Jornada científica NanoFrontMag - 21 de junio de 2016 - IMDEA Nanociencia Presentación grupo GBE-CSIC José L. Carrascosa

1. Objetivo 6
Análisis de la influencia de de los mecanismos de internalización de NPM
en diferentes líneas celulares tumorales sobre sus propiedades magnetotérmicas
Actividad 2
Estudios de internalización y distribución intracelular de NPMs
Grupo GBECNB
José L. Carrascosa

2. Superparamagnetic OD15 Vs MCF-7
MCF7 +
[0,5μg/μl] SPIONs
24h
MCF7 human
breast cancer cells
control
Calero, M. Chiappi, M. et al., J.Bionanotech. ,
2015

3. ≤200 nm
CLATHRIN-MEDIATED
ENDOCYTOSIS
Macropinosome
Early endosome
Late endosome
(multivesicular bodies)
Lysosome
Clathrin-coated
vesicles
200 nm
200 nm
MACROPINOCYTOSIS
200 nm
200
nm
GOLGI NETWORK
pH 6.2
pH 5.2
Calero, M. Chiappi, M. et al., J.Bionanotech. , 2015

4. Cryo-Soft X-ray tomography
SXT
Super-Resolution
Confocal
cryo-ET
SXT
Resolution in Z (nm)
Thickness(μm)

5. ALBA - MISTRAL
Xray-source
CCD
Zone Plate
Objective diffractive lens
Central stop
Capillary
condenser
BESSY II - U41-TXM
Cryo-Soft X-ray tomography

6. CRYO EPI-FLUORESCENT
microscopy
cryo-stage
XRAY CRYO-
TOMOGRAPHY
MCF7 CELL
CULTURE+SPIONs
ON FINDER GRIDS &
Plunge freezing
BESSY II U41-TXM
Grid design
ALBA MISTRAL
EM Grid design
Soft X-ray cryo-tomography Workflow
Plunge freezing
10x transmited light on-line

7. VLM-SXTM Correlative workflow

8. • There is an Increase in volume occupied by endocytic organelles containing SPION
• The nanoparticles travel from the exterior to a perinuclear area
• The size of the endocytic organelles containing SPION increases with incubation time, but cellular division
alters this progression
Cryo-Soft X-ray tomography

9. Energy Stack: 700 to 730 eV
700 eV; Pre-edge 709 eV; L3 iron edge 720 eV; L2 iron edge 730 eV; Post-edge
O.D.

10. 700 eV 709 eV
Ratio (709/700) projection image
Iron chemical map (3D volume)
2D projection images
X-ray mosaicin vivo fluorescent
2 µm2 µm
8 µm25 µm
Near Edge Absorption Soft X-ray Tomography
Conesa JJ. et al., Scientific Reports (2016)

11. 2 µm500 nm
Conesa JJ. et al., Scientific Reports (2016)
Near Edge Absorption Soft X-ray Tomography

12. Structure of Macromolecular
Complexes
José L. Carrascosa
Javier Chichón
María José Rodríguez
Michele Chiappi
Javier Conesa
Unidad de Biocomputación
Carlos O. Sanchez
Joaquín Oton
ALBA, Barcelona
Eva Pereiro
Instituto de Ciencia
de Materiales de Madrid
Puerto Morales
IMDEA Nanociencia
Gorka Salas