Nanoparticles for in-vitro and in-vivo biosensing and imaging

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4 CONTENTS 4.9 Fluorescence Spectroscopy: Burst analysis . . . . . . . . . . . . . . . . . . 114 4.9.1 Aggregates size estimate . . . . . . . . . . . . . . . . . . . . . . . . 114 4.9.2 Excited state lifetime in the presence of BSA . . . . . . . . . . . . 115 4.10 Basic gold nanocrystal protein sensor . . . . . . . . . . . . . . . . . . . . . 120 4.11 p53 protein detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 4.12 Fluorescence burst analysis in solution . . . . . . . . . . . . . . . . . . . . 121 4.13 Dependence of the FITC Lifetime on the p53 Concentration . . . . . . . . 123 4.14 In Vitro Selectivity of the p53 Assay . . . . . . . . . . . . . . . . . . . . . 125 4.15 In Vivo Test of the p53 Assay . . . . . . . . . . . . . . . . . . . . . . . . . 126 4.16 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 5 Anisotropic nanoparticles 132 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 5.2 Gold nanoparticles luminescence . . . . . . . . . . . . . . . . . . . . . . . 133 5.2.1 Two-photon luminescence (TPL) . . . . . . . . . . . . . . . . . . . 135 5.3 Experimental details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 5.3.1 Nanoparticles synthesis . . . . . . . . . . . . . . . . . . . . . . . . 137 5.3.2 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 5.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 5.4.1 UV-Vis characterization . . . . . . . . . . . . . . . . . . . . . . . . 139 5.4.2 Transmission Electron Microscopy (TEM) characterization . . . . 141 5.4.3 Z-potential characterization . . . . . . . . . . . . . . . . . . . . . . 144 5.5 Dynamic Light Scattering characterization . . . . . . . . . . . . . . . . . . 144 5.5.1 Solutions of LSB (LSB micelles) . . . . . . . . . . . . . . . . . . . 144 5.5.2 Dynamic light scattering of the NPs . . . . . . . . . . . . . . . . . 146 5.6 FCS experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 5.7 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 5.8 Concentration evalutation . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 5.9 TPL dependence on excitation power . . . . . . . . . . . . . . . . . . . . . 157 5.10 Emission Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 5.11 Excitation spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 5.12 Dependence of TPL on the Polarization of the exciting field light beam . 161 5.12.1 Citotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 5.13 Cellular uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 5.14 Photothermal effects of gold NRs . . . . . . . . . . . . . . . . . . . . . . . 171 5.15 Experimental details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 5.15.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 171
CONTENTS 5 5.15.2 Spectral characterization . . . . . . . . . . . . . . . . . . . . . . . 173 5.15.3 Fluorescence Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 173 5.15.4 Dye-Lifetime measurement . . . . . . . . . . . . . . . . . . . . . . 173 5.16 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 5.17 Rhodamine-B characterization . . . . . . . . . . . . . . . . . . . . . . . . 174 5.17.1 Fluorescence emission measurement . . . . . . . . . . . . . . . . . 174 5.17.2 Lifetime measurement . . . . . . . . . . . . . . . . . . . . . . . . . 175 5.18 Assay characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 5.19 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 6 In-vivo microscopy 182 6.1 Intravital microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 6.2 Problems in IVM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 6.3 The biological problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 6.3.1 NK-DC interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 6.3.2 Sample preparation methods . . . . . . . . . . . . . . . . . . . . . 191 6.4 Data acquisition and analysis . . . . . . . . . . . . . . . . . . . . . . . . . 192 6.5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 6.5.1 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 192 6.5.2 Lymph nodes topography . . . . . . . . . . . . . . . . . . . . . . . 200 6.5.3 Validation of the experimental setup performances: T and DC cells 201 6.5.4 NKs in steady state conditions . . . . . . . . . . . . . . . . . . . . 202 6.6 DC-NK cells dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 6.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 6.8 Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 6.9 Immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 6.10 PRRs and control of adaptive immunity . . . . . . . . . . . . . . . . . . . 218 6.10.1 TLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 6.11 Dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 6.12 Natural Killer cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 6.13 Lymph-nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 6.13.1 Lymph-node architecture . . . . . . . . . . . . . . . . . . . . . . . 224 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 6.14 Laser sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 6.14.1 Millennia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 6.14.2 Titanium-Sapphire (Ti-Sa) optical resonant cavity . . . . . . . . . 236 6.15 Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 6.15.1 Nikon TE300 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Page 1 and 2: UNIVERSITÀ DEGLI STUDI DI MILANO B
Page 3: CONTENTS 3 3.2 Dynamic Light scatte
Page 7 and 8: Introduction In the last two decade
Page 9 and 10: Chapter 9 then we have applied the
Page 11 and 12: Chapter 1 Metal nanoparticles 1.1 N
Page 13 and 14: Chapter 1 13 carry out reactions in
Page 15 and 16: Chapter 1 15 donor and the metal at
Page 17 and 18: Chapter 1 17 Figure 1.4: Surface pl
Page 19 and 20: Chapter 1 19 where the scattering a
Page 21 and 22: Chapter 1 21 screens the surface ch
Page 23 and 24: Chapter 1 23 easily be seen from eq
Page 25 and 26: Chapter 1 25 The first is equal to
Page 27 and 28: Chapter 1 27 dipole approximation a
Page 29 and 30: Chapter 1 29 used for disease or ca
Page 31 and 32: Bibliography [1] Nanotoxicology: na
Page 33 and 34: Chapter 1 33 [30] Bohren CF; Huffma
Page 35 and 36: Chapter 2 Principles 2.1 Basics of
Page 37 and 38: Chapter 2 37 A direct consequence o
Page 39 and 40: Chapter 2 39 be considerably more c
Page 41 and 42: Chapter 2 41 Figure 2.5: Effect of
Page 43 and 44: Chapter 2 43 2.4 Two-photon excited
Page 45 and 46: Chapter 2 45 m = λV MI hc 2 N A (2
Page 47 and 48: Chapter 2 47 P SF OP E (u, v) = |h(
Page 49 and 50: Chapter 2 49 renditions of the spec
Page 51 and 52: Bibliography [1] Lakowicz J.R. In p
Page 53 and 54: Chapter 2 53 [29] Born M. and Wolf
Page 55 and 56:
Chapter 3 55 3.2 Dynamic Light scat
Page 57 and 58:
Chapter 3 57 where n is the refract
Page 59 and 60:
Chapter 3 59 with g V H (t) ∝ β
Page 61 and 62:
Chapter 3 61 κ m (Γ) = dm K(−τ
Page 63 and 64:
Chapter 3 63 that plagues conventio
Page 65 and 66:
Chapter 3 65 set equal to the new p
Page 67 and 68:
Chapter 3 67 the molecules. In orde
Page 69 and 70:
Chapter 3 69 number of molecules in
Page 71 and 72:
Chapter 3 71 γ 1 G(τ) = V ex 〈C
Page 73 and 74:
Chapter 3 73 Figure 3.2: Auto-corre
Page 75 and 76:
Chapter 3 75 electronic distortions
Page 77 and 78:
Chapter 3 77 Figure 3.5: left panel
Page 79 and 80:
Chapter 3 79 p 1 (k, V 0 , ɛ) = 1
Page 81 and 82:
Chapter 3 81 3.9.3 Photon Moment An
Page 83 and 84:
Chapter 3 83 Figure 3.6: Schematic
Page 85 and 86:
Bibliography [1] Chu B. In Laser Li
Page 87 and 88:
Chapter 3 87 [27] Eigen S.M. and Ri
Page 89 and 90:
Chapter 3 89 [53] Chen Y., Tekmen M
Page 91 and 92:
Chapter 4 91 • L.Sironi, S.Freddi
Page 93 and 94:
Chapter 4 93 die. Mutations in the
Page 95 and 96:
Chapter 4 95 arm of chromosome 17.
Page 97 and 98:
Chapter 4 97 shown that one of the
Page 99 and 100:
Chapter 4 99 DNA replication while
Page 101 and 102:
Chapter 4 101 4.2). The ratio R = [
Page 103 and 104:
Chapter 4 103 The polydispersity of
Page 105 and 106:
Chapter 4 105 The fitting of the FC
Page 107 and 108:
Chapter 4 107 the streptavidin (4.5
Page 109 and 110:
Chapter 4 109 29 nm for the 10 nm s
Page 111 and 112:
Chapter 4 111 A 1 Γ 1 (nm) λ 1 (n
Page 113 and 114:
Chapter 4 113 Figure 4.8: (Left: Fl
Page 115 and 116:
Chapter 4 115 been systematically i
Page 117 and 118:
Chapter 4 117 Figure 4.12: Left: Me
Page 119 and 120:
Chapter 4 119 at the basis of the o
Page 121 and 122:
Chapter 4 121 presence of p53, comp
Page 123 and 124:
Chapter 4 123 Figure 4.17: The figu
Page 125 and 126:
Chapter 4 125 from probably aspecif
Page 127 and 128:
Chapter 4 127 Figure 4.21: Panel A:
Page 129 and 130:
Bibliography [1] Anker J. N.; Hall
Page 131 and 132:
Chapter 4 131 [47] Das P.; Metiu H.
Page 133 and 134:
Chapter 5 133 In this chapter the c
Page 135 and 136:
Chapter 5 135 Figure 5.1: Left:Symm
Page 137 and 138:
Chapter 5 137 5.3 Experimental deta
Page 139 and 140:
Chapter 5 139 back aperture of the
Page 141 and 142:
Chapter 5 141 5.4.2 Transmission El
Page 143 and 144:
Chapter 5 143 For anisotropic branc
Page 145 and 146:
Chapter 5 145 coefficient D is then
Page 147 and 148:
Chapter 5 147 Figure 5.9: ACF of th
Page 149 and 150:
Chapter 5 149 measured through a ve
Page 151 and 152:
Chapter 5 151 objective in epifluor
Page 153 and 154:
Chapter 5 153 The fitting of FCS cu
Page 155 and 156:
Chapter 5 155 Sample Population (%)
Page 157 and 158:
Chapter 5 157 5.8 Concentration eva
Page 159 and 160:
Chapter 5 159 Figure 5.17: Emission
Page 161 and 162:
Chapter 5 161 5.12 Dependence of TP
Page 163 and 164:
Chapter 5 163 Figure 5.22: Histogra
Page 165 and 166:
Chapter 5 165 5.13 Cellular uptake
Page 167 and 168:
Chapter 5 167 Figure 5.29: NPs obta
Page 169 and 170:
Chapter 5 169 Figure 5.33: Autofluo
Page 171 and 172:
Chapter 5 171 5.14 Photothermal eff
Page 173 and 174:
Chapter 5 173 Figure 5.37: ζ-poten
Page 175 and 176:
Chapter 5 175 Temperature [ ◦ C]
Page 177 and 178:
Chapter 5 177 In figure 5.39 the fl
Page 179 and 180:
Chapter 5 179 P [mW] < τ > [ns] 10
Page 181 and 182:
Chapter 5 181 The dependence of the
Page 183 and 184:
Chapter 6 183 Currently, only alumi
Page 185 and 186:
Chapter 6 185 tact organ studies, a
Page 187 and 188:
Chapter 6 187 with liquid flow chan
Page 189 and 190:
Chapter 6 189 Figure 6.1: DC in clo
Page 191 and 192:
Chapter 6 191 • New contrast agen
Page 193 and 194:
Chapter 6 193 experiments by flowin
Page 195 and 196:
Chapter 6 195 it has discontinuitie
Page 197 and 198:
Chapter 6 197 P t. rection. If all
Page 199 and 200:
Chapter 6 199 Figure 6.6: Commonly
Page 201 and 202:
Chapter 6 201 Figure 6.9: 3D and 2D
Page 203 and 204:
Chapter 6 203 Figure 6.11: (A) NK c
Page 205 and 206:
Chapter 6 205 are somehow confined,
Page 207 and 208:
Chapter 6 207 Figure 6.15: Distance
Page 209 and 210:
Chapter 6 209 Figure 6.18: (A)The s
Page 211 and 212:
Chapter 6 211 Figure 6.21: The figu
Page 213 and 214:
Chapter 6 213 Figure 6.24: Paramete
Page 215 and 216:
Chapter 6 215 of the natural killer
Page 217 and 218:
Chapter 6 217 and CD8 + T cells, is
Page 219 and 220:
Chapter 6 219 6.10.1 TLRs TLRs are
Page 221 and 222:
Chapter 6 221 arise from myeloid pr
Page 223 and 224:
Chapter 6 223 of infection, primari
Page 225 and 226:
Chapter 6 225 Figure 6.30: Lymph-no
Page 227 and 228:
Bibliography [1] A. Diaspro, G. Chi
Page 229 and 230:
Chapter 6 229 [25] E. O. Long. Regu
Page 231 and 232:
Chapter 6 231 [47] S. H. Wei, M. J.
Page 233 and 234:
Chapter 6 233 [72] S. Fossum and W.
Page 235 and 236:
Chapter 235 mode-locked cavity. Thi
Page 237 and 238:
Chapter 237 Figure 6.34: Energy lev
Page 239 and 240:
Chapter 239 Figure 6.37: Prisms seq
Page 241 and 242:
Chapter 241 part of the TE300 and t
Page 243 and 244:
Chapter 243 Figure 6.42: The Olympu
Page 245 and 246:
Chapter 245 a f=100 mm bispherical
Page 247 and 248:
Chapter 247 are just events’ dete
Page 249 and 250:
Chapter 249 smaller quartz cell (He
Page 251 and 252:
Chapter 251 needed to a linear corr
Page 253 and 254:
Chapter 253 Figure 6.48: Channel ar
Page 255 and 256:
Chapter 255 ming for burst height a
Page 257:
Chapter 257 • M. Caccia, T. Gorle
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