Japanese Researchers Develop New Optical Clearing Agent; Neurolucida Used For 3D Imaging in Study

Volume rendering of mouse cerebral cortex and hippocampus. Adult Thy1-YFP-H line mouse brain was cleared with SeeDB and imaged using two-photon microscopy. Imaging area shown is 4 x 5 mm (8 x 10 tiles), 2mm thick. We could easily make a volume rendering from a large set of 3D data (in this case, 9GB two-photon data).

Volume rendering of mouse cerebral cortex and hippocampus. Adult Thy1-YFP-H line mouse brain was cleared with SeeDB and imaged using two-photon microscopy. Imaging area shown is 4 x 5 mm (8 x 10 tiles), 2mm thick. We could easily make a volume rendering from a large set of 3D data (in this case, 9GB two-photon data).

A new optical clearing agent developed by scientists in Japan clears brain tissue samples with greater transparency and less time than other clearing agents, according to a paper published in Nature Neuroscience.

“Combined with two-photon microscopy, SeeDB allowed us to image fixed mouse brains at the millimeter-scale level,” say the authors, who after clearing the brain tissue with SeeDB, captured images with a multiphoton Olympus microscope, and visualized 3D reconstructions with Neurolucida.

A solution of fructose, water, and alpha-thioglycerol, SeeDB cleared gray and white matter brain tissue samples in three days without affecting the volume or morphology of the tissue. Dendritic spines of pyramidal neurons in the cerebral cortex was one aspect of fine morphological architecture that the authors note remained intact after SeeDB treatment.

Reconstruction of lateral dendrites of sister mitral cells. Fluorescent neuronal tracer (Alexa647 dextran amine) was electroporated into a single glomerulus to label 'sister' mitral cells associated with a common glomerulus. After optical clearing of the olfactory bulb with SeeDB, the olfactory bulb was imaged using confocal microscopy. Lateral dendrites of labeled mitral cells were reconstructed using Neurolucida. This reconstruction was used for quantitative analysis of 'sister' mitral cell distribution.

Reconstruction of lateral dendrites of sister mitral cells. Fluorescent neuronal tracer (Alexa647 dextran amine) was electroporated into a single glomerulus to label ‘sister’ mitral cells associated with a common glomerulus. After optical clearing of the olfactory bulb with SeeDB, the olfactory bulb was imaged using confocal microscopy. Lateral dendrites of labeled mitral cells were reconstructed using Neurolucida. This reconstruction was used for quantitative analysis of ‘sister’ mitral cell distribution.

Continue reading “Japanese Researchers Develop New Optical Clearing Agent; Neurolucida Used For 3D Imaging in Study” »

Researchers at Stanford use confocal stereology to study neurodevelopment

A Stereo Investigator system for confocal stereology was installed in Dr. Michelle Monje’s lab in the Department of Neurology and Neurological Sciences at Stanford University School of Medicine. Dr. Monje and her lab members will use the system to investigate the molecular and cellular mechanisms of postnatal neurodevelopment.

Dr. Julie Korich, staff scientist at MBF, installed Stereo Investigator on a Zeiss laser scanning confocal microscope and trained the lab members on how to use the system.

From top left: Chris Mount, Grant Lin, Ingrid Inpma, David Purger From bottom left: Elizabeth Qin, Viola Caretti, Lauren Wood

From top left: Chris Mount, Grant Lin, Ingrid Inpma, David Purger
From bottom left: Elizabeth Qin, Viola Caretti, Lauren Wood

During the training, Dr. Korich discussed how Stereo Investigator integrates with Zeiss’ microscope software, explained the Cavalieri probe for estimating regional volume, and showed the lab how to use Stereo Investigator to collect confocal image stacks in the systematic and random way that’s necessary for unbiased stereology. She also explained how to count cells from those image stacks and from 3D virtual slides with Stereo Investigator on a computer away from the microscope.

Click here to learn more about how researchers are using Stereo Investigator in their labs.

On-Site: First MBF Bioscience System Installed in Africa

From left: Jacquie, Tyrone, Susan, Liz, Mauritz, Hayley, Jose

Africa’s first MBF Bioscience system was installed at the University of Cape Town in South Africa last month.  The head of MBF Bioscience Latin America, Dr. Jose Maldonado, integrated Stereo Investigator with a Zeiss 510 Confocal microscope in the Confocal and Light Microscope Imaging Facility at Dr. Dirk Lang’s lab.  Jose also trained the team on how to use the system and gave a talk on stereology theory during his visit.

Comprising a wide range of research areas including neuroscience, cell biology, and applied anatomy, the University of Cape Town’s Confocal and Light Microscope Imaging Facility provides state-of-the-art equipment for advanced fluorescent image acquisition and analysis.

We’re pleased to be able to provide tools for Dr. Lang and his team’s research, and thrilled to have a system in South Africa! Here are a few photos from Jose’s training session.

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Our Confocal Stereology Webinar Recording is Available to View Online

Confocal microscopy offers clear access to the tiniest biological elements. Stereology provides an exceptional way to quantify them. Combine the two and you’ve got an unparalleled tool for conducting research.

Our latest webinar “Confocal Stereology: A Technical Innovation from MBF Bioscience and Carl Zeiss Microscopy” provides an in-depth look at confocal microscopy, stereology, and how the two can work together. MBF Bioscience Staff Scientists Dr. Susan Hendricks and Dr. Jose Maldonado demonstrate how a Zeiss LSM confocal microscope powered by Zeiss Zen and Stereo Investigator® is a powerful tool for quantifying spines, synapses, co-labeled cells, or fibers in a variety of biological tissues.

Watch the webinar for step-by-step instructions on how to acquire and analyze confocal SRS Image Stacks, see how much easier it to see bright tightly packed small objects in a confocal image, and find out why Stereo Investigator along with Zeiss Zen is the most tightly integrated and easy to use solution for confocal systematic random sampling stereology.

To find out about upcoming MBF Bioscience instructional webinars, “Like” us on Facebook and follow us on Twitter.

 

Upcoming Webinar: Confocal Stereology – A Technical Innovation from MBF Bioscience and Carl Zeiss

On Thursday, October 6, Dr. Jose Maldonado and Dr. Susan Hendricks present a live webinar on confocal stereology with Stereo Investigator and the ZEISS LSM confocal microscope.

Stereological quantification of cells, synapses, or other very small structures benefit from the high resolution imaging of laser scanning confocal microscopes. MBF Bioscience and Carl Zeiss have partnered to bring you the most user-friendly and efficient solution for laser scanning confocal stereology.

Stereo Investigator combined with the latest ZEISS LSM confocal microscopes is the world’s most fully integrated system for using stereology to quantify co-labeled objects with the precision of laser scanning confocal microscopy. Additionally, the off-line capabilities of Stereo Investigator workstations permit more effective use of your confocal system by making it available for other users.

Click here to register for Thursday’s webinar.

Q&A: Dr. Daniel Peterson Discusses His Courses on Microscopy and Stereology

Talk of zebrafish and Spaceballs probes make Dr. Daniel Peterson’s microscopy and stereology courses sound intriguing. But Dr. Peterson says it’s the hands-on instruction that students enjoy most about the biannual workshops he teaches in Chicago. From August 15-19, 2011, the Associate Professor and Executive Director at the Center for Stem Cell and Regenerative Medicine at The Rosalind Franklin University of Medicine and Science, welcomes students to Chicago’s Club Quarters for five days of  instruction on all aspects of state-of-the-art microscopy, stereology, and histological analysis.

He spoke to us about the atmosphere in the classroom, what kinds of students take his course, and what aspects of the workshops participants get most excited about. Continue reading “Q&A: Dr. Daniel Peterson Discusses His Courses on Microscopy and Stereology” »

In Focus: Dr. Clara Thore

Who: Clara R. Thore, Ph.D., Computer Imaging Specialist

Where she works: The Microvascular Research Lab, Department of Diagnostic Radiology, Wake Forest University School of Medicine , Winston-Salem, North Carolina

Research focus: Microvascular changes in the brain in diseases of aging, such as vascular dementia, leukoaraiosis, and Alzheimer’s disease.

MBF Bioscience software used: Stereo Investigator

Research methods at a glance: Dr. Thore and her fellow researchers have come a long way from their early studies involving scanning AP stained sections on a flatbed scanner to measure vessel density. Today, they use stereological methods, which provide more accurate data and take less time.

What she does: Dr. Thore studies the brain’s tiniest blood vessels. She examines human autopsy material and highly sensitive archival tissues for her research on Alzheimer’s disease, vascular dementia, and other diseases of aging.

At her lab, The Brain Microvascular Pathology Laboratory, she and her colleagues use Stereo Investigator to quantitate vascular densities in the human brain and the brains of experimental animals. One hundred-micrometer-thick sections of celloidin-embedded tissue blocks with arteries, arterioles, and capillaries marked with AP staining provide Dr. Thore with a 3D view of her subject. By implementing stereology, immunohistochemistry, and confocal microscopy techniques, she studies changes to the cerebral microvascular system.

Previously, Dr. Thore and her team measured vessel density by scanning the AP stained sections on a flatbed scanner, which she said provided crude area fraction data. They then captured digital images from sections and used algorithms to process the images to binary in order to quantitate vessel density as area fraction. “The image processing introduced variance, was time consuming, and did not account for the depth of the captured field,” she explained. Dr. Thore and her colleagues began using stereological methods, which they found avoided these problems by providing random systematic sampling.

“The installation of the Stereo Investigator system by MBF Biosciences has made a huge difference in our quantitative approach,” said Dr. Thore. “By using the built-in Stereo Investigator Space Balls probe, we no longer have to be concerned with the directionality of vessels in our sections. Stereological quantization is done real-time, which has decreased the time needed per specimen and allowed us to increase the number of areas and slides sampled.”

“In Focus” is a new series that spotlights scientists who use MBF Bioscience products in their work. Thank you Dr. Clara Thore for participating.

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Confocal Microscopy and Stereology Courses

Dr. Daniel A. Peterson’s practical training courses offer excellent, hands-on instruction in the use of confocal microscopes and design-based stereology. Each year, Dr. Peterson, a Chicago-based neuroscientist and Executive Director of the Center for Stem Cell and Regenerative Medicine at Rosalind Franklin University holds two training sessions. This year’s events take place March 8-12 and August 16-20.

From graduate students, to post doctorates, to advanced laboratory technicians, anyone in the biomedical field who uses qualitative and quantitative microscopy in their research would benefit from the week-long courses. Attendees will gain a comprehensive overview of  modern histological preparation and microscopic analysis, and are encouraged to bring their own material for discussion and customized consultation.

Registration for the March course ends February 26. Go to neurorenew.com to enroll.

{Image courtesy of Neuro Renew Inc.}

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Integration with the CARV II and Qioptiq OptiGrid

Carv II

We are pleased to announce that we have added support for two leading confocal devices for microscopy. The CARV II and the Qioptiq OptiGrid are now completely integrated with the MBF family of software solutions. These devices enable Stereo Investigator to perform stereology in a confocal environment and allow Neurolucida, AutoNeuron, and Virtual Slice to benefit from better visualization due to the thin depth of field.

Both devices are microscope independent; there is no need to worry about losing the investment you already have in your current working microscope. You can integrate the microscope of your choice. This means added support for acquisitions of multichannel images, image stacks, and virtual slides.

The CARV II (pictured above), currently offered by BD Biosciences, includes a spinning disk with multiple sets of pinholes arranged in a spiral and placed in the image plane of the objective lens. According to the product web site, “the column of excitation light is projected through 1,000 pinholes to simultaneously scan the entire field once every millisecond, thereby creating a full image of the focal plane in real time.”

Qioptiq Optigrid

The Qioptiq OptiGrid Structured Illumination Microscopy system affords “benchtop access to confocal-quality imaging right from the convenience of your own lab.” It enables multichannel fluorescence and real-time 3D imaging with a lower cost. The OptiGrid also integrates standard illumination and operation with much of your existing instrumentation and software. Programmable focus and experiment automation mean simpler multi-channel imaging.

First published in The Scope, fall 2008.