1. Single Ion Bombardment of Living Cells at LIPSION - Status Report -
Tilman Butz
Universität Leipzig
Germany
CELLION Kick-Off Meeting
Uppsala, February 2004

2. Introduction

3. What is LIPSION? Ion beam 3.5 MV SingletronTM Accelerator (H+, He+)
highly motivated
Scientist
target chamber
with irradiation platform

4. Our Main Objectives
to develop our focused ion beam for radiobiological applications
to improve the target accuracy to better than 1 µm
to cultivate and irradiate cells
to study the radiation related responses

5. Technical Set-Up

6. Cell Dish and Dish Holder
mini Petri dish (35 mm )
Irradiation Window
200 nm thick Si3N4 (2 mm × 2 mm)
Dish Holder
for vertical position
with particle detector (pin diode)
light guide for illumination

7. Irradiation Platform Tunnel Ion Exit Window Mini-Petri Dish Holder
attached to the target chamber
Ion Exit Window
100 nm thick Si3N4 (1 mm x 1 mm)
Mini-Petri Dish Holder
can be slid into the tunnel with minimised air gap between exit window and irradiation window

8. Hit Verification Test

9. Hit Accuracy Test Test Pattern Hit Accuracy written into CR-39
2.25 MeV H+
step width 2 µm
10 × 10 points
each point 1 ms 3000 H+/s
Hit Accuracy
better than 0.5 µm

10. Hit Accuracy
“LIPSION”
written into CR-39
2.25 MeV H+
step width 2 µm

11. High precision irradiation
Endothelial cell
with nucleus of about 15 µm × 10 µm

12. Biological Tests

13. Cell Culture Laboratory
Anja, our biological expert
Workbench
Incubator
Fluorescence Microscope

14. Sham Irradiation as Control
medium removal
vertical positioning
sham irradiation for 15 minutes
horizontal repositioning
restoring the usual medium level
How many cells survive this procedure?
Have the cells enough sedentariness to stay at the same place in vertical position?

15. Medium Removal
horizontal beamline -> irradiation of the mini-Petri dishes in vertical position
particle detector behind the cells -> remove medium as much as possible
using a micro-pipette for medium removal and wash the cells with PBS buffer
a thin film remained in the centre of the window, whereas a significant meniscus remained at the edges of the frames

16. cell culture before sham irradiation
Survival of the Cells
cell culture before sham irradiation
(FDA and PI staining)
field of view
out of

17. Survival of the Cells 30 min after sham irradiation:
no significant increase in mortality

18. Sedentariness Study
Accuracy of the Cell Positions: comparison before and after this procedure (determined by Hoechst 33258)
Cell Positions: nearly unchanged
15 min after
15 min before
Sedentariness of the Cells: quite sufficient

19. First Irradiation

20. Irradiation and Observation
1 proton / 4 µm² (about 100 protons per cell)
17.5 h after irradiation
(~0.5 Gy)
Observation
almost all cells survived
540 µm
540 µm
Energy loss image
(STIM)
low
high

21. Irradiation with Increased Dose
Growth Rate after Irradiation (2 Gy)
(control: td = 33 h)
seems to be a delayed cell doubling time

22. Pattern Irradiation
Irradiation 1 proton / 4 µm² three times with increasing areas
as well as crossing lines
Observation
17.5 h after irradiation
540 µm
540 µm ?
Energy loss images
(STIM)
low
high
remarkable increase
in mortality

23. Conclusions

24. Conclusions Work has to be continued... Technically
Sub-micron hit precision is possible
Micron (sub-micron) target precision is desired
Cell recognition / beam control software is needed
Biologically
Cells grow on Si3N4
Cells do not care for vertical positioning
Cells survive short term medium removal
Irradiation pattern not identifiable (ROS?)
Work has to be continued...

25. Near future irradiation plans
Cultivation of
HeLa (human cervix carcinoma)
CHO (chinese hamster ovary)
Fibroblasts
Irradiation
high doses
successively lower doses

26. Technical Development
Construction of a new sample chamber (completion Oct. 2004)
can be used as:
standard IBA chamber
irradiation platform
(with replaced back)
on demand beam positioning and blanking system

27. Thanks to Thank You Tilo Reinert Anja Fiedler Jiři Škopek
Judith Tanner Jürgen Vogt
Thank You