At Dr. Patrick R. Hof’s lab at the Icahn School of Medicine at Mount Sinai, researchers imaged the cerebral cortex using light-sheet fluorescence microscopy and quantified the number of neurons, including those that express proteins involved in Alzheimer’s disease and schizophrenia, using the Image Volume Fractionator¹. This work marks the beginning of an important and ambitious project to build an atlas of cortical cells, using a multi-resolution imaging pipeline. At the pipeline’s highest level of resolution, both the Image Volume Fractionator, for use with thick sections of cleared tissue, and the Optical Fractionator, for much thinner sections, are being used to estimate cell number. The researchers will also use automatic cell detection and plan to compare results obtained using the three methods.

In the paper A Multimodal Imaging and Analysis Pipeline for Creating a Cellular Census of the Human Cerebral Cortex¹, the authors describe the beginning of the effort to build a census of the human cerebral cortex, a laminar structure, that contains layers comprised of different cell types visible using high resolution microscopy. There are a number of different neuronal cell types in each layer, including projection neurons and interneurons, as well as excitatory and inhibitory neurons. Layers can be identified based on different proteins contained in certain cells using fluorescence immunohistochemistry. Calretinin, a calcium-binding protein, is found in a subpopulation of the inhibitory interneurons that contain GABA. Neurofilament protein, which can be found in the cytoskeleton, makes up 30 percent of cortex cells. Parvalbumin, another calcium-binding protein, is also found in a subset of cortical cells.

The cells in the cortex have a purpose that is supported by their neurochemical and anatomical characteristics. Here are two examples involving Alzheimer’s disease and schizophrenia. Calretinin-positive cells in the cortex are spared in Alzheimer’s disease², but neurofilament protein-positive cells degenerate, and that degeneration may predict cognitive decline³. The parvalbumin-containing basket cell is an inhibitory GABAergic interneuron in the cortex that inhibits the main output cell—the pyramidal neuron. Problems with this cell type may affect gamma oscillations, leading to the deficits in cognitive control that accompany schizophrenia⁴.

Wouldn’t it be valuable to have an atlas or census of the cortex that is “zoomable” like a GPS map, and shows the cell types and their connections? Hof. et.al., demonstrate that this is possible using three imaging techniques at increasing resolutions (Fig. 1).

Fig. 1 The three imaging modalities used in this study. Magnetic Resonance Imaging (MRI) is the lowest resolution. Optical Coherence Tomography (OCT) is the mid-resolution. Light Sheet Fluorescence Microscopy (LSFM) is the highest resolution. The Image Volume Fractionator probe is carried out using LSFM. Those images are registered to eliminate distortion to help match them to OCT and the MRI images.

At the most highly resolved level, LSFM, two unbiased stereology techniques are used to build a census inside the atlas: the Optical Fractionator and the new, Image Volume Fractionator. The latter is made possible by tissue clearing methods, which in turn allows for the use of tissue sections that are, in this case, 10 times thicker than for the Optical Fractionator (Fig. 2).

Fig. 2 The Image Volume Fractionator (IVF) was designed to be used on thick sections or large intact specimens. It is much more efficient than working on traditional histological sections that were not cleared and therefore need to be, in this case, 10 times thinner. N is the estimate of number of cells. Systematic random sampling and disector rules are followed while counting.

Since the LSFM images are ten times thicker than the thinner sections needed in the absence of tissue clearing, counting with the Image Volume Fractionator probe can be done more quickly. It is much more efficient to count cells in one large image than in ten separate thinner tissue sections. There is also less sectioning artifact, which helps with registering the higher resolution LSFM images back to the larger volume MRI images.

We are excited to see this new use of the Image Volume Fractionator, which increases efficiency and reduces imaging distortions from physical sectioning. The potential that cleared tissue offers for increasing efficiency is great, but is still largely untapped. As this method is used more frequently, we look forward to hearing feedback from the research community to further improve the capabilities and usability of the Image Volume Fractionator in Stereo Investigator – Cleared Tissue Edition.

References:

1) A Multimodal Imaging and Analysis Pipeline for Creating a Cellular Census of the Human Cerebral Cortex 2021, Constantini, et al., https://www.biorxiv.org/content/10.1101/2021.10.20.464979v1

2) Hof, P. R., Nimchinsky, E. A., Celio, M. R., Bouras, C. & Morrison, J. H. Calretinin, Immunoreactive neocortical interneurons are unaffected in Alzheimer’s disease. 861 Neurosci Lett 152, 145-148 (1993).

3) Bussiere, T. et al. Progressive degeneration of nonphosphorylated neurofilament protei enriched pyramidal neurons predicts cognitive impairment in Alzheimer’s disease: Stereologic analysis of prefrontal cortex area 9. Journal of Comparative Neurology (2003).

4) Glausier, J. R., Fish, K. N. & Lewis, D. A. Altered parvalbumin basket cell inputs in the dorsolateral prefrontal cortex of schizophrenia subjects. Mol Psychiatry 19, 30-36 (2014). Lewis, D. A., Curley, A. A., Glausier, J. R. & Volk, D. W. Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci 35, 57-67 (2012).

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Williston, VT (December 10, 2020) — The ability to image large, intact biological specimens is about to get a whole lot better. Developed in collaboration with Columbia University, MBF Bioscience’s revolutionary light sheet theta microscope system, ClearScope®, has the ability to image whole tissue quickly and gently, in high resolution 3D.  

The ClearScope system features a unique, two-axis scanning mode that goes beyond the capabilities of typical light sheet microscopy. The patent pending design ensures the specimen is constantly illuminated using the thinnest section of the light sheet optimizing axial resolution. Shadow effects and uneven illumination, which hindered analysis in the past, are eliminated by the new technology, so that even the smallest subcellular structures within the tissue can be visualized with precision.  

Compatible with a range of tissue clearing techniques, including CLARITY, iDISCO, uDISCO, SeeDB and Sca/e, ClearScope has the ability to capture the details of very thick, very large specimens, such as intact tissue from human and primate brains. President and co-founder of MBF Bioscience, Jack Glaser says, “With low photo-bleaching, fast imaging speed, and high image quality, ClearScope is the most advanced tissue-imaging system in development today, and is set to revolutionize brain research.” 

Designed to work with a wide range of cleared specimens, the system is completely customizable and easily adapted to address the specific needs of researchers across a variety of disciplines.  

If you are interested in learning more about MBF Bioscience’s advanced new system for imaging large tissue specimens, visit: https://www.mbfbioscience.com/clearscope  

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About MBF Bioscience: MBF Bioscience integrates the world’s leading microscope systems with our revolutionary quantitative imaging and visualization software to accelerate research in the fields of: stereology, neuron and microvasculature reconstruction, vascular analysis, worm tracking, brain mapping and big image data management in medical research and education. 

Since 1988, MBF Bioscience has forged a rich history of creating innovative products to empower biological researchers with the quantitative analysis tools they need to obtain accurate, unbiased results. With offices in North America, Europe, Japan, and South Korea, MBF Bioscience has helped researchers across the globe publish over 15,000 peer-reviewed papers in peer reviewed journals. MBF Bioscience partners with the NIH and distinguished scientists across the world to continue their commitment to neuroscience research with their software technology, and also in the fields of stem cells, pulmonology, oncology, and toxicology. For more information visit www.mbfbioscience.com or follow MBF Bioscience on Facebook, Twitter, and LinkedIn. 

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Media Contact: 

Pasang Sherpa 
Marketing Manager 
1-802-288-9290 
pasang@mbfbioscience.com 

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Williston, VT (February 04, 2020) — MBF Bioscience’s revolutionary light sheet microscope system, ClearScope, sets a new standard for microscopic imaging.

The new decade is poised to bring about incredible scientific innovations, and MBF Bioscience is leading the charge in 2020 with the creation of the “light sheet theta microscope” system, ClearScope.

MBF Bioscience secured exclusive license from Columbia University to develop the light sheet theta microscope technology invented by Dr. Raju Tomer. This patent-pending technology in ClearScope leap frogs a number of inherent limitations of other light sheet microscopes. ClearScope will be commercially released this year.

ClearScope performs high-resolution, 3D imaging of intact, cleared specimens that are larger than any other light sheet microscope is capable of imaging. The technology permits fast imaging speed, high- quality imaging and low photo-bleaching. With the exclusive dual, oblique light sheets providing homogeneous illumination, MBF Bioscience has created a microscope system superior to all existing commercial light sheet microscopes. It is compatible with tissue clearing techniques including CLARITY, uDISCO, SeeDB, Scale and Binaree.

President and co-founder of MBF Bioscience, Jack Glaser says, “Dr. Tomer’s, revolutionary design of light sheet theta microscopy, and the new capabilities it provides, will have a dramatic impact in scientific research. We are excited to bring this technology to market in ClearScope. ”

Dr. Raju Tomer, assistant professor of biological sciences at Columbia University says, “I am very excited about the licensing of our light sheet theta microscope technology to MBF Bioscience for developing a robust and user-friendly ClearScope product. This technology has clear potential to revolutionize high-resolution, quantitative imaging of very large cleared samples. It will also be a valuable instrument for dynamic imaging of live specimens. I think MBF Bioscience, with its extensive expertise in neuroscience imaging and data analytics, is a natural partner to take it beyond a lab instrument stage, to benefit the scientific community at large.”

With the ability to image cleared tissue of virtually any XY size, ClearScope has the ability to completely revolutionize the way researchers image and collect data of tissue specimens. ClearScope image data can be analyzed with MBF’s flagship software solutions, Neurolucida 360, Vesselucida 360 and Stereo Investigator.

To learn more about ClearScope visit: www.mbfbioscience.com/clearscope

About MBF Bioscience: MBF Bioscience integrates the world’s leading microscope systems with our revolutionary quantitative imaging and visualization software to accelerate research in the fields of: stereology, neuron and microvasculature reconstruction, vascular analysis, worm tracking, brain mapping and big image data management in medical research and education.

Since 1988, MBF Bioscience has forged a rich history of creating innovative products to empower biological researchers with the quantitative analysis tools they need to obtain accurate, unbiased results. With offices in North America, Europe, Japan, and South Korea, MBF Bioscience has helped researchers across the globe publish over 15,000 peer-reviewed papers in peer reviewed journals. MBF Bioscience partners with the NIH and distinguished scientists across the world to continue their commitment to neuroscience research with their software technology, and also in the fields of stem cells, pulmonology, oncology, and toxicology. For more information visit www.mbfbioscience.com or follow MBF Bioscience on Facebook, Twitter, and LinkedIn, or compete in our image contest at www.neuroart.com

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Media Contact:
Pasang Sherpa
Marketing Manager
1-802-288-9290/ pasang@mbfbioscience.com

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The importance of studying the brain in three dimentions is something we understand at MBF Bioscience. Every day scientists around the world use our products to reconstruct neurons and analyze brain cells in 3D. That’s why we’re excited to hear about the new possibilities for whole brain analysis coming out of Dr. Karl Deisseroth’s lab at Stanford University.

A press release issued last week describes a whole-organ imaging process called CLARITY that made a postmortem whole mouse brain transparent.

The researchers used chemical engineering to replace lipids (the fatty elements of brain tissue that give the organ its form) with a hydrogel solution that congeals into a supportive mesh. After removing the opaque, impermeable lipids, Deisseroth’s team was left with a transparent brain with all its important structures intact.

After rendering the mouse brain transparent, the scientists used fluorescent antibodies to light up individual neural circuits, allowing for focused analysis of cellular structures as well as relationships between cells and their circuits.

“Studying intact systems with this sort of molecular resolution and global scope — to be able to see the fine detail and the big picture at the same time — has been a major unmet goal in biology, and a goal that CLARITY begins to address,” Deisseroth said in the press release.

Here at MBF Bioscience, we tested our Neurolucida software for neuron reconstruction with a sample image stack from a brain processed with a similar method. “We used Neurolucida perform automatic neuron reconstruction on a cell in an image stack representing a distance of over 1mm. Neurolucida was able to reconstruct dendritic arbors through the 1mm-stack in minutes. We’re excited to see how researchers in other labs will use our software along with whole-brain imaging in the future,” said MBF Bioscience President Jack Glaser.

Watch a video about the technique at nature.com.

Chung, K., Wallace, J., Kim, S. Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., … & Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems. Nature. doi: 10.1038/nature12107

Images provided by Dr. Kwanghun Chung, first author of the study.

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