Cleared Tissue and Whole Organ Imaging
Recently, there have been significant advances in tissue clearing techniques that have allowed scientists to reconstruct and map neural tissues with higher optical resolution and at greater tissue depth. These technological advances have led to a number of large-scale collaborations among research scientists and institutions focusing on constructing maps of neural circuits to better understand functional connectivity and anatomical factors relating to neurological disorders and diseases. MBF Bioscience has established collaborations with scientists at Columbia University to license a new microscope technology called Light Sheet Theta Microscopy (LSTM) that produces higher resolution images of cleared tissue and can image larger tissues at higher speeds than other Light Sheet technologies available.
Tissue clearing is a chemical technique to make biological tissues transparent to allow for high resolution microscopic imaging of structures deep within the specimen without sectioning the tissue into thinner slices. The advantage of this is that the specimen retains its original structure and cytoarchitecture allowing researchers to investigate spatial relationships between structures, cells, and/or molecules. This is particularly valuable in research investigations involving long neural projections, vasculature, and airways. Connectomics research has been advanced significantly through the utilization of tissue clearing methods to map neural networks and trace projections.
Thy1 GFP labeling in a cleared mouse brain showing dendritic spines at 63x. The image stack was acquired with Vesalius
There are a wide variety of tissue clearing methods but in general clearing a tissue specimen involves fixating objects of interest such as neurons, microvessels, and proteins while removing light-scattering molecules such as lipids to improve transparency of the specimen. Molecular labelling is then used to attach fluorophores to objects of interest for fluorescent imaging with laser microscopy tools. Lastly, the specimen is submerged in a liquid media with a specific refractive index matched to the imaging technique being utilized to scan the specimen. The final product is a semi-transparent specimen that retains the original structure and cytoarchitechture of the original organism.
Alexa Fluor 555 labeling in a cleared mouse brain showing extensive neuronal connections at 63x. The image stack was acquired with Vesalius.
Tissue clearing is being used extensively to image and quantify neural networks, dendritic spines, axonal projections between nuclei, and vascular networks in the kidney, lungs, cardiovascular system and lymphatic system. Obtaining specimen transparency is only one of the challenges faced by scientific researchers. There are technological limitations such as the maximum size of specimens that can be imaged, the enormous amount of image data collected when scanning a specimen, challenges in reconstructing large image volumes precisely, and in quantifying objects of interest to an investigation. Watch a video of a 3D Whole Brain Reconstruction Acquired with LSTM.
Having extensive knowledge of the challenges associated with cleared tissue imaging, we set out to create a complete cleared tissue imaging and analysis system capable of imaging deeper, faster, and more efficiently than the fluorescent imaging technologies currently on the market. In collaboration with Dr. Raju Tomer at Columbia University we have created ClearScope®, a Light Sheet Theta Microscope (LSTM) with imaging capabilities far beyond commercial light sheet systems currently on the market. In addition, MBF Bioscience has partnered with Caliber ID to create Vesalius®, a resonant scanning laser confocal microscope system that performs fluorescent slide scans as well as imaging of cleared tissue specimens up to 6mm thick. Each system includes MBF’s acclaimed image data analysis software to provide researchers with a complete hardware+software solution for completing entire research investigations from start to finish.
ClearScope® is a complete state-of-the-art microscope system utilizing LSTM technology that includes every tool needed to conduct a tissue scan from start to finish. The Clear Scope system allows researchers to:
- Acquire 3D image montages automatically, either whole tissues or discrete sections
- Store and remotely access massive 3D image files efficiently, minimizing system resource consumption
- Access data remotely and collaborate with others
- Analyze scanned images on any dedicated PC using MBF's full suite of included software tools such as Neurolucida 360, Vesselucida 360, and Brain Maker.
- Create captivating images and video for publications
- Custom specimen chambers for imaging larger specimens, including whole mouse brains or tissue slabs up to 6mm thick
- CLARITY, iDISCO, uDISCO, CUBIC, SeeDB, Sca/e and more
- Objective lenses are available in magnifications including: 4x, 10x, 16x, 20x, and 25x
- Fully automated stitching algorithms efficiently create high-resolution mosaics of thick and thin tissues
- An additional 4x optical zoom within the microscope head allows for better finer imaging of small structures
Neurolucida 360 is the premier tool used by neuroscientists to quickly and accurately reconstruct intricate neuronal structures that range in scale from complex, multicellular networks of neurons to sub-cellular dendritic spines and putative synapses. Using Neurolucida 360 in combination with expansion microscopy and light sheet microscopy allows researchers obtain comprehensive morphometric data with more accuracy than ever before.
Stereo Investigator – Cleared Tissue Edition is the gold standard in unbiased stereology for use on cleared tissue. It allows researchers to use the world’s most cited stereology software — MBF Bioscience’s Stereo Investigator — to analyze intact, cleared tissue specimens imaged with light sheet or confocal microscopes. Stereo Investigator – Cleared Tissue Edition is a software tool specifically engineered for analyzing cleared tissue image data accurately and efficiently. It includes the Image Volume Fractionator, a new, state-of the-art stereological probe developed by MBF Bioscience to count cells in large, 3D images of intact specimens.