In LSFM, the specimen is no longer positioned on a microscope slide, but placed in a liquid filled specimen chamber. Four main specimen preparation techniques have been developed and used to perform LSFM: hooking, embedding, containing, or flat mounting (Fig. ).
Different specimen preparation techniques for LSFM.
The first and simplest way consists of “hooking” the specimen using a clip, tweezers, or a hook made of glass, stainless steel, or plastic material [Fig. ]. This is particularly suitable to image large specimens such as organs (e.g., mouse brain), adult insects, but also yeast cells dispersed in an agarose droplet, as the hook provides a higher stability of the specimen than holding as a block of agarose (Taxis et al. 2006
). However, one has to consider that mechanical contact can partially damage the specimen or interfere with imaging.
The second and most common technique is embedding. The specimen is embedded in a gelling agent, usually low melting agarose, using a specimen holder such as a glass capillary that shapes a cylinder [Fig. ]. This agarose cylinder is pushed out of the capillary and placed in front of the objective lens. This provides a complete access to the specimen. The chosen gelling agent has a refractive index, which is close to that of water and strong enough not to break during translation or rotation. This specimen preparation technique has been extensively used for imagingD
(embryo, larvae, pupa, and adult) (Swoger et al., 2007
, fixed cysts and cell aggregates (Pampaloni et al., 2007
(Huisken et al., 2004
; Swoger et al., 2007
, yeast cells (Taxis et al., 2006
), or zooplankton. One limit of embedding is the effects of the gelling agent on the specimen. It may exert compression forces on the specimen during the gelling process, especially at high concentrations (e.g., 2% agarose). It can also restrain the specimen movements and this can be crucial when imaging developing samples such as embryos.
The third mounting technique consists of holding the specimen in a container. This container can be made from a gelling agent using a special moulding device or suitable polymers (refractive index, thickness . . .) [Fig. ]. These containers can be held using suitable clips. This technique is convenient to image living cells embedded in three-dimensional extracellular matrices, compression-sensitive specimens (e.g., developing embryos), as well as in vitro
assays (Engelbrecht et al., 2007
; Keller et al., 2008
). The main limitation of this approach is the capacity to tailor the chamber size to the specimen. It is difficult to prepare agarose chambers with an inner diameter of less than 0.5 mm and a beaker wall thin enough not to affect the imaging quality. In addition, suitable polymers are often difficult to find and to shape to accommodate small specimens such as sea urchin eggs (100 μm).
The fourth technique is to mount an object on a flat surface. This is mandatory for the ultramicroscope as the vertical detection axis allows depositing the object underneath the objective lens (Dodt et al., 2007
) but in the case of a horizontal detection axis as in the SPIM, the flat surface must be mounted on a holder [Fig. ].
In summary, the method of specimen preparation for LSFM must respect three very important criteria. It must be mechanically stable. The specimen must be well supported to avoid distortion due to movement during imaging. It must support the physiological aspects of the specimen (e.g., development). The mounting system should be flexible enough to allow the specimen to develop and should not change its mechanical properties during observation or dissolve in the buffer. Finally, it must support good imaging (e.g., no mechanical interference during the imaging). For example, the refractive index of the mounting medium should be as close as possible to the refractive index of the buffer filling the imaging chamber in order not to scatter light. Both optical and physiological aspects have to be brought together, especially in live imaging where biocompatibility has to be considered.
Like in any other microscopy technique, the specimen preparation must be carefully considered, as badly labeled and deformed specimens will not take full advantages of the microscope. This is especially important in LSFM as the specimen handling is performed in a very different way (rotation, incubation chamber . . .). In general, however, the attitude should be that LSFM provide an entirely new means for specimen preparation. The fact that illumination and detection occur along different directions should be regarded as an opportunity to reconsider the means according to which specimens can be prepared. After all, we want to prepare specimens in a manner that maintains it close to its actual physiological appearance.