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Three-Dimensional Approaches in Histopathological Tissue Clearing System

Three-dimensional microscopic approaches in histopathology display multiplex properties that present puzzling questions for specimens as related to their comprehensive volumetric information. This information includes spatial distribution of molecules, three-dimensional co-localization, structural formation and whole data set that cannot be determined by two-dimensional section slides due to the inevitable loss of spatial information. Advancement of optical instruments such as two-photon microscopy and high performance objectives with motorized correction collars have narrowed the gap between optical theories and the actual reality of deep tissue imaging. However, the benefits gained by a prolonged working distance, two-photon laser and optimized beam alignment are inevitably diminished because of the light scattering phenomenon that is deeply related to the refractive index mismatch between each cellular component and the surrounding medium. From the first approaches with simple crude refractive index matching techniques to the recent cutting-edge integrated tissue clearing methods, an achievement of transparency without morphological denaturation and eradication of natural and fixation-induced nonspecific autofluorescence out of real signal are key factors to determine the perfection of tissue clearing and the immunofluorescent staining for high contrast images. When performing integrated laboratory workflow of tissue for processing frozen and formalin-fixed tissues, clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue hydrogel (CLARITY), an equipment-based tissue clearing method, is compatible with routine procedures in a histopathology laboratory.


INTRODUCTION
Presently, the investigation of whole intact tissue based on three-dimensional (3D) histology is available thanks to the modern advances in optical instrument, tissue clearing and novel molecular labeling techniques.
Dense and tight texture formed by chemical tethering of hydrogel can enhance the preservation of molecular tissue components in addition to electrophoretic delipidation by which the opaque surface turns into transparent substance enabling clear observation of entire tissue sample even with naked eyes [1]. Threedimensional reconstruction of intact tissue always gives comprehensive information for better understanding of structural and interactional whole data set which cannot be achieved by two-dimensional (2D) serial section slides due to the inevitable loss of information caused by unnatural artificial distortion: serial sectioning, of intact tissue [2]. Continuing efforts to explore the unseen world hidden by limitation of technology in tissue clearing, optics and computational 3D rendering, opened a new era of detailed explicit visualization of whole tissue imaging. Development of confocal microscopy and relevant optical advancement allowed researchers to access high resolution images acquired along the z-axis direction in a precise step dividing by several hundred nanometers. However, the benefits gained from the objectives with long travel range and a high power laser with optimized beam condition are naturally diminished because of the light scattering phenomenon and that is incremented by refractive index (RI) mismatch of each cellular component [3]. In history, there have been many attempts to overcome this physical hindrance by matching RIs, replacing body fluid with plastic monomer, hyperhydration with aqueous-based clearing solution and tissue-gel hybridization followed by electrophoretic lipid removal [4]. Autofluorescence is one of the matter should be concerned in order to make sure of clarified visualization with much reduced interference from unwanted fluorescent signals following the tissue clearing [5].
Bleaching of the tissue is often requested before tissue clearing to get rid of endogenous fluorescent biomolecules or autofluorescence acquired during fixation, staining process and sample preservation with diverse chemical reagents [6][7][8]. Immunolabeling of target molecules is a

History of tissue clearing
The purpose of tissue clearing is to make the objects transparent without morphological denaturation which may cause misunderstanding of intact natural environment of tissue sample, in order to achieve comprehensive three dimensional constitution of tissue sample [9]. The 3D visualization of molecular localization and interactions without distortion by serial section of sample tissue allows medical technologists to explore the realm of hidden nature that contemporarily used conventional 2D imaging techniques in histopathology may barely impart. The first approach of tissue clearing was attempted by Spalteholz, a German anatomist [10], after realizing that contemporary histological methods were no longer effective for his new discoveries in macroscopic structures [11,12]. The clearing solution invented by Spalteholz was a crude mixture of organic solvents: methyl salicylate and benzyl benzoate, in the wintergreen oil so as to match RI to 1.55 [13]. The protocol could be briefly condensed with several steps: formalin fixation, pigment bleaching with hydrogen peroxide, gradient dehydration by serially increased ethanol concentration and incubation in the clearing solution [14]. This method, however, rarely introduced due to its incremented damages on the superficial layers of the tissue up to few centimeters as well as tissue shrinkage besides the limitations which solvent-based clearing protocols mainly have: a) tissue transparency, b) immunolabeling and c) endogenous fluorescence stability [15]. Canada balsam is, a hydrophobic oleoresin found in the bark of the fir Abiesbalsamea, widely used as a transparent mounting media in histopathology laboratory due to its very close  [16]. More recently, plastination has been introduced for long term preservation of tissues, organs, systems and whole body by replacement of water and lipid with plastic monomers which impart transparency to the objects [17]. Plastination was rather suitable for establishing educational materials than used for biomedical research in histopathology due to its penetrating limitation less than 3 mm of thickness after plastic polymerization [18][19][20] exposure to the clearing mixture and RI matching [31].
Bleaching of the tissue is often requested before tissue hours of exposure to LEDs [34]. Another intriguing method to avoid the interference from unwanted autofluorescence is to use an alternative protocol which adopts use of minimal concentration of aldehyde.
Tokumasu and Dvorak introduced the autofluorescence compromising fixation protocol for immunocyto-chemistry of erythrocyte fixation, using dimethyl suberimidate (DMS); diimidoester cross-linking agent, with 2% paraformaldehyde (PFA) so that aldehydeamine reaction; the main cause of fixation-induced autofluorescence, could be reduced [35]. The spectral unmixing is the computational spectrum decomposition technique for separating overlapping emission spectra from multiple fluorophores including autofluorescence [36]. The diffraction grating and multiple-anode photo-   [39]. Although the outline of tissue is swollen or shrunk depending on the Scale methods applied, the overall tissue shape and internal structures do not seem too much distorted from the original shape of the sample tissue. However, those methods intrinsically have a critical drawback that longer incubation time is needed for complete tissue clearing up to several weeks compared to the tissue-gel hybridization also known as "CLARITY".

CLARITY
Over the past decades, multifarious experimental approaches for novel tissue clearing methods had been   [70,71] Abbreviations: FLIM, fluorescence lifetime imaging; ADOTA, azadioxatriangulenium; LED, light emitting diode.  [38]. Simply, PBST (0.1% Triton X-100 in PBS) propane]dihydrochloride), 0.05% Saponin and PBS at 4°C for 24 hours. Then, tubes containing ovaries were de-gassed to remove oxygen and polymerize hydrogel monomers at 37°C for 3 hours using ETC system; X-CLARITY TM (Logos Biosystem, Korea). Polymerized ovaries were transferred into 1 L of clearing solution containing 200 mM boric acid and 4% SDS dissolved in distilled water with addition of NaOH to reach pH 8.59. Delipidation was accomplished by electrophoresis under 1.0∼1.5 A, at 37°C for 16 hours. The clearing solution was automatically circulated through the buffer drain controlled by peristaltic pump in the ETC system and ovaries gradually became transparent after 8 hours depending on sample size. (A) A polymerization system accelerating hydrogel infusion into the corner to corner of the sample tissue under vacuum condition before electrophoretic delipidation. A hydrogel polymerized tissue is ready for delipidation in the electrophoresis chamber where lipid micelles are actively removed along the electric field under the electric current around 1.0∼1.5 A at 37∼45°C consecutively, for 16 hours in the case of mouse ovary. (B) Formaldehyde in a fixed tissue is cross-linked with hydrogel. Electrophoresis of the polymerized tissue allows charge dependent lipid removal via SDS micelle. Proteins and DNAs remain in the cleared tissue as conserved 3D networks. (C) Before and after tissue clearing of mouse ovary. can be used as per the entire clearing procedure: washing, antibody dilution and immersion for matching RI; however, several drawbacks still remain such as poor transparency and over swollen of the tissue [59].
Even though the RI inhomogeneity of biological tissue is removed by the tissue clearing followed by treatment of RI matching medium, the RI mismatch between objective lens and cleared tissue still remains and that spawns an imaging issues; distortion of point-spread function (PSF). To this end, all the optical elements Figure 2. Example of three dimensional approach in histopathology. Serially sectioned sample tissue can be individually imaged under wide-field microscope with motorized stage for z-stacking which is followed by regeneration of XYZ-section image for presumption of three dimensional information by interrogating XZ and YZ plane view. On the other hand, a sample tissue can be solely imaged after tissue clearing without sectioning step artificially given under current histotechnology. Cleared ovaries were transferred into 15 mL conical tubes containing PBS with 0.1% Triton-X before serial rinsing with PBS for 1 hour to remove residual SDS. After which, samples were incubated with primary antibodies (anti-Mullerian hormone; red and platelet endothelial cell adhesion molecule-1; green) for 24 hours, and then washed with buffer for 1 hour, followed by fluorescent secondary antibodies for 6 hours and nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; blue). Intact ovaries were mounted on the glass bottom dishes (WillCo Wells, Amsterdam, Netherlands) using FocusClear solution (CelExplorer Labs, Hsinchu, Taiwan) for RI matching. Transparent tissue with successful immunofluorescent staining was subjected to confocal imaging for 3D reconstruction and image analysis. Images were obtained using Leica TCS SP8 confocal microscope (Mannheim, Germany). Two-and three-dimensional images, as well as data analysis were obtained using Spots and Isosurfaces function based on the Imaris algorithm (Bitplane, UK).