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Microtomy with Femtosecond Lasers

BioPhotonics
May 2007
Using a laser to perform microtomy can allow noncontact sectioning of biological tissue.

Peggy Menne, Rowiak GmbH

A microtome is an instrument used to prepare thin sections of human, animal or plant tissue for microscopic examination. Usually, sectioning biological specimens is performed by microtomes that work mechanically. These instruments require that tissue undergo special preparations, such as fixation in formalin or liquid nitrogen and embedding in resin or paraffin to preserve its structure and to gain stability. These treatments can change the biological material in ways that are not always desirable.

A laser microtome is a novel sectioning instrument that can solve this problem. In contrast with conventional microtomes, it cuts with the help of photons, is contact-free and enables the cutting of tissue in its native state. Special preparation techniques are not required.

The heart of the laser microtome is a femtosecond laser that emits light in the near-infrared range. Ultrafast laser technology has become much easier to use because of the recent rapid development of commercially available turnkey laser systems.

The near-infrared range is well suited for processing biological material because most biological tissues have a very low absorption coefficient in this part of the spectrum. Thus, manipulation of tissue can even be done inside the material.

Rowiak_Figure1.jpg

Figure 1. Laser microtomy uses a tightly focused beam to section a tissue specimen.

To generate a cut, the laser beam is tightly focused into the specimen by a high-numerical-aperture objective. Intensities at the focus can rise to 1 TW/cm2. These extreme intensities lead to the ionization of the illuminated material by multiphoton absorption and to the formation of a plasma. The explosive expansion of the plasma causes photodisruption of the material (Figure 1).

If the pulse duration is sufficiently short (100 to 400 fs), only very low pulse energies of ~10 nJ are needed to produce such high intensities in the focal region and to induce multiphoton absorption. Because of the low energy deposition into the tissue, mechanical and thermal side effects are reduced to a minimum, limiting the range of collateral damage to diameters below 1 μm.

The laser microtome is designed as a stand-alone device. Its main component is a high-power femtosecond oscillator emitting ultrashort laser pulses with a wavelength of 1030 nm, a pulse duration of ~300 fs and a pulse repetition rate of about 10 MHz. After propagation of the light through an attenuator, delivery optics and a special coated objective with a 0.6 numerical aperture, pulse energies of up to 100 nJ can be delivered to the sample, which corresponds to an average power of 1 W. However, many types of tissue can be cut with much lower pulse energy.

To produce tissue slices, the laser beam and the specimens are moved simultaneously — the laser beam via a fast scanner and the specimen by a 3-D piezo-driven positioning stage. Users can vary parameters such as pulse overlap, slice thickness and the size of the cutting area. With the current setup, we can cut samples as large as 14 × 14 mm. The optimum speed depends on the properties of the tissue, but a typical speed is 1 mm2/s.

Sectioning with the laser microtome is not as fast as with traditional microtomes, but it is fast enough to process fresh tissue samples in an uncritical time period — an important fact if further physiological investigations are desired. Slices of 5 to 100 μm thickness have been generated. Thinner slices are possible, but handling the tissue slice after separation is more difficult. For light microscopic examinations, section thicknesses of 5 to 10 μm are sufficient in most cases. Thicker slices are especially interesting for tissue cultivation for further investigations or for neurological experiments.

Native tissue

Because no mechanical forces are applied to the sample, native tissue can be processed without being fixed and embedded in resin or paraffin. The only requirement is to prepare small tissue pieces with millimeter-scale dimensions, as defined by the maximum cutting area. The tissue sample must be placed on a conventional microscopic glass slide. A good optical connection between glass slide and tissue surface is necessary to achieve a high cutting quality. Therefore the supported tissue surface must be flat. If needed, a small drop of liquid is helpful for index matching. Saline solutions work, as do most media used for specimen storage or nutrition.

Once the glass slide is placed in the sample holder and the parameters are set, the cutting process can start. The specimen should be centered above the objective. During the cutting, a live video of the sample — more precisely, the cutting plane — is shown. This allows the user to observe whether the procedure is successful or not. A fast-growing field of microbubbles, for example, is typical when photodisruption occurs and, therefore, is a sign that cutting is happening. After the cutting, the tissue slices must be separated manually from the bulk with tweezers. It is recommended that this be done under a stereomicroscope. Finally, the slices are placed on a glass slide and can be stained and covered if necessary.

Fresh tissue sections are not always easy to handle, so the slice separation requires some experience. One of our current research activities is to optimize this work with the help of special adhesives and micro tools.

In general, there is a difference between scattering and nonscattering tissue. Scattering tissue causes photons to be lost while propagating through the tissue; consequently, the cutting process and the bubble formation are less regular than in nonscattering tissue. The pulse energy must be higher to exceed the threshold for cutting, or the pulse overlap must be higher. The latter reduces the cutting speed.

Rowiak_Figure2.jpg
Figure 2. Laser microtomy can be used on the nonscattering tissue of a pig’s eye. A cornea slice 10 to 20 μm thick is shown (left, 10x objective; right, 40× objective).

One example of nonscattering tissue processed by the laser microtome is the corneal tissue of a pig eye (Figure 2). The high level of transparency is ideal for cutting (which is known in corneal surgery).

The system has been used to successfully section scattering tissue as well. For example, good results have been achieved with pig kidneys. The capsule — the smooth outer side of the kidney — was used as an interface to the glass slide. In tests with different settings, the laser pulse energy varied between 80 and 120 nJ and the slice thickness between 5 and 30 μm, and the speed of the translation stage was around 1 to 2 mm/s (Figure 3).

Rowiak_Figure3.jpg
Figure 3. The technique also can be used on scattering tissue such as from a kidney. Parts of the tubulus system are visible in the top left image of a kidney slice from a pig (~20 μm thick, 10× objective). The thinner slice on the top right (20 μm, 40× objective) shows connective tissue with emerging single-cell nuclei. The bottom images compare a kidney slice 10 to 20 μm thick made with laser microtomy (left) with a paraffin slice of the same tissue sample that was prepared with a mechanical microtome (right). In conventional microtomy, the first slices cannot be used in most cases; these slices came from regions deeper in the sample than the slices that were created via laser microtomy; therefore, the tissue structure is somewhat different. In the paraffin slices, the glomeruli, for example, can be seen. Because the laser microtome slices are on glass slides, covering them is much more difficult, and the slices do not appear as even as the conventionally prepared ones.

We also have tested cartilage tissue taken from pig larynx. The slices were good quality and the chondroblasts can be seen clearly (Figure 4).

Rowiak_Figure4.jpg
Figure 4. Laser microtomy was performed on a cartilage slice from a pig, producing a slice 10 to 20 μm thick (left, 20× objective; right, 100× objective with oil).

We have had less experience with lung and heart tissue. The preparation of lung tissue is somewhat challenging because of the large amount of air inside the lung. The air leads to reflection and scattering losses at the interfaces between tissue and air cavities and deteriorates the laser focus. However, filling the lung with nutrition solution or with agar gel as an index matcher reduces the scattering and allows cutting of slices thicker than 10 μm. In experiments with heart tissue, the muscle fibers can be seen, but cutting quality must be optimized (Figure 5).

Rowiak_Figure5.jpg
Figure 5. Using mouse samples, lung slices 10 to 20 μm thick were produced from a mouse (left, 40× objective), as were heart slices 10 to 20 μm thick (right, 20× objective).

A challenge in microtomy is cutting hard biological material, such as bones and teeth. Usually, the material must be decalcified to obtain proper slices for light microscopy. Many samples cannot be sectioned by a mechanical microtome. Often, the samples must be ground down to the desired thickness of around 1 μm. This technique is time-consuming, and it complicates posttreatment. Laser microtomy has great potential in this field of research. However the cutting parameters must be optimized because sectioning of bones and teeth requires more pulse energy and the tissue itself is quite irregular.

One of our current development activities is the integration of 3-D imaging with the microtomy system. Cutting flat sections via femtosecond laser technology is only the first step. The next step is an upgrade to 3-D processing of specimens. But that makes sense only if the 3-D structure of the tissue is known.

This can be achieved by optical coherence tomography — a method for imaging different layers of transparent or scattering tissue by scanning the area of interest with a low coherent light source.

The measuring principle is similar to ultrasound imaging. As a typical light source, superluminescent diodes — or even the femtosecond laser pulses themselves with a broad emission band and a corresponding low coherence length — can be used. The penetration depth of the NIR radiation is up to 2 mm. Because of differences in their optical path lengths, photons reflected from different layers inside the tissue can be distinguished by interference measurements with an external reference plane.

With such a tomographic imaging system, real 3-D cutting near the surface of the tissue sample is possible, targeting specific areas or volumes of interest.

Laser microtomy is not a device for routine pathology because the procedure of tissue separation is too slow. The technology’s advantage is in preparing tissue in its native state. For immunohistological investigations, it is sometimes desirable to keep tissue alive. Furthermore, there are several applications that are, at present, difficult or impossible to perform with conventional sectioning methods. This includes cutting hard tissue, plants, wood and other materials. The laser microtome might be a good option for these applications.

Meet the author

Peggy Menne is public relations and quality manager at Rowiak GmbH in Hannover, Germany; e-mail: pm@rowiak.de.


GLOSSARY
microscopic
Characteristic of an object so small in size or so fine in structure that it cannot be seen by the unaided eye. A microscopic object may be rendered visible when examined under a microscope.
microtome
A device used for slicing very thin specimens in preparation for mounting on a microscope slide.
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