharose; (8) dehydration of the sample with 30% acetone; (9) dehydration with 50% acetone; (10) dehydration with 70% acetone; (11) dehydration with 90% acetone; (12) dehydration with 100% acetone and resin infiltration; (13) resin infiltration; (14) resin polymerization.

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Fig. 2
: (A) Conventional technique: ultrathin section of a compressed dengue virus-1 (DENV-1) infected C6/36 cell. It shows an ellyptical cell outline and laterally located nucleus (Bar = 1 μm); (B-F) the non-conventional technique; (B) ultrathin section of a non-compressed DENV-1 infected C6/36 cell. It shows a rounded cell and a centralised nucleus (Bar = 1 μm); (C) semithin section of a C6/36 cell pellet (Bar = 50 μm); (D) semithin section of C6/36 cells infected with DENV-4 (Bar = 10 μm); (E) ultrathin section of a C6/36 cell fragment showing nucleus (N), mitochondria (M), lysosome (L) (Bar = 200 nm); (F) ultrathin section of C6/36 cell mitochondria (Bar = 100 nm).

Fig. 1 : protocol of low impact fixation of cells. (1-2) The culture medium was changed by cacodilate buffered 1% glutaraldehyde and aliquots of 1.5 mL of the cell suspension were kept overnight at 8ºC in 2 mL Eppendorf tubes; (3) a portion of the fixative was gently removed and the cell-fixative-suspension was transferred to smaller vials of 500 µL capacity; (4-5) the fixative was changed by 0.2 M cacodilate buffer added to 7% saccharose; (6) 0.2 M cacodilate buffer added to 7% saccharose was changed by 1% aqueous osmium tetroxide; (7) washing with distilled water and 0.2 M cacodilate buffer with 7% saccharose; (8) dehydration of the sample with 30% acetone; (9) dehydration with 50% acetone; (10) dehydration with 70% acetone; (11) dehydration with 90% acetone; (12) dehydration with 100% acetone and resin infiltration; (13) resin infiltration; (14) resin polymerization.

Fig. 2 : (A) Conventional technique: ultrathin section of a compressed dengue virus-1 (DENV-1) infected C6/36 cell. It shows an ellyptical cell outline and laterally located nucleus (Bar = 1 μm); (B-F) the non-conventional technique; (B) ultrathin section of a non-compressed DENV-1 infected C6/36 cell. It shows a rounded cell and a centralised nucleus (Bar = 1 μm); (C) semithin section of a C6/36 cell pellet (Bar = 50 μm); (D) semithin section of C6/36 cells infected with DENV-4 (Bar = 10 μm); (E) ultrathin section of a C6/36 cell fragment showing nucleus (N), mitochondria (M), lysosome (L) (Bar = 200 nm); (F) ultrathin section of C6/36 cell mitochondria (Bar = 100 nm).

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Instituto Oswaldo Cruz, Ministério da Saúde Av. Brasil, 4365 - Pavilhão Mourisco, Manguinhos, 21040-900 Rio de Janeiro RJ Brazil, Tel.: (55 21) 2562-1222, Fax: (55 21) 2562 1220 - Rio de Janeiro - RJ - Brazil
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Low impact to fixed cell processing aiming transmission electron microscopy

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[1] + Corresponding author: barth@ioc.fiocruz.br