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The OPFOS Microscopy Family: High-Resolution Optical-Sectioning of Biomedical Specimens

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The OPFOS Microscopy Family: High-Resolution Optical-Sectioning of Biomedical Specimens 1 2 3 4 5 The OPFOS microscopy family: 6 High-resolution optical-sectioning 7 of biomedical specimens 8 9 10 1, 2 2 11 Jan A.N. Buytaert °, Emilie Descamps , Dominique Adriaens , 1 12 and Joris J.J. Dirckx 13 14 15 16 17 18 19 20 21 1 Laboratory of BioMedical Physics – University of Antwerp, 22 Groenenborgerlaan 171, B-2020 Antwerp, Belgium 23 2 Evolutionary Morphology of Vertebrates – Ghent University, 24 K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 ° Corresponding author: 40 email [email protected]; 41 telephone 0032 3 265 3553; 42 fax 0032 3 265 3318. 43 44 45 46 OPFOS microscopy family Buytaert et al. 47 Abstract 48 49 We report on the recently emerging (Laser) Light Sheet based Fluorescence Microscopy field 50 (LSFM). The techniques used in this field allow to study and visualize biomedical objects non- 51 destructively in high-resolution through virtual optical sectioning with sheets of laser light. 52 Fluorescence originating in the cross section of the sheet and sample is recorded 53 orthogonally with a camera. 54 55 In this paper, the first implementation of LSFM to image biomedical tissue in three 56 dimensions – Orthogonal-Plane Fluorescence Optical Sectioning microscopy (OPFOS) – is 57 discussed. Since then many similar and derived methods have surfaced (SPIM, 58 Ultramicroscopy, HR-OPFOS, mSPIM, DSLM, TSLIM, …) which we all briefly discuss. All these 59 optical sectioning methods create images showing histological detail. 60 61 We illustrate the applicability of LSFM on several specimen types with application in 62 biomedical and life sciences. 63 64 65 66 Keywords 67 68 serial sectioning, OPFOS, LSFM, optical sectioning, fluorescence, 69 three-dimensional imaging, biomedical 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 1 OPFOS microscopy family Buytaert et al. 94 Introduction 95 96 Serial (Mechanical) Histological Sectioning (SHS) creates physical slices of fixed, stained and 97 embedded tissues which are then imaged with an optical microscope in unsurpassed sub- 98 micrometer resolution. Obtaining these slices is however extremely work-intensive, requires 99 physical (one-time and one-directional) slicing and thus destruction of the specimen. A 2-D 100 sectional image reveals lots of histologically relevant information, but a data stack and its 3- 101 D reconstruction are even more essential for the morphological interpretation of complex 102 structures, because they give additional insight in the anatomy. The SHS method requires 103 semi-automatic to manual image registration to align all recorded 2-D slices in order to get 104 realistic 3-D reconstructions. Often dedicated image processing of the sections is needed 105 because of the geometrical distortions from the slicing. 106 107 A valuable alternative to achieve sectional imaging and three-dimensional modeling of 108 anatomic structures can be found in the little known and relatively recent field of 109 microscopy called (Laser) Light Sheet based Fluorescence Microscopy or LSFM. These non- 110 destructive methods generate registered optical sections in real-time through bio(medical) 111 samples ranging from microscopic till macroscopic size. LSFM can reveal both bone and soft 112 tissue at a micrometer resolution, thus showing a large amount of histological detail as well. 113 114 The first account of the LSFM idea was published by Voie, Burns and Spelman in 1993 and 115 applied to image the inner ear cochlea of guinea pig [1]. Their method was called 116 Orthogonal-Plane Fluorescence Optical Sectioning (OPFOS) microscopy or tomography. The 117 motivations for the OPFOS invention were (1) the above mentioned disadvantages of serial 118 histological sectioning, (2) the typical photo-bleaching of fluorophores in conventional or 119 confocal fluorescence microscopy, and (3) the fact that samples are optically opaque which 120 means a limited penetration depth and inefficient delivering and collecting of light. 121 122 Surprisingly, all these problems can be avoided by combining two old techniques. Voie and 123 colleagues first combined the Spalteholz method of 1911 [2] with the even older 124 Ultramicroscope method of 1903 [3]. In most microscopy techniques, the same optical path 125 and components are used for the illumination and the observation of light. Siedentopf and 126 Nobel Prize winner Zsigmondy made a simple change of the optical arrangement in their 127 Ultramicroscopy setup by separating the illumination and viewing axis [3]. Furthermore, 128 their illumination was performed by a thin plane or sheet of light. Orthogonal viewing or 129 observation of this sheet offers full-field and real-time sectional information. Their method 130 was originally developed for gold particle analysis in colloidal solutions with sunlight. OPFOS 131 used the same optical arrangement but for tissue microscopy. The separation of the 132 illumination and imaging axis combined with laser light sheet illumination only illuminates 133 the plane that is under observation (in contrast to confocal microscopy) and thus avoids 134 bleaching in sample regions that are not being imaged. Generally, samples are optically 135 opaque so the plane of laser light cannot section the sample. Spalteholz introduced a 136 clearing method which dates back exactly 100 years [2]. His museum technique is capable of 137 making tissue transparent by matching the refractive index throughout the entire object 138 volume by means of a mixture of oils with refractive indices close to that of protein. 139 Submerged in this Spalteholz fluid, a prepared specimen appears invisible, with light passing 140 right through it unscattered and without absorption. This clearing or refractive index 2 OPFOS microscopy family Buytaert et al. 141 matching is essential for the OPFOS technique to achieve a penetration depth of several 142 millimeters. This procedure is followed by staining of the sample with fluorescent dye or just 143 by just relying on naturally occurring auto-fluorescence. The sectioning laser plane activates 144 the fluorophores in the cross section of sheet and sample, which are finally orthogonally 145 recorded by a camera. 146 147 OPFOS utilizes yet a third method in conjunction with the two previous techniques when the 148 specimen contains calcified tissue or bone. In this case, the calcium first needs to be 149 removed before the Spalteholz procedure is applied. Bone cannot be made transparent, as 150 the calcium atoms strongly scatter light. 151 152 Since 1993, many OPFOS-like derived methods were developed for tissue microscopy, all 153 based on light sheet illumination. ‘LSFM’ has become a broadly accepted acronym to cover 154 the whole of these techniques, coined in Dresden (Germany) 2009. In the discussion, we will 155 give a short overview of this OPFOS-derived LSFM microscopy family. First, we will explain in 156 detail the specimen preparation and the optical arrangement of the original OPFOS setup. 157 The remainder of this paper will serve to demonstrate some applications of OPFOS. 158 159 160 Materials and Methods 161 162 Specimen preparation 163 In most LSFM methods, the biomedical tissue samples are severely limited in size, though for 164 instance the LSFM implementations of Ultramicroscopy, HR-OPFOS and TSLIM (cf. the 165 discussion section) are capable of imaging macroscopic samples up to tens of millimeters [4]. 166 In all cases, an elaborate specimen preparation is required: 167 Euthanasia: Living animals cannot be used in combination with clearing solutions. In 168 general, LSFM is thus mainly used in vitro. Clearing can be omitted and living animals 169 can be used if the species possesses a natural transparency at a certain 170 developmental stage, for instance fish embryos [5,6]. The embryos are immobilized 171 by embedding in agarose. 172 Perfusion: Before dissecting a sample to the required dimensions, transcardial 173 perfusion with phosphate buffered saline is useful as coagulated blood is difficult to 174 clear with Spalteholz fluid [7-9]. If perfusion is omitted, bleaching is required. 175 Fixation: Immersion in 4% paraformaldehyde (10% formalin) during 24h for 176 preservation and fixation of the specimen. 177 Bleaching: Optional bleaching in 5% to 10% hydrogen peroxide for one or more days 178 can be performed when the sample contains dark pigmented tissue (e.g. black skin, 179 fish eyes) [10]. This step can also be applied after decalcification [11]. 180 Decalcification: When the specimen contains calcified or mineralized tissue, such as 181 cartilage or bone, decalcification is in order. A 10% demineralized water solution of 182 dihydrate ethylenediaminetetraacetic acid (EDTA) slowly diffuses calcium atoms from 183 the sample through a chelation process. Low power microwave exposure (without 184 heating) drastically accelerates the decalcification process from a month to several 185 days [12,13]. 3 OPFOS microscopy family Buytaert et al. 186 Dehydration: Immersion in a graded ethanol series (f.i. 25%, 50%, 75%, 100%, 100% 187 each for 24h) removes all water content from the sample [12]. In the final 100% step, 188 optional addition of anhydrous copper sulfate at the bottom of the ethanol bath 189 might improve the dehydration [14]. 190 Hexane or benzene: The optional immersion in a graded series of hexane or benzene 191 is said to improve dehydration further [8,11,14,15]. Furthermore, hexane might assist 192 in clearing myelin present in the tissue sample. Nerve axons are surrounded by 193 myelin sheets which do not easily become transparent with Spalteholz fluid. 194 Clearing: To achieve large volume imaging in inherently less transparent samples, 195 clearing is needed. The specimen are to be immersed in clearing solution, either 196 through a graded series (f.i. 25%, 50%, 75%, 100%, 100% each for 24h) when the 197 hexane or benzene step was skipped [12], or directly in 100% pure clearing solution 198 when hexane or benzene were applied [8,11].
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