imaging-the-aging-cochlea-with-light-sheet-fluorescence-microscopy

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

University of Minnesota

We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.

Thin-sheet laser imaging microscopy for optical sectioning of thick tissues.

We report development of a continuous scanning procedure and the use of a time delay integration (TDI) line scan camera for a light-sheet based microscope called a thin-sheet laser imaging microscope (TSLIM). TSLIM is an optimized version of a light-sheet fluorescent microscope that previously used a start/stop scanning procedure to move the specimen through the thinnest portion of a light-sheet and stitched the image columns together to produce a well-focused composite image. In this paper, hardware and software enhancements to TSLIM are described that allow for dual sided, dual illumination lasers, and continuous scanning of the specimen using either a full-frame CCD camera and a TDI line scan camera. These enhancements provided a ~70% reduction in the time required for composite image generation and a ~63% reduction in photobleaching of the specimen compared to the start/stop procedure.

Implementation of a continuous scanning procedure and a line scan camera for  thin-sheet laser imaging microscopy.

Thin-sheet laser imaging microscopy (TSLIM) was used to serially section five whole cochleas from 4-wk-old CBA/JCr mice. Three-dimensional reconstructions of Rosenthal's canal (RC) were produced in order to measure canal length and volume, to generate orthogonal cross sections for area measurements, and to determine spiral ganglion neuron (SGN) number. RC length averaged 2.0 mm ± 0.04 (SEM) as measured along the centroid of the canal compared to an average basilar membrane (BM) length of 5.9 ± 0.05 (SEM). RC volume averaged 0.036 mm(3) ± 0.009 (SEM). Significant increases in the radial area of RC were observed at the base (13%), middle (62%), and apex (90%) of its length. The total number of spiral ganglion neurons (SGNs) in RC in each of the five animals averaged 8626 ± 96 (SEM). SGN number increased at the expanded regions of RC. Increased area and cell number at the base and apex are likely related to extensions of the organ of Corti past the length of RC in these areas. The increase in area and cell number in the middle of the RC appears to be related to the most sensitive frequency region of the organ of Corti. Volume imaging or tomography of the cochlea as provided by TSLIM has the potential to be an efficient and accurate semi-automated method for the quantitative assessment of the number of SGNs and hair cells of the organ of Corti.

TSLIM imaging and a morphometric analysis of the mouse spiral ganglion.

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts.

Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection.

Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to "float" in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.

Decellularized ear tissues as scaffolds for stem cell differentiation.

We made a qualitative and quantitative comparison between a state-of-the-art implementation of micro-Computed Tomography (microCT) and the scanning Thin-Sheet Laser Imaging Microscopy (sTSLIM) method, applied to mouse cochleae. Both imaging methods are non-destructive and perform optical sectioning, respectively, with X-rays and laser light. MicroCT can be used on fresh or fixed tissue samples and is primarily designed to image bone rather than soft tissues. It requires complex back-projection algorithms to produce a two-dimensional image, and it is an expensive instrument. sTSLIM requires that a specimen be chemically fixed, decalcified, and cleared; but it produces high-resolution images of soft and bony tissues with minimum image postprocessing and is less expensive than microCT. In this article, we discuss the merits and disadvantages of each method individually and when combined.

MicroCT versus sTSLIM 3D imaging of the mouse cochlea.

Administration of an aminoglycoside antibiotic and loop diuretic causes damage to hair cells in the organ of Corti, resulting in their death and the death of their corresponding spiral ganglion neurons. While this phenomenon has been studied previously, analysis of its effects in the whole cochlea has not been reported. The authors sought to evaluate the effects of a combination dose of kanamycin and furosemide in mice cochlea using an imaging system and computer analysis that allowed for nondestructive, whole-cochlea visualization.

Kanamycin-furosemide ototoxicity in the mouse cochlea: a 3-dimensional analysis.

Thin-sheet laser imaging microscopy (TSLIM) optical sectioning can be used to assess temporal bone soft tissue morphology before celloidin sectioning.

Article	Year
Thin-sheet laser imaging microscopy for optical sectioning of thick tissues. BioTechniques\| PubMed ID: 19450235	2009
Implementation of a continuous scanning procedure and a line scan camera for  thin-sheet laser imaging microscopy. Biomedical optics express\| PubMed ID: 21258493	2010
TSLIM imaging and a morphometric analysis of the mouse spiral ganglion. Hearing research\| PubMed ID: 21420476	2011
Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection. Biomedical optics express\| PubMed ID: 22254177	2012
Decellularized ear tissues as scaffolds for stem cell differentiation. Journal of the Association for Research in Otolaryngology : JARO\| PubMed ID: 23085833	2013
MicroCT versus sTSLIM 3D imaging of the mouse cochlea. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society\| PubMed ID: 23360693	2013
Kanamycin-furosemide ototoxicity in the mouse cochlea: a 3-dimensional analysis. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery\| PubMed ID: 24415490	2014
Comparison of traditional histology and TSLIM optical sectioning of human temporal bones. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology\| PubMed ID: 24914787	2014

Shane B. Johnson

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