Laser Nanosurgery

Originally built at EMBL in 2003, the Laser Nanosurgery platform was developed to perform cellular and subcellular ablation
in living cells and organisms under fluorescence excitation. The system is available at IRB Barcelona since 2009.

The combination of a pulsed UV laser, a laser scanning device and an optimized optical path for fluorescence excitation and diffraction
limited beam focussing has brought many applications for the study of cellular processes in living cells.
The system is versatile, includes a dedicated CW laser beam for combined  FRAP and Nanosurgery, and outperforms commercial systems
which are usually optimized for fixed tissues microsurgery.

 Technical Specifications

 Laser for Nanosurgery

Q-switched tripled Nd:YAG   
 Wavelength:            355 nm
 pulse width:             470 ps
 Repetition Rate:       1000 Hz
 Energy per pulse:      max. 10 µJ
 Average Power:        max. 10 mW
 Peak Power:             max. 20 kW
 JDS Uniphase (today: Teemphotonics)
 Laser for F.R.A.P.
Multiline Argon laser
 Wavelengths (nm):   456, 476, 488, 501, 514
 Power:                    max. 150mW @ 514 nm
 National Laser
 Lasers Power control
  special AOTF for UV (350-400nm)
   AOTF 8 channels for VIS range 400 - 700nm
 AA optoelectronics
 Laser Scanning
Fast Galvo-mirrors   
 XY10 + MiniSax galvos, +/- 6 degrees
 GSI Lumonics
 Focus control
 Piezo Objective stage 0-100µm
 Physik Instrumente
 Objective lens
 63x / 1.2 Water Immersion
 working distance 280 µm
 Carl Zeiss
 Fluorescence Filters

 Excitation Filters:
 Emission Filters:
 AHF AnalysenTechnik

 Stage Insert for glass-bottom 35mm Petri-dish,                                     slides, multiwell plates.
 Temperature control:     Heat from 26ºC to 45ºC
                                     Cool  from 0ºC to 10ºC
 CO2                              from 0% to 10%.

 Known Applications

 Application Details Example publications
 Cell/Organ  Ablation
 Single or multiple cell(s) ablation by plasma formation
 2 mechanisms:
 - Cell membrane perforation, instant cell removal. large action radius.
 - Cell Apoptosis induction, slow process. Confined to cell volume.
Hoijman et al Nat Commun (2015)
Saias et al  Dev Cell (2015)
Pujol-Martí et al Curr Biol (2014)
Brugues et al, 2014
Jekely et al, Nature 2008.
 Solon et al, Cell 2009.
 Caussinus et al, Curr Biol 2008.

 Subcellular Ablation
  Confined damage of subcellular structures and organelles
 Ex: Actin Stress Fibers, Microtubules, Mitochondria, Golgi compartments.
Vignaud et al JCS 2012
Colombelli et al, Traffic 2005, JCS 2009.

 Cell fission    
  Separation of a cell in two parts: Karyoplast and Cytoplast.
 - specific to adherent cells (cytoskeletal tension required)
 Taengemo et al, JCS 2011.
 DNA Damage
 Double Strand Breaks (DSBs) in cultured cells nuclei
 Roukos et al , JCS 2011
 Hard Material/Glass Ablation
 Inscription inside material volumes by plasma-ablation, confined in 3D.
 Data storage possible.
 2 levels:
    - change in refractive index, very confined (about 450x450x700nm)
    - Microexplosion, larger effect (from 1 to 10µm).
 Colombelli et al, RSI 2004.
 Correlative Microscopy
 Use the possibility to inscribe specific landmarks into the coverslip
 to find a specific cell after removal of the sample for fixation.
 2 possible modes:
    - Correlative Light Microscopy with Electron Microscopy.
    - Correlative Live Microscopy with Immunolabelling Microscopy. 

 Colombelli et al, JCS 2009.
 Colombelli et al, Meth in Cell Biol 2008.
 Taengemo et al, JCS 2011.