Quantitative optical nanoscopy of infrared absorbing nano-markers : Toward colon cancer tissue microarrays characterization

Transcription

1 Laurent COGNET Institut d optique, CNRS, Univ. Bordeaux Quantitative optical nanoscopy of infrared absorbing nano-markers : Toward colon cancer tissue microarrays characterization E. S. Shibu 1, Z. Gao 1, N. Varkentina 1, I. Soubeyran 3, F. Ichas 2, F. De Giorgi 2, B. Lounis 1, L. Cognet 1 1 Institut Optique - LP2N, CNRS and Université de Bordeaux 2 Fluofarma, Pessac 3 Institut Bergonié, Bordeaux

2 Microscopy is at the heart of cancer diagnostics L anatomo-pathology : visual inspection of tissue/cell morphologies hematoxilin and eosine staining Tumeur intestinale Immunohistochemistry : functionnal analysis of the presence of deregulated proteins enzyme immuno-labelling + enzymatic amplifications or Immunofluorescence Antigene of cancer target Cell or tissue Exemple: Chromogranine A. 2

3 Beyond medical diagnostics Immunohistochemistry for guiding therapeutic choices Towards personalized therapies - Issue of efficiency and comfort of treatments - Economic issue (cost of a targeted therapy / year: 50k *) *Source : Centre de soutien et d accompagnement thérapeutique aux personnes atteintes du cancer, 27/04/2010 Immunohistochemistry for the pharmaceutical industry Development of medicines - 1 in screened molecules leads to a drug, one third will pay - Average time on the market 12 years - Average cost of 800 million development / molecule (x10 in 20 years) * *Source : Les industries de médicament, 03/05/2006 3

4 Current methods of immunohistochemistry (based on fluorescence or enzymatique staining) are neither quantitative, nor reproducible Non linear signals, photobleaching, non specific signals etc enzyme Antigene Of cancer target Antigene of cancer target Cell or tissue Cell or tissue Innovative optical microscopy based on particle imaging are needed 4

5 Photothermal imaging using gold nanoparticles The idea: replace fluorescent molecules by gold nanoparticles detected by photothermal heterodyne imaging Antigene Of cancer target Cell or tissue Optical image of 10 nm gold nanoparticles Compatibility with standard sample preparations Images rendering identical to conventional immunohistochemistry Easy comparison with standard protocols 5

6 First tests on colon cancer tissues (TMA) (p53 gold labelling) White light Photothermal imaging (control) Intrinsic signals from tissues provide deleterious background 6

7 Replace gold nanoparticles by NIR absorbers Tiny gold nanorods (1) Need to be produced below 10nm (2) Compare with spherical gold nanoparticles Ultrashort carbon nanotubes 1 dimensional object with NIR absorption Diameter : 1-5 nm Length: 300 nm to 10s of µm (1) Need to be shortened: ultrashort CNTs (2) Can we detect them with photothermal imaging? 7

8 Production of tiny gold nanorods Shibu et al to be submitted Mono-disperse samples of tiny gold nanorods 8

9 Dual color / NIR photothermal microscope 9

10 Imaging individuel tiny gold nanorods in cells Shibu et al to be submitted NIR imaging of tiny gold nanorods reduces intrinsic cellular background in PhI imaging 10

11 Project NanoDiag: Replace gold nanoparticles by NIR absorbers Tiny gold nanorods (1) Need to be produced below 10nm (2) Compare with spherical gold nanoparticles Ultrashort carbon nanotubes 1 dimensional object with NIR absorption Diameter : 1-5 nm Length: 300 nm to 10s of µm (1) Need to be shortened: ultrashort CNTs (2) Can we detect them with photothermal imaging? 11

12 Production of Ultrashort CNTs 10 mm x 10 mm Nanotube shortening and length sorting Length characterization (AFM) 12

13 yposition (µm) Cumulative Frequency yposition (µm) yposition (µm) PhI Signal yposition (µm) yposition (µm) Detection of Ultrashort CNTs (a) C04 toto046.dat C1 C06 toto024.dat mm x 10 mm C Gao et al Sci. Rep. accepted AFM length characterization vs Photothermal imaging C08 toto077.dat 45 0 C10 toto030.dat xposition (µm) xposition (µm) C C CoMoCAT C4 C5 76 C14 toto030.dat xposition (µm) xposition (µm) C xposition (µm) C1 C2 C3 C4 C PhI signal (b) C2 C3 C1: 13 nm AFM Length (nm) 13

14 Imaging individuel ultrashort CNTs in cells 5 µm 5 µm 10 µm 10 µm Ultrashort NTs: 4h incubation, 37 C, 5% CO 2 Control: no nanotubes 14

15 NIR nano-absorbers Photothermal imaging using gold nanoparticles The idea: replace fluorescent molecules by NIR nano-absorbers detected by photothermal heterodyne imaging Tiny gold nanorods Antigene Of cancer target Ultrashort carbon nanotubes Cell or tissue Small nano-absorbers (<20 nm) for good penetration in fixed tissues Compatibility with standard sample preparations Images rendering identical to conventional immunohistochemistry Easy comparison with standard protocols Work in progress: nanoparticle biofunctionalization 15