Nanodissection With Femtosecond Laser Technology

Analysis of cells and cellular processes in biological and medical research requires systems for imaging as well as tools for manipulating and dissecting cells and subcellular structures. Femtosecond laser technology is applicable for both. Imaging using femtosecond lasers is known as multiphoton microscopy, and is already well established. Near-infrared radiation is also very well suited for gentle manipulation of living cells and subcellular structures. Different research groups are engaged in using ultrashort laser pulses for nanodissection; however, only a few commercial systems are available, including the system described here. The CellSurgeon laser nanodissection system (Rowiak GmbH, Hanover, Germany) has been developed in collaboration with the Medical Technology Department of the Laser Centre Hanover (Germany). It can be used to manipulate cell dynamics, deactivate cell organelles, or influence cellular processes such as metabolism and apoptosis. An overview of the technology follows, including information on its potential for two different applications.

Principle and advantages

The main component of the CellSurgeon system is a tunable NIR femtosecond laser coupled to a microscope objective. Depending on the configuration, the pulse repetition rate varies between several kilohertz and 90 MHz. The laser is moved by software-controlled scan mirrors, enabling various cutting geometries such as single point and lines as well as predefined or freehand shapes. A three-axis piezo-driven microscope stage permits positioning of samples with a resolution of 20 nm in each direction.

Figure 1 - Nanodissection is induced within the focal volume of a tightly focused femtosecond laser.

Nanodissection with ultrashort pulses is based on nonlinear absorption effects, which occurs when high laser intensities are confined to a very small volume (Figure 1). This is realized by tight focusing of the laser beam using high-numerical-aperture objectives. If the energy per laser pulse increases above a certain threshold, the photon density in the focal volume rises high enough to induce multiphoton absorption and avalanche ionization. These processes lead to a very high concentration of free electrons within the focal region, resulting in plasma-mediated ablation of material.1

Depending on the numerical aperture of the microscope objective and the laser pulse energy, the lateral extent of the focal volume and thus the interaction with the sample can be limited to less than 1 μm. That allows the manipulation or dissection of single-cell organelles, which have a typical size of a few micrometers. For precise dissection, the energy has to be as low as possible, while the numerical aperture of the objective has to be high. One key advantage of femtosecond lasers is that at a pulse duration of 140 fsec, only a few nanojoules of energy are necessary for dissection. In contrast, dissection with UV laser systems requires energies that are at least two or three orders of magnitude higher. The low laser energies allow very precise cuts with minimum dimensions of approx. 100 nm and cause negligible collateral damage, reducing the risk of injuring or killing the cell.

Applications

The laser nanodissection system is especially suited for applications in medical and biological research. Manipulation and dissection of cells or subcellular structures can be useful for investigations of cellular processes such as metabolism, apoptosis, or cell dynamics. An understanding of cellular and subcellular mechanisms is important for the development of drugs and stem cell or gene-based therapies against diseases such as cancer or Alzheimer’s and Parkinson’s disease.

Two examples of experiments are given in order to help the reader visualize potential applications. All experiments were conducted at the Laser Centre Hanover by the Biophotonics Research Group (part of the Biomedical Optics Dept. of the Laser Centre Hanover).

Figure 2 - Fluorescence image of endothelial cells. Left: cells before manipulation; single mitochondrion to be ablated is marked by yellow circle. Right: cells after manipulation; the ablation was realized at a pulse energy of 1 nJ, and the ablated mitochondrion is not visible. (Figure courtesy of J. Baumgart, Laser Centre Hanover; reproduced with permission from Ref. 2.)

The first experiment demonstrates the deactivation of specific cell structures. In this case, mitochondria in living endothelial cells were disrupted to study the induction of cell death. The mitochondria disruption was realized at pulse energies between 0.7 and 1.0 nJ and with a cutting speed of 14 mm/sec at a pulse repetition rate of 90 MHz. Fluorescence imaging was used to investigate changes of the mitochondria arrangement. For this microscopic examination, the cells were stained with MitoTracker Orange (Molecular Probes, Eugene, OR). Figure 2 shows an example of a treated endothelial cell before and after the disruption of a single mitochondrion. To receive a first impression of the viability after the laser manipulation, the treated cells were observed over a period of 1 hr with both fluorescence imaging and brightfield microscopy imaging. Usually, apoptotic or necrotic cells change their volume, which can be seen under the brightfield microscope. In none of the studies did the ablation of a single mitochondrion lead to the induction of cell death during the observation time. Certainly, further study with longer observation time is required to draw a solid conclusion. First results, however, are promising that cell organelles can selectively be ablated by laser nanodissection without inducing further cell damage or even cell death.2

Another possible application of laser nanodissection is the optical perforation of cell membranes to enable the transfer of genes into living cells. By focusing the laser beam for 20–40 msec on the membrane, transient pores are created so that fluorescent molecules or nucleic acids can enter into the cell. Conventional techniques to permeabilize the cell membranes involve the use of viral vectors, chemical carriers, or electroporation. However, the application of these techniques for primary cells or stem cells, especially with limited populations, can be critical or impossible due to low success rate or severe side effects. For these cell types, optical membrane perforation provides an interesting alternative because it is a very gentle and precise method, allowing targeted perforation of single cells.

DNA transfection by laser nanodissection requires a detailed examination of the laser parameters for the cell type used. The motivation for the following set of experiments was to find out the optimum laser parameters for membrane perforation in order to induce an uptake of fluorescent molecules, more precisely propidium iodide (PI). This fluorochrome is commonly used to stain DNA and to differentiate necrotic, apoptotic, and normal cells.

Figure 3 - Opto-perforated granulosa cells in the presence of PI. a) Fluorescence image of granulosa cells during opto-perforation; the treated cells are highlighted by the dashed circles. The cells were treated at a pulse energy of 0.9 nJ and an irradiation time of 40 msec. All manipulated cells are fluorescent. b) Brightfield image of the same cells. c) Fluorescence image of the cells after 90-min incubation in PBS. The cells were restained with PI to verify the viability. The cell pointed out by the arrow is representative for a cell whose membrane is damaged and therefore still permeable for the fluorophore. d) Brightfield image after the incubation time. (Figure courtesy of J. Baumgart, Laser Centre Hanover; reproduced with permission from Ref. 3.)

The aim of the experiments was to analyze the relationship between pulse energy, irradiation time, repetition rate, and transfection efficacy as well as the viability of the cells. For the laser manipulation, a coverslip with GFSH-R17 granulosa cells of rat was transferred into a perfusion chamber containing 0.5 mL of phosphate-buffered saline (PBS) and 1.5 µm PI. By focusing the laser with a 0.8-NA NIR water immersion objective (Achroplan, Carl Zeiss AG, Jena, Germany), the focus had a theoretical size of approx. 600 nm. The cells were perforated at a central wavelength of 800 nm and a repetition rate of 90 MHz. Perforation of the granulosa cells was realized at pulse energies in the range of 0.7–1.1 nJ at irradiation times between 30 and 60 msec. Every parameter combination was tested with 40–60 cells. After manipulation, the cells were observed by fluorescence microscopy to verify the induced fluorescence of the PI, and then washed with PBS and incubated in PBS for 90 min. Finally, the viability of the treated cells was controlled by relabeling with PI and comparing the fluorescence intensity before and after restaining. Cells with an intact physiologic structure are impermeable for PI molecules. They show only very low fluorescence, emitted by fluorophores uptaken directly after the optoperforation. In contrast, the membrane of damaged cells becomes completely permeable for fluorophores and can be identified by its higher fluorescence intensities (Figure 3).

The efficiency of dye uptake increased with the pulse energy and the irradiation time, while the viability observed 90 min after the treatment decreased. In detail, the viability varied between 20 and 90% and the efficiency between 10 and 90%. The best compromise between efficiency and viability was found at a pulse energy of 0.9 nJ and an irradiation time of 40 msec. These parameters led to an efficiency of 70% and a viability of 80%. Certainly, this is a satisfactory result.

As a proof of principle, these optimized parameters were successfully used to transfect canine mammary cells (MTH53a) with a vector coding for a GFPHMGB1 fusion protein. The transfected cells were observed up to 48 hr after the treatment and the cells did not show any signs of apoptosis or necrosis.3 These first results display the potential of laser nanodissection for cell transfection.

Summary

The CellSurgeon is a versatile tool that can open new horizons for scientists who are primarily interested in the investigation of cells and cellular processes. Nanodissection with femtosecond lasers can help solve various problems in cell and molecular biology as well as in medicine or pharmacy. Cells, their organelles, and structures can be selected with high accuracy and high reproducibility. The technology allows targeted manipulation of cell parts without damaging others. This considerably improves the success rate of cell manipulation and thus the efficiency of laboratory work.

References

  1. Vogel, A.; Noack, J.; Huettman, G.; Paltauf, G. Mechanisms of femtosecond laser nanosurgery of cells and tissues. Appl. Phys. B 2005, 81(8), 1015–47.
  2. Heisterkamp, A.; Baumgart, J.; Maxwell, I.Z.; Ngezahayo, A.; Mazur, E.; Lubatschowski, H. Fs-Laser Scissors for Photobleaching, Ablation in Fixed Samples and Living Cells, and Studies of Cell Mechanics. In Laser Manipulation of Cells and Tissues; Elsevier Inc.: New York, NY, 2007.
  3. Baumgart, J.; Bintig, W.; Ngezahayo, A.; Willenbrock, S.; Murua Escobar, H.; Ertmer, W.; Lubatschowski, H.; Heisterkamp, A. Femtosecond laser based opto-perforation of living GFSHR-17 and MTH53a cells. Optics Express 2008, 16, 3021–31. www.opticsinfobase. org/abstract.cfm?URI=oe-16-5-3021.

Ms. Menne is with Rowiak GmbH, Garbsener Landstrasse 10, D-30419 Hanover, Germany; tel.: +49 511 277 2952; fax: +49 511 277 2959; e-mail: pm@rowiak.de.

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