Serial (mechanical) histological sectioning (SHS) creates physical slices of fixed, stained, and embedded tissues which are then imaged with an optical microscope in unsurpassed submicrometer resolution. Obtaining these slices is however extremely work intensive, requires physical (one-time and one-directional) slicing and thus destruction of the specimen. A 2D sectional image reveals lots of histologically relevant information, but a data stack and its 3D reconstruction are even more essential for the morphological interpretation of complex structures, because they give additional insight in the anatomy. The SHS method requires semiautomatic to manual image registration to align all recorded 2D slices into order to get realistic 3D reconstructions. Often dedicated image processing of the sections is needed because of the geometrical distortions from the slicing.
A valuable alternative to achieve sectional imaging and three-dimensional modeling of anatomic structures can be found in the little known and relatively recent field of microscopy called (laser) light-sheet-based fluorescence microscopy or LSFM. These nondestructive methods generate registered optical sections in real-time through bio(medical) samples ranging from microscopic till macroscopic size. LSFM can reveal both bone and soft tissue at a micrometer resolution, thus showing a large amount of histological detail as well.
The first account of the LSFM idea was published by Voie et al. in 1993 and applied to image the inner ear cochlea of guinea pig [1
]. Their method was called orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy or tomography. The motivations for the OPFOS invention were (1) the above-mentioned disadvantages of serial histological sectioning, (2) the typical photobleaching of fluorophores in conventional or confocal fluorescence microscopy, and (3) the fact that samples are optically opaque which means a limited penetration depth and inefficient delivering and collecting of light.
Surprisingly, all these problems can be avoided by combining two old techniques. Voie was the first to combine the Spalteholz method of 1911 [2
] with the even older Ultramicroscope method of 1903 [3
]. In most microscopy techniques, the same optical path and components are used for the illumination and the observation of light. Siedentopf and Nobel Prize Winner Zsigmondy made a simple change of the optical arrangement in their ultramicroscopy setup by separating the illumination and viewing axis [3
]. Furthermore, their illumination was performed by a thin plane or sheet of light. Orthogonal viewing or observation of this sheet offers full-field and real-time sectional information. Their method was originally developed for gold particle analysis in colloidal solutions with sunlight. OPFOS used the same optical arrangement but for tissue microscopy. The separation of the illumination and imaging axis combined with laser light sheet illumination only illuminates the plane that is under observation (in contrast to confocal microscopy) and thus avoids bleaching in sample regions that are not being imaged. Generally, samples are optically opaque so the plane of laser light cannot section the sample. Spalteholz introduced a clearing method which dates back exactly 100 years [2
]. His museum technique is capable of making tissue transparent by matching the refractive index throughout the entire object volume by means of a mixture of oils with refractive indices close to that of protein. Submerged in this Spalteholz fluid, a prepared specimen appears invisible, with light passing right through it unscattered and without absorption. This clearing or refractive index matching is essential for the OPFOS technique to achieve a penetration depth of several millimeters. This procedure is followed by staining of the sample with fluorescent dye or just by relying on naturally occurring autofluorescence. The sectioning laser plane activates the fluorophores in the cross-section of sheet and sample, which are finally orthogonally recorded by a camera.
OPFOS utilizes yet a third method in conjunction with the two previous techniques when the specimen contains calcified tissue or bone. In this case, the calcium first needs to be removed before the Spalteholz procedure is applied. Bone cannot be made transparent, as the calcium atoms strongly scatter light.
Since 1993, many OPFOS-like derived methods were developed for tissue microscopy, all based on light sheet illumination. “LSFM” has become a broadly accepted acronym to cover the whole of these techniques. In the discussion, we will give a short overview of this OPFOS-derived LSFM microscopy family. First, we will explain in detail the specimen preparation and the optical arrangement of the original OPFOS setup. The remainder of this paper will serve to demonstrate some applications of OPFOS.