Fluorescence Optical Sectioning Research Articles

A qualitative comparison of 3D visualization in Xenopus laevis using a traditional method and a non-destructive method

Many tools are currently available to investigate and visualize soft and hard tissues in animals both in high-resolution and three dimensions. The most popular and traditional method is based on destructive histological techniques. However, these techniques have some specific limitations. In order to avoid those limitations, various non-destructive approaches have surfaced in the last decades. One of those is micro-CT-scanning. In the best conditions, resolution achieved in micro-CT currently approaches that of standard histological protocols. In addition to bone, soft tissues can also be made visible through micro-CT-scanning. However, discriminating between structures of the same tissue and among different tissue types remains a challenge. An alternative approach, which has not yet been explored to its full potential for comparative anatomy studies, is Orthogonal-Plane Fluorescence Optical Sectioning (OPFOS) microscopy or tomography, also known as (Laser) Light Sheet based Fluorescence Microscopy (LSFM). In this study, we compare OPFOS with light microscopy, applying those techniques to the model organism Xenopus laevis. The potential of both methods for discrimination between different types of tissues, as well as different structures of the same tissue type, is tested and illustrated. Since the histological sections provided a better resolution, adjacent structures of the same tissue type could be discerned more easily compared to our OPFOS images. However, we obtained a more naturally-shaped 3D model of the musculoskeletal system of Xenopus laevis with OPFOS. An overview of the advantages and disadvantages of both techniques is given and their applicability for a wider scope of biological research is discussed.

Simultaneous multi-color optical sectioning fluorescence microscopy with wavelength-coded volume holographic gratings.

Optical sectioning fluorescence microscopy provides high contrast images of volumetric samples and has been widely used for many biological applications. However, simultaneously acquiring multi-color fluorescence images require additional optical elements and devices, which are bulky, wavelength specific, and not cost-effective. In this paper, wavelength-coded volume holographic gratings (WC-VHGs) based optical sectioning fluorescence microscopy is proposed to simultaneously offer multi-color fluorescence images with fine out-of-focus background rejection. Due to wavelength degeneracy, multiplexed WC-VHGs are capable of acquiring multi-wavelength fluorescence images in a single shot, and displaying the laterally separated multi-wavelength images onto CCD. In our system optical sectioning capability is achieved through speckle illumination and HiLo imaging method. To demonstrate imaging characteristics of our system, dual-wavelength fluorescence images of both standard fluorescent microspheres and ex vivo mT/mG mice cardiac tissue are presented. Current results may find important applications in hyperspectral imaging for biomedical research.

Correlative fluorescence and atomic force microscopy to advance the bio-physical characterisation of co-culture of living cells

An understanding of the cell mechanical properties involved in numerous cellular processes including cell division, cell migration/invasion, and cell morphology, is crucial in developing and informing cell physiology and function. Atomic force microscopy (AFM) offers a powerful biophysical technique that facilitates the imaging of living cells under physiological buffer conditions. However, AFM in isolation cannot discriminate between different cell types within heterogeneous samples for example in a solid biopsy. The current studies demonstrate the potential of AFM in combination with correlative fluorescence optical sectioning microscopy for live cell imaging. Furthermore, this work establishes the advantage of fluorescence-AFM imaging to distinguish and analyse single-cell bio-physical properties in mixed human cell populations, in real-time. Critically, our results show that correlative fluorescence-AFM imaging allows the simultaneous co-localised detection of fluorescence coupled with nano-mechanical mapping. The findings from this work contribute to the promotion and dissemination of correlative multimodal imaging in life sciences, providing a platform for further investigations in biological and pre-clinical research.

Fast method of speckle suppression for reflection phase microscopy

Light propagating in turbid medium is randomly altered by optical inhomogeneities, which not only change the momentum and polarization of light but also generate a speckle pattern. All these effects strongly limit capabilities of laser based, quantitative phase–sensitive optical biomedical imaging modalities by hindering a reconstruction of phase distribution. Here we introduce the method of rapid incident light modulation, which allows to suppress speckle noise and preserve the spatial phase distribution. We implement this approach in the full-field Michelson interferometer, where the incident light is modulated using the digitalmicromirror device (DMD). Full Text: PDF ReferencesF. Zernike, "Phase contrast, a new method for the microscopic observation of transparent objects part II," Physica 9, 974-986 (1942). CrossRef M. C. Pitter, C. W. See, and M. G. Somekh, "Full-field heterodyne interference microscope with spatially incoherent illumination," Opt. Lett. 29, 1200-1202 (2004). CrossRef N. B. E. Sawyer, S. P. Morgan, M. G. Somekh, C. W. See, X. F. Cao, B. Y. Shekunov, and E. Astrakharchik, "Wide field amplitude and phase confocal microscope with parallel phase stepping," Review of Scientific Instruments 72, 3793-3801 (2001). CrossRef G. W. John and I. H. Keith, "A diffuser-based optical sectioning fluorescence microscope," Measurement Science and Technology 24, 125404 (2013). CrossRef S. Lowenthal and D. Joyeux, "Speckle Removal by a Slowly Moving Diffuser Associated with a Motionless Diffuser," J. Opt. Soc. Am. 61, 847-851 (1971). CrossRef S. Kubota and J. W. Goodman, "Very efficient speckle contrast reduction realized by moving diffuser device," Applied Optics 49, 4385-4391 (2010). CrossRef Y. Li, H. Lee, and E. Wolf, "The effect of a moving diffuser on a random electromagnetic beam," Journal of Modern Optics 52, 791-796 (2005). CrossRef C.-Y. Chen, W.-C. Su, C.-H. Lin, M.-D. Ke, Q.-L. Deng, and K.-Y. Chiu, "Reduction of speckles and distortion in projection system by using a rotating diffuser," Optical Review 19, 440-443 (2012). CrossRef J. Lehtolahti, M. Kuittinen, J. Turunen, and J. Tervo, "Coherence modulation by deterministic rotating diffusers," Opt. Express 23, 10453-10466 (2015). CrossRef J.-W. Pan and C.-H. Shih, "Speckle reduction and maintaining contrast in a LASER pico-projector using a vibrating symmetric diffuser," Opt. Express 22, 6464-6477 (2014). CrossRef J. I. Trisnadi, "Hadamard speckle contrast reduction," Optics Letters 29, 11-13 (2004). CrossRef M. Szkulmowski, I. Gorczynska, D. Szlag, M. Sylwestrzak, A. Kowalczyk, and M. Wojtkowski, "Efficient reduction of speckle noise in Optical Coherence Tomography," Opt. Express 20, 1337-1359 (2012). CrossRef J. W. Goodman, Speckle phenomena in optics: theory and applications (Roberts and Company Publishers, 2006). DirectLink Y. Choi, P. Hosseini, W. Choi, R. R. Dasari, P. T. C. So, and Z. Yaqoob, "Dynamic speckle illumination wide-field reflection phase microscopy," Opt. Lett. 39, 6062-6065 (2014). CrossRef Y. Choi, T. D. Yang, K. J. Lee, and W. Choi, "Full-field and single-shot quantitative phase microscopy using dynamic speckle illumination," Opt. Lett. 36, 2465-2467 (2011). CrossRef R. Zhou, D. Jin, P. Hosseini, V. R. Singh, Y.-h. Kim, C. Kuang, R. R. Dasari, Z. Yaqoob, and P. T. C. So, "Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy," Optics Express 25, 130-143 (2017). CrossRef M. Schmitz, T. Rothe, and A. Kienle, "Evaluation of a spectrally resolved scattering microscope," Biomedical optics express 2, 2665-2678 (2011). CrossRef P. Judy, The line spread function and modulation transfer function of a computer tomography scanner, Med. Phys (1976), Vol. 3, pp. 233-236. CrossRef

Turnover and flow of the cell membrane for cell migration

The role of cell membrane dynamics in cell migration is unclear. To examine whether total cell surface area changes are required for cell migration, Dictyostelium cells were flattened by agar-overlay. Scanning electron microscopy demonstrated that flattened migrating cells have no membrane reservoirs such as projections and membrane folds. Similarly, optical sectioning fluorescence microscopy showed that the cell surface area does not change during migration. Interestingly, staining of the cell membrane with a fluorescent lipid analogue demonstrated that the turnover rate of cell membrane is closely related to the cell migration velocity. Next, to clarify the mechanism of cell membrane circulation, local photobleaching was separately performed on the dorsal and ventral cell membranes of rapidly moving cells. The bleached zones on both sides moved rearward relative to the cell. Thus, the cell membrane moves in a fountain-like fashion, accompanied by a high membrane turnover rate and actively contributing to cell migration.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

DRAQ5 and Eosin ('D&E') as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues.

Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin (“D&E”) enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service.

Simultaneous Advanced Microscopies for Live Cell Signaling Dynamics Investigations

We present a new type of combined microscopy for the life sciences based on the integration of differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping Atomic Force Microscopy (AFM).[1] Our hardware platform creates the opportunity for simultaneous acquisition of membrane receptor recognition maps and fluorescence optical sectioning images of live cells, providing new experimental capabilities in cell signaling investigations.Simultaneous spatio-temporal generation of AFM and fluorescence microscopy data is made possible through time-independent illumination at low light intensities of the AFM cantilever that strongly reduces the interaction of AFM cantilever motion with fluorescence excitation light as compared to existing hardware integration platforms based on confocal laser scanning microscopy and AFM. Namely, fluorescence excitation light has the capability to induced cantilever heating and subsequent sample heating through heat conduction. We identify several system specific noise-sources that can interfere with live cell investigations and describe solutions to mitigate them.[1] Miranda, A., Martins, M. & De Beule, P. A. A. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy. Rev. Sci. Instrum. 86, 093705 (2015).

Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy.

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

A diffuser-based optical sectioning fluorescence microscope

A novel design for an optical sectioning microscope is described. The microscope uses laser illumination of a diffuser to illuminate a sample with a rapidly-changing image-plane speckle pattern. The fluorescent light from the sample is then imaged back onto the same diffuser before being imaged onto an integrating detector array. The diffuser is moved so as to illuminate each part of the specimen with a very large number of independent speckles while an image is captured. This arrangement employs a simple rotating diffuser and should allow a wide-field optically-sectioned image to be recorded in a shorter time than can be achieved with a scanning confocal system. An analysis is presented to show that the arrangement can produce an image with a sectioning capability similar to that of a conventional scanning confocal microscope. The quality of the images produced is assessed through computer simulation.

Two-Photon Immunofluorescence Characterization of the Trabecular Meshwork In Situ

To develop an in situ model to study biological responses and glaucoma pathology in the human trabecular meshwork (TM). Characteristic TM cell- and glaucoma-associated markers were localized in situ in relation to the tissue's autofluorescent structural extracellular matrix (ECM) by two-photon excitation fluorescence optical sectioning (TPEF). Human donor corneoscleral (CS) tissue containing the intact aqueous drainage tract was incubated with dexamethasone (Dex) or TGF-β1, and immunostained for epifluorescence (EF) microscopy with antibodies to myocilin and alpha smooth muscle (α-SMA). Separate specimens were labeled for Type-IV collagen and fibronectin. Nuclei were stained with Hoechst 33342. Multimodal TPEF was used to visualize EF, intravital dyes, and autofluorescence (AF) in situ. Three-dimensional (3D) localization of fluorescence within the TM was analyzed using reconstruction software. Autofluorescent beams, perforated sheets, and fibers, consistent with the uveal (UV), CS, and juxtacanalicular (JCT) meshwork, respectively, were captured at different depths of the TM. Type-IV collagen EF distinctly outlined the AF beams in a location consistent with basement membrane. Fibronectin EF showed a diffuse reticular pattern throughout the TM. TGF-β1-induced α-SMA expression, which was distributed perinuclearly in cells among autofluorescent structures. Dex-induced myocilin expression had both cytosolic and extracellular distributions. The authors have localized markers that are characteristic of TM cells and relevant to glaucoma pathogenesis in situ using multimodal TPEF without conventional histological embedding and sectioning. Protein expression was inducible in situ and could be analyzed with respect to cells and the ECM within the 3D environment of the human TM.

Three-Dimensional Imaging and Modeling of Anatomic Structures, Sectional and Radiological Anatomy, and Staining Techniques

The first three-dimensional (3D) reconstruction was performed by Born (1883) and His (1885) using serial sections. Since then, a variety of reconstruction techniques have been developed, and the construction of physical models has become important in anatomy. The first computer-aided 3D reconstruction was achieved by Glaser and Van der Loos in 1965. With the improvements in both computer hardware and software tools, computerized modelling of anatomical structures has become very useful for visualizing complex 3D forms. Three-dimensional visualization of various microanatomic structures using special preparation and staining methods is important. Devices used for this purpose such as MicroCT, microMRI, SEM, and Confocal microscopy and so forth. are development parallel to the technological advances and give excellent 3D images, and it allows a better understanding of gross and microanatomic structures. On the other hand, first studies on cross-sectional anatomy date back to the 16th century. The widespread use of cross-sectional imaging techniques such as CT and MRI has gained an increasing importance in sectional anatomy. The most known cadaveric cross-sectional study, the visible human project, is an outgrowth of the National Library Medicine. In this special issue, we reviewed and edited seven articles on three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. K. K. Schmid (University of Nebraska, USA) Marx and Samal (University of Nebraska-Lincoln, USA) discussed three-dimensional regression, a technique that can be used for mapping images and shapes that are represented by sets of three-dimensional landmark coordinates, for comparing and mapping 3D anatomical structures in their research paper. Y. Hoshino and coworkers (University of Pittsburgh, USA) reviewed previous research about image analysis of the anterior cruciate ligament (ACL) anatomy and its application to ACL reconstruction surgery. They concluded that three-dimensional image analysis of the ACL anatomy and its application to the navigation system is becoming more prevalent and reliable for advancing the anatomic studies related to the native ACL and the ACL reconstruction procedure. A. Arencibia et al. (Veterinary Faculty, University of Las Palmas de Gran Canaria) investigated imaging features of the temporomandibular joint (TMJ) using computed tomography and magnetic resonance imaging in two normal camels. They stated in conclusion that CT is an excellent method for the detailed assessment of the bony structures, and MRI is a valid imaging modality for the evaluation of the soft tissues. Processing of CT and MR images allowes to appreciate different bone structures and soft tissues of the TMJ, assisting in the interpretation of the images. J. A. N. Buytaert and colleagues (University of Antwerp and Ghent University, Belgium) discussed the first implementation of (laser) light-sheet-based fluorescence microscopy (LSFM) to image biomedical tissue in three-dimensional orthogonal plane fluorescence optical sectioning microscopy (OPFOS). They have shown with several applications that the OPFOS (and derived) methods are a valuable addition for sectional imaging and three-dimensional modeling of anatomic structures. The review paper entitled “Ultrahigh voltage electron microscopy (UHVEM) links neuroanatomy and neuroscience/neuroendocrinology” by H. Sakamoto (Okayama University, Japan) and M. Kawata (Kyoto Prefectural University of Medicine, Japan) stressed that UHVEM and light microscopy are useful and powerful tools for studying molecular and/or chemical neuroanatomy at the ultrastructural level. D. R. Marker et al. (The Johns Hopkins Hospital and University School of Medicine, Weill Cornell Medical College, USA) discussed strategic improvements for gross anatomy web-based teaching in their research paper. To better meet the expectations of gross anatomy students, electronic radiology teaching files for first-year coursework were organized into a web site. The web site was custom designed to provide material that directly correlated to the gross anatomy dissection and lectures. Quick links provided sets of images grouped by anatomic location. Their findings suggest that a well-organized web portal can provide a user-friendly, valuable educational resource for medical students who are studying gross anatomy. Finally, Herber and coworkers (University of California, USA) in their study entitled “Imaging an adapted dentoalveolar complex” used micro-X-ray computed tomography combined with 3D modeling using image processing, scanning electron microscopy, fluorochrome labeling, conventional histology (H&E, TRAP), and immunohistochemistry (RANKL, OPN) to elucidate the dynamic nature of bone, the periodontal ligamentspace, and cementum in the rat periodontium. They concluded that studies focusing on a functional rat dentoalveolar complex should acknowledge the baseline phenomena of bone-PDL-cementum adaptation, especially in regards to the physiological distal drift, before additional experimental variables are imposed. We hope that this special issue will contribute as a stimulus for further research into three-dimensional imaging and modeling of anatomic structures, sectional and radiological anatomy, and staining techniques. We are very grateful to the contributing authors for their scientific contributions to this special issue. We also thank the reviewers who spend their valuable time and thoughts on each paper. We appreciate the Hindawi Publishing Corporation for their help in the guidance of the procedure. Tuncay Peker Nadir Gulekon Ilkan Tatar Levent Sarikcioglu David Kachlik

Real-time in vivo imaging of the beating mouse heart at microscopic resolution

Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying the complex biology of live animals. Here we present a technique based on a novel stabilizer setup combined with a gating acquisition algorithm for the imaging of a beating murine heart at the single-cell level. The method allows serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes over the course of several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischaemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.

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

We report on the recently emerging (laser) light-sheet-based fluorescence microscopy field (LSFM). The techniques used in this field allow to study and visualize biomedical objects nondestructively in high resolution through virtual optical sectioning with sheets of laser light. Fluorescence originating in the cross-section of the sheet and sample is recorded orthogonally with a camera. In this paper, the first implementation of LSFM to image biomedical tissue in three dimensions—orthogonal-plane fluorescence optical sectioning microscopy (OPFOS)—is discussed. Since then many similar and derived methods have surfaced, (SPIM, ultramicroscopy, HR-OPFOS, mSPIM, DSLM, TSLIM, etc.) which we all briefly discuss. All these optical sectioning methods create images showing histological detail. We illustrate the applicability of LSFM on several specimen types with application in biomedical and life sciences.

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate.

Meeting report: First light sheet based fluorescence microscopy workshop

In Jena, at the turn of the century, Richard Zsig-mondy (1865–1929), working together with thephysicist Heinrich Siedentopf, had the brilliantidea to use a light sheet to illuminate from the sidethe colloid solution they were investigating [1].From there the idea of light sheet illumination van-ished from the scientific spotlight until it was re-vived it in the 90’s as Orthogonal-Plane Fluores-cence Optical Sectioning (ORFOS) [2] and thetaconfocal microscopy [3]. At the beginning of thenew millennium, Ernst Stelzer and colleagues de-veloped Selective Plane Illumination Microscopy(SPIM) and applied it to live imaging of model or-ganism embryos [4,5].Since then the light-sheet il-lumination approach proved instrumental for awide variety of biological disciplines ranging frommarine biology [6], to fundamental developmental[7,8] and cell biology research questions [9,10].Yetthe technology remains largely confined to the lab-oratories that orchestrated its revival and the ben-efits of light sheet illumination are inaccessible tothe broad scientific community. It was the purposeof the First Light Sheet based Fluorescence Mi-croscopy workshop to bring together the key play-ers in the light sheet technology and to establish ef-fective communication among them that will hope-fully lead to better integration of light sheet mi-croscopy into scientific mainstream. It is symbolicthat the workshop took place at the Max Planck In-stitute of Molecular Cell Biology and Genetics(MPI-CBG) in Dresden only some 100 miles awayfrom Jena where it all started.The workshop aimed at founding a communityand to encourage communication and sharing be-tween the different groups developing, using orsimply interested in this technology for their re-search. In preparation for the workshop we estab-lished a Wiki where every invited participant coulddescribe his research and sign up for a presenta-tion in one of the sessions (https://spim.mpi-cbg.de).This self-organizing approach worked verywell, as most of the participants were in fact pre-senting in the session of their choice and all speak-ers received equal amount of time to present theirdata.The presentations were divided in three mainsections:

Three-dimensional reconstruction of the pigeon inner ear

Three-dimensional reconstructions of the inner ear of the pigeon (Columba livia domestica), from two-dimensional images, obtained with (conventional) light microscopy or orthogonal-plane fluorescence optical sectioning (OPFOS), are presented. The results are compared with available information on the dimensions and orientation of the semicircular canals.

Tomographic imaging of macroscopic biomedical objects in high resolution and three dimensions using orthogonal-plane fluorescence optical sectioning

A new optical-fluorescence microscopy technique, called HR-OPFOS, is discussed and situated among similar OPFOS-implementations. OPFOS stands for orthogonal-plane fluorescence optical sectioning and thus is categorized as a laser light sheet based fluorescence microscopy method. HR-OPFOS is used to make tomographic recordings of macroscopic biomedical specimens in high resolution. It delivers cross sections through the object under study with semi-histological detail, which can be used to create three-dimensional computer models for finite-element modeling or anatomical studies. The general innovation of this class of microscopy setup consists of the separation of the illumination and observation axes, but now in our setup combined with focal line scanning to improve sectioning resolution. HR-OPFOS is demonstrated on gerbil hearing organs and on mouse and bird brains. The necessary specimen preparation is discussed.

Three‐dimensional reconstruction of the guinea pig inner ear, comparison of OPFOS and light microscopy, applications of 3D reconstruction

Three-dimensional (3D) reconstruction of anatomical structures can give additional insight into the morphology and function of these structures. We compare 3D reconstructions of the guinea pig inner ear, using light microscopy and orthogonal plane fluorescence optical sectioning microscopy. Applications of 3D reconstruction of the inner ear are further explored. For each method two bullas were prepared for 3D reconstruction. Both methods are explained. In general, the 3D reconstructions using orthogonal plane fluorescence optical sectioning microscopy are superior to light microscopy. The exact spiral shape of the cochlea could be reconstructed using orthogonal plane fluorescence optical sectioning microscopy and the length of the basilar membrane measured. When a resolution of 20 microm is sufficient, orthogonal plane fluorescence optical sectioning microscopy is a superior technique for 3D reconstruction of inner ear structures in animals.