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

Introduction

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

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

Technical Set-Up

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

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

Hit Verification Test

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 irradiated @ 3000 H+/s Hit Accuracy better than 0.5 µm

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

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

Biological Tests

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

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?

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

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

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

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

First Irradiation

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

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

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

Conclusions

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...

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

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

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