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
|Component || Specifications || Company|
| 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
| Laser Scanning|
| 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:|
| AHF AnalysenTechnik|
| Environmental |
| 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%.
| Application|| Details|| Example publications|
| Cell/Organ Ablation|| Single or multiple cell(s) ablation by plasma formation|
- 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
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.
- 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.