Radiation Effects in Life Sciences oocyte eggs in uterus spermatheca gonad Quality of Radiation Biological Effects Applications of SSD in Life sciences Nanodosimetry Particle Microscope (pct) vulva
Radiation in Life Sciences Why Should Biologists Care about Radiation? Imaging and Therapy of Cancer Space Radiation Environment Our Origins ( Evolution) Our Future?? Radiation targets DNA influences the Gene Pool can be used for Cancer Research to induce DNA Faults
Charged Particles in Radiobiology Much of Radiobiology is done with Photons (UV, X-and γ-rays) Protons & Light Ions Physical Properties are Superior for Cancer Treatment Defined Range Track Structure Magnetic and Electrical Confinement Main Component of Space Radiation Trapped Belts, Sun and Galactic Sources Energy Range Similar to Synchrotron Responses of Normal Tissues to Radiation Limit Medical Treatment and Space Mission Operations Cell Inactivation Cell and Tissue Changes of State
Energy Loss by Radiation in Matter Photons interact catastrophically N(x)/N o =e -µx N(x)/N o is fraction of photons left at depth x µis linear attenuation coefficient Mass attenuation coefficient (cm 2 g 1 ) 1000 100 10 1 0.1 0.01 Rayleigh (coherent) Compton total photoelectric Silicon At high E, small Conversion Prob. in thin Materials pair production 0.001 0.01 0.1 1 10 100 1,000 10,000 Photon energy (MeV) Charged Particles leave structured track 1/ρ(dE/dx) = 4πr o 2 N e z 2 m o c 2 /β 2 [ln(2m o c 2 β 2 /I(1-β 2 ) -β 2 -Σ i (c i /Z)]» N e is electrons per gram of medium, ρ is density of medium, β is v/c, z is charge of particle» The important component is : de/dx ρ z 2 /β 2 Energy loss de/dx (MeV cm 2 g 1 ) 50 30 20 10 7 5 4 3 2 Silicon ~1/β 1.5 measure p MIP Bethe-Bloch 1 0.1 1 10 100 1000 10000 p/m = βγ e ± Rad µ ± Without radiative losses
Photon and Proton: Dose vs. Depth X-rays & Gamma Rays: Exponential D o s e Depth D o s e Protons: Bragg Curve (derives from negative slope of Bethe-Bloch) Depth
Parameters to Describe Quality of Radiation Dose & Dose Rate Fluence & Fluence Rate Linear Energy Transfer (LET) Relative Biological Effectiveness (RBE) or Quality Factor (Q) Cellular Responses to Charged Particles Fluence (E) is Better than Dose as an Independent Variable LET is Insufficient to Describe Track Structure (Track dimensions vs. Target geometry)
Proton Radiobiology in Perspective n = 416 50 MeV protons D = 1 Gy de/dx per µm 1.3 kev 36 ionizations RBE* 1.1 n = 112 10 MeV protons 4.7 kev 134 ionizations 1.4 n = 54 4 MeV protons 10 µm 10 kev 2.0 276 ionizations * rel to 60 Co γ rays
Nano Dosimetry Are there different classes of damage depending on the Linear Energy Transfer (LET) and number of ionizations/dna molecule? LET # of Ionizations (Cluster Size) Damage Example Low 1-5 Repairable? Single Strand Break High 6-12 Irreparable? Double Strand Break Very High >12 Recombination & Saturation? Effect of OH - radicals in the damage process Improve dosimetry of proton beam for cancer therapy??
Radiation Damage DNA Ionization event (formation of water radicals) Primary particle track Light damage- reparable delta rays Water radicals attack the DNA e - OH Clustered damageirreparable The mean diffusion distance of OH radicals before they react is only 2-3 nm Ionization in 1 mm of low pressure gas = Ionization in 2nm of DNA
Schematic of Nanodosimeter low pressure gas particle E 1 δ electron ion SSD E 2 (strong) SSD vacuum ion counter differential pumping
ND Ion Cluster Spectra (LLUMC Weizmann group) Ion Cluster Spectra 100 Ave. Clustersize as a function of LET 10 1 0.1 P (22Mev) He (4.5MeV) C (70MeV) C (20MeV) 0.01 10 1 10 2 10 3 10 4 de/dx [MeV/(g/cm 2 )] Ion cluster spectra depend on particle type and energy Need to correct for the position of the primary particle track The average cluster size increases with increasing LET Protons have small chance to generate large LET: lucky for us!
Quality of Radiation Comparison of unc-22 Mutation Rates for Low and High Energy Protons and Gamma Rays 1.00e-3 unc-22 Mutation Frequency vs. Dose Arithmetic Mutation Frequency 8.00e-4 6.00e-4 4.00e-4 2.00e-4 0.00 250 MeV Bragg Peak p + 250 MeV p + 60 Co Gamma -2.00e-4 0 10 20 30 40 50 60 70 Dose in Gray
Rat Skin Tumorigenesis Argon ions vs. electrons From Burns & Albert, 1986
Low Dose: Atomic Bomb Risk Data Departs from Linearity at Low Doses Pierce & Preston (2000) Rad Res. 154:178
Low-Dose Cell Survival Low-dose studies with a proton microbeam precise lowdose/fluence cell survival curves hypersensitive region at low doses more pronounced at higher proton energies (3.2 MeV vs. 1 MeV) Dose (Gy) 3.2 MeV protons Schettino et al. Radiation Res. 156, 526-534, 2001
Adaptive Response & Bystander Effect Low-dose studies with an alpha particle microbeam only 10% of cells exposed more cells inactivated than traversed (bystander effect) previous exposure to low level of DNA damage increases resistance (Adaptive response) --- expected -o- 0 hrs after priming γ dose -- 6 hrs after priming γ dose Sawant et al. Radiation Res. 156, 177-180, 2001
Cross Section vs. LET Probability of killing a cell per particle vs. a measure of track structure (LET) scales with geometric cross section of the target but is not a unique function. Kiefer
Biological Effectiveness: Colony Formation Assay The Standard Cell Killing Method Colony formation method and example of CHO cell colonies. A 70 colonies in control culture resulting from 100 seeded cells. Plating efficiency is 70%. B 32 surviving colonies from 2000 cells irradiated with 8 Gy of x-rays. Surviving fraction is: 32/0.7*2000 = 0.023 From Hall, Radiobiology for the Radiologist
Relative Biological Effectiveness Densely ionizing Sparsely ionizing RBE is used to compare different qualities of radiation. Heavy ions X-Rays J. Kiefer (1990)
Scales and Structures of DNA and Chromatin DNA in cells is always in the form of chromatin which is a highly-ordered DNA-protein complex that is constantly being remodeled. Charged particle tracks would be expected to cause patterns of molecular damage that reflect the ordered packing.
Particle Tracks Place Clusters of Ionizations in Volumes on the Scale of Chromatin
The Fundamental Concept of Dose is Misleading Dose is defined as energy absorbed per unit mass (irrespective of the spatial distribution of the absorbed energy) 1 Dose Unit 1 Dose Unit Low LET radiation deposits energy in a uniform pattern High LET radiation deposits energy in a non-uniform pattern Structured Energy Deposition Patterns Interact with Target Structure to Produce Correlated Damage
Correlated Damage The organization of chromatin favors the production of multiple damage sites within loops and nucleosomes. Such patterns are unique to charged particle radiation. A. Chatterjee, LBNL
Risk Mitigation through Shielding? Fragmentation May Enhance Damage Physical vs Biological Assessment of Shielding Dose Eq., msv/day GCR Dose Equivalent on ISS 0.8 0.7 0.6 Aluminum 0.5 0.4 Water 0.3 0.2 Liquid hydrogen Polyethylene 0.1 0.0 0 10 20 30 40 50 Depth, g/cm 2 Dicentrics per Cell per Quarter Chromosome Aberrations on ISS 0.006 Aluminum 0.004 0.002 Liquid hydrogen 0.000 0 10 20 30 40 50 Depth, g/cm 2 Track Model Water Polyethylene F. Cucinotta
Cell Reaction to Radiation Damage Repair (redundant information in DNA), Annealing Death loss of genetic material» metabolic death» delayed apoptotic death genetic instability» delayed apoptotic death Reproductive Death long-term arrest (exit cell cycle, senesce, differentiate) remain metabolically viable for long time Cell Death May Actually Be Controlled Self-Disassembly Apoptosis (Programmed Cell Death) eliminates Damaged Cells
Physiologic Functions of Apoptosis A. Fingers B. Gut C. Tadpole Tails D. Sex Differentiation E. Proliferating Cells F. Self-reactive Lymphocytes G. Irradiation CELL 88:350 1997
Microenvironment & Communication α & p+ Cells Are Not Autonomous and Sometimes Aren t Even Targets Irradiated Unirradiated Precision Field Experiments needed
The Bystander Effect
The Bystander Effect Too Many Cells Respond Soluble Factors Implicated
New Understanding of Cellular Radiation Damage Target in Cell Cell Death Dose Dependence Charged particles vs. X-ray effects Time scale of change in Genome Conventional Wisdom Only DNA in the cell nucleus Random ~ DSB in DNA ~ Dose Dose Equivalent Assumption is Valid Immediate, to all dependents New Principles Also cell membranes & control systems Also controlled disassembly(apoptosis May not ~ Dose (Track Structure, RBE) Dose Equivalent & normalization factors (RBE) not always valid May not appear for up to 50 cell generations (genomic instability)
New Understanding of Repair Mechanisms Mitigation of Cell and Tissue Damage Management of Damage and Survival Conventional Wisdom Repair of DNA damage Autonomy of Individual Cells New Principles Control of signal transduction pathways Apoptosis may be advantageous Distribution of damage to neighbors (Bystander Effect) Cells in tissues respond differently than individuals (Microenvironment Effects)
Specific Experiments to track the damage Tracking Resolution limited to the few um level. Selected biological systems are of that size: LLU- Particle Tracking Silicon Microscope a versatile and inexpensive broad-beam and microbeam particle tracking system for protons and alpha particles wide range of energies (1 MeV - 250 MeV protons) in vitro and in vivo radiobiological studies research studies for radiation therapy and protection support of DOE and NASA low-dose research programs
Gametogenesis in the adult hermaphrodite of C. elegans oocyte eggs in uterus spermatheca gonad vulva
Chromosome structures in the gonad of the adult hermaphrodite
Silicon Strip Detectors in Life Sciences X-rays are difficult for silicon detectors (Eff or Energy low) BUT charged particles are perfect: Combined measurement of position, angle and energy or LET of single particles High spatial resolution (microns) Support high particle rate Wide dynamic energy range Fast response (self-trigger possible) Radiation hardness Adaptation to most geometries Compatibility with physiological conditions of cells Simple operation (e.g. low voltages) No consumables, compact
Specific Experiments to track the damage 244 Cm Alpha Irradiator + SSD Objective Helium Aperture 32 mci 244 Cm Condenser Mylar Bottom Petri Dish He Slider
Particle Tracking Silicon Microscope (PTSM) Protons produce damage AND identify damaged molecule Biological Sample (directly on SSD) Transfer to Automated Microscope when Occupancy ~ 10% Double-sided SSD: x-y coordinate, Energy, Cluster characteristics. Assay with Automated Microscope using stored x-y coordinates
Statistical vs. Specific Radiobiology Global (Nanodosimetry): Well in Hand? Ionization Cluster Spectra Radiation Plasmid Sample Nanodosimeter Incubation with Base Excision Enzymes mobility 0 minutes 15 minutes 30 minutes 60 minutes 120 minutes ssb dsb intact Relative frequency %90 %88 %86 %16 %14 %12 %10 %8 %6 %4 %2 %0 Gel Electrophoresis protons 4 MeV α 5 MeV 0 1 2 3 4 5 6 7 8 9 1011121314151617181920 Cluster size Frequency of lesions of different complexities Local: Needs Improvement No Radiometry Measurement Correlated with Damage on individual DNA Molecule Correlation needed! Tag individual Interaction, Investigate Damage in detail on struck Structures
Particle Tracking Silicon Microscope Conventional radiobiological experiment: statistical random traversal of cells by a broad particle beam only average number of hits per cell is known Particle-tracking radiobiological experiment: specific number of particles per cell is exactly known broad beam or microbeam setup λ = 1.5 P(n) = λ n /n! e -λ SSD n = 2 1 3 0 collimator SSD n = 0 0 3 0
Particle Tracking Silicon Microscope MCM Conceptual design biological targets located on detector surface single-particle tracking energy or LET measurement ASIC and controller design adapted to application dedicated data acquisition system DSSD ASIC RO Control Cables DAQ
Radiation Effects in Life Sciences vs. in Materials Many Similarities: Fluence or Dose Low Dose effects Rate effects LET & NIEL RBE & Kerma Microenvironment: Bystander Effect & Latch-up (SEE) Adaptive Response & Annealing Cell behavior vs. Circuit Living cells are well engineered (many cycles of design rule and VHDL verification!) and have mechanisms to deal with radiation damage: Repair or Apoptosis. In special cases, we will be able to supply specific data from particle-tracking measurements to replace the conventional statistical radiobiological approach.