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13 4 2016 laser research | about the same number of lethal cells. This tempera- tureof56.5 °CcorrespondedtotheLD50value(50 % lethality).Nocellsurvivedthermaltreatmentat58 °C. HSP production Examinations with regard to the production of HSP via light microscope or transmission laser microscopy showed a slight, unspecific colouring of the cells after incubation of 37 °C (control, Fig. 2). An increase in HSP production(intensecolouring)wasnotedatatempera- ture of 50 °C, while thermal treatment at 60 °C again resultedinslight,unspecificcolouringofthecells. REM Scanning electron microscopy showed a typical flat, long distribution of the control cells (37 °C cells, Fig. 3). These cells exhibited many processes and mi- crovilli-like structures. In addition, cell-to-cell con- nections with neighbouring cells were observed. The successive rise in temperature resulted in the first critical temperature level of 46.5 °C ± 0.5 °C. From this level onwards, significant initial changes of the cells were registered via light and electron micro- scope,especiallyaninitialdeformationandrounding of the cells. The cell structure (microvilli-like struc- tures) was reduced. However, microvilli were ob- servedattemperaturesofupto50 °C(Fig.4).At50 °C (chance of survival > 70 °C according to Life/Dead Assay), the cells left distinct cytoplasm protuber- ancesonthebaseofthecoverslip(Fig.4,arrow),prob- ably caused by a rapid contraction or rounding. Incubationatatemperatureof60 °C,atwhichnone of the cells survived, resulted in a different outcome. There was no apparent deformation or rounding of thecells,withtheoriginalcellshaperemainingmostly intact and some small reductions. The cells appeared to have been “thermally fixed” instantly. Neither mi- crovilli nor other surface structures were visible. Cell processes in contact with the coverslip remained in- tact, but exhibited denaturation and fixation caused by rapid heating (Fig. 5). TEM Thefibroblast-likeDPSCcells(Fig.6)exhibitedlong, extended mitochondria (M) within the 3-D network ofthecellat37 °C(control).Thenucleus(K)appeared to be undivided and to have a normal nuclear enve- lope (arrows). ER/RER, free ribosomes as well as the Golgi apparatus did not show any anomalies. A sig- nificantly expressed cytoskeleton (Z) whose fila- ments were aligned parallely to the longitudinal axis (probably microfilaments) was observed. The cells featured a number of inclusions. At 50 °C, cell rounding became irreversible (Fig. 7). Mitochondria(M)exhibitedstructuralchanges,espe- cially an inflation which concurred with the destruc- tion of the christae alignment, the parallelism of which got lost. There was no longer a three-dimen- sionalnetwork.TheGolgiapparatuswassignificantly deformed and hardly any vesicles were constricted. The cytoskeleton was partially disintegrated and could no longer be detected. The cell membrane ap- peared to have increases vacuolisation. The nucleus (K) appeared to be damaged irreversibly. The nuclear envelope was inflated and partially disintegrated (*). The nuclear plasma condensed at the chromatin, re- sultinginareductionoftheeuchromatin-areaswhich condensed at the heterochromatin. The nucleus ex- hibited segmented chambering (arrow). Contrarily, the external shape of DPSC cells incu- bated at 60 °C (Fig. 8) remained mostly intact. How- ever, cytoplasm was hardly detectable. Mitochondria (M) were destroyed, membranes and christae were partially wound up (arrows). Golgi apparatus and cy- toskeletonwerenotdetected.Theeuchromatinareas werereducedatthenucleus(K)andcondensedatthe heterochromatin (*). The nuclear membrane was significantly vesiculated. Discussion Thefirstindicationstoatemperature-relateddam- age of the DPSC were seen in the Life/Dead Assay. Fig. 6 Fig. 6: TEM: Control cells at 37 °C. K: Nucleus; ER: endoplasmatic reticulum, RER: rough endoplasmatic reticulum; M: mitochondria; Z: cytoskeleton; arrows: markers of the nuclear membrane. 42016