Life Science
Laser cutting/ablation
Coupling of pulsed lasers to a microscope for selective photomanipulation of samples
Pulsed lasers enable photomanipulation of various structures within tissues and cells, even down to the molecular level, as well as the interference with cellular processes. The selective laser cutting of cellular structures like microtubules or the removal of whole cells by ablation permit to study cell and developmental biology. This technique provides new insights into the architecture of the mitotic spindle, the process of chromosome segregation as well as cell locomotion. Furthermore, it gives new information about origin, fate, or function of individual cells in the developing organism and can also be used for gene activation.
As a primary advantage, laser-based cutting / ablation is a very flexible method that can be performed at any cellular site, in any cell pattern and at any time in development within living tissues and cells. It reaches submicrometer resolution thus allowing to target specific cell organells. The ablation efficiency depends on the fluorecent label. Efficient ablation requires pulsed laser excitation to reach high peak intensities with moderate average power. In this way, cell damage is minimized in untreated regions. In addition, the heat generation is significantly reduced due to the short pulse duration. Thus, by using pulsed laser light, the necessary ablation illumination times are very short without causing visible collateral damage.
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For laser cutting or ablation experiments, a pulsed laser must be connected to the Laser Scanning Microscope (LSM). This interfacing is currently realized for the Olympus FluoView FV1000 / FV1200 via the standard or the SIM scanner. There are in principle three different options to couple the pulsed lasers to the LSM:
- Direct connection of one laser to the SIM scanner. This configuration is usually used for laser cutting with pulsed excitation at 375 nm or 405 nm. It enables to micromanipulate living cells or tissues and simultaneously analyze the consequences via imaging.
- Connection of several pulsed lasers to the SIM scanner. This allows for synchronous laser cutting / bleaching and uninterrupted, fast live cell FLIM imaging. This setup requires suited ADM beamsplitters for the overlay of the SIM and main beam path in the LSM. In this way it is possible to photo-manipulate one region-of-interest (ROI) and measure FLIM in another area when the excitation is carried over larger distances.
- A third configuration uses pulsed diode laser at 375 nm or 405 nm that are coupled to both SIM and standard scanner. This is realized by using a dedicated switching option in the PicoQuant Laser Combining Unit (LCU) along two fibers.
The LSM FluoView software allows to activate the pulsed lasers and to adjust the scan range. Confining the scanning or bleaching area to a defined ROI requires an exact timing of the PicoQuant pulsed diode laser, which is realized by externally gating the lasers (i.e. switching “on” and “off”) on a nanosecond time scale. Thus, the pulsed lasers excite the sample only within the ROI, avoiding photo damage in other regions of the sample. In order to integrate the gating option of the pulsed lasers into the Olympus FV1000 / FV1200 setup, special electronic modules were developed by Olympus and PicoQuant. Using the Olympus external laser control module FV10-LDIF and the Trigger Module Fastgate TMF400, the fast gating can be precisely controlled by the Olympus LSM software laser management system allowing for gating times of less than 10 ns, i.e. in between two laser pulses.
Related technical and application notes:
LSM Upgrade Kit
