Controlled damage of subcellular structures

We used optical scalpel to study a damage of living cells. These experiments were performed in cooperation with the group of Prof. R. Janisch from the Faculty of Medicine of the Masaryk University in Brno. The effects of pulse laser irradiation were used to study of the infusorians Paramecium tetraurelia and Blepharisma undulans undulans. Destruction of the cytoskeleton by laser irradiation was detected by immunofluorescence staining¹. A difference in the development and healing of the wound was observed between Paramecium and Blepharisma cells. A more immediate reaction was recorded in Blepharisma cells containing blepharismin, a red pigment, known to absorb light energy. The damage to the infusorian cortex due to laser irradiation was compared with that produced by mechanical devices.

Fig. 1 Expresure of the Blepharisma cortex to a laser beam. Place of hitting marks with green pointer.

Experiments:

The sizes of these protozoa range from tens to hundreds of micrometers. Therefore they were immobilized by sucking into a microcapillary (TransferMan NK mechanical micromanipulator with a CellTram Air micropipette Eppendorf, Germany). The interventions inside immobilized objects were regulated by a spatial positioning of the optical scalpel controlled by the computer.

Fig. 2 Irradiation of the Blepharisma in detail. Place of hitting marks with red pointer.

Time span between A and J pictures was 8 sec.

Laser Minilite II (Continuum) was used to generate UV laser pulse with the following properties: wavelength, l=355 nm; maximal pulse energy, 8 mJ; pulse length, 5 ns. The laser beam was enlarged by a system of lenses, directed to an Olympus IX70 microscope by dichroic mirrors and focused by Olympus Plan 20X and Plan 40X microscope objectives. The position of the beam in the viewing field of the microscope was adjusted laterally and longitudinally by movable lenses^2,3. Specific areas of the surface or selected structures close below the surface of an immobilized cell were irradiated by laser pulses of energy ranging from 4 to 15 mJ. Influences of the laser pulses on selected cytoplasmic structures were studied together with the reparation of damaged cytoskeleton using imunofluorescent methods.

Fig. 3 Laser-induced wound in Blepharisma cortex visualized by immunofluorescence staining. 

Defect in  cytosceleton is marked by red arrow.

Results:

The response of Paramecium tetraurelia to the damage caused by a laser is different comparing to the response to mechanical damage. The margins of the wound contract rapidly and reparation rate of membrane decreases due to local temperature increase. The cell of Blepharisma undulans undulans contains a red pigment in the margin layer which absorbs laser energy better and therefore the cell damage by the pulse has more serious consequence comparing to the Paramecium tetraurelia. The increased reproduction rate is an interesting result of cell damage.

References:

1. Z. Moravčík, R. Janisch, J. Ježek, P. Zemánek, “Response of infusorian cells to injury caused by a laser microbeam.” Scripta Medica 76, pp. 149-162, 2003.   ABSTRACT

2. P. Zemánek, L. Šrámek, A. Jonáš, Z. Moravčík, R. Janisch, M. Liška, “Standing wave trap and single beam gradient optical trap - experiments and biological applications.” Proc. SPIE 3820, pp. 401–410, 1999.  ABSTRACT

3. J. Ježek, A. Jonáš, M. Liška, P. Jedlička, E. Lukášová, S. Kozubek, P. Zemanek. “Combined system for optical cutting and multiple-beam optical trapping.” Proc SPIE 4016, 303–308, 2000.  ABSTRACT