Stereology Courses Offered this Fall

Researchers around the globe use stereology to efficiently and effectively gather quantitative data on the cellular level. The method is considered one of the most accurate ways to determine cell volume in 3D renderings of two-dimentional images. Two upcoming courses sponsored by MBF Bioscience offer hands-on instruction in the latest stereological methods.

At the Sixth Course “Morphometry and Stereology in Neurosciences,” taking place October 18-22 in Amsterdam, students will use Stereo Investigator to run stereological probes on virtual tissue files of a section of a mouse brain and a mouse striatum. Professors from the Department of Anatomy and Neuroscience at the VU University Medical Center will guide students through the steps of design-based stereology. Topics like morphometry, confocal microscopy, and histological shrinkage will be presented in lectures, exercises and demonstrations.

On Thursday, November 11, and Friday, November 12, Dr. Mark West will conduct an “Introduction to Stereology for Neuroscientists.” The workshop occurs in conjunction with the Society for Neuroscience’s annual meeting in San Diego. Limited to 30 students, the course focuses on stereological studies of the nervous system.

For more information, and to register for these courses, go to stereology.info.

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Register for Dr. Dan Peterson’s Practical Training Course in Confocal Microscopy and Stereology

There’s still time to enroll in Dr. Dan Peterson’s course, Practical Training Course in Confocal Microscopy and Stereology offered August 16-20, 2010 in downtown Chicago.

Course content includes:

  • Comprehensive and practical, “hands-on” instruction in state-of-the-art microscopy.
  • Spinning disk and laser scanning confocal microscopes equipped for stereology along with brightfield stereology stations are available for student use and instruction. Substantial amounts of course time are spent using the equipment.
  • Lectures on the fundamentals of tissue preparation and staining, microscopy, and digital imaging.
  • Theory and practice of design-based stereology.
  • Tutorial on the new application of confocal stereology, illustrated through the quantitation of neural stem cells.
  • Extensive group discussion- participants are encouraged to bring their own material and to discuss their projects.

Dr. Peterson is widely regarded as the leading expert in the field of confocal stereology. He designed the course for anyone who would benefit from a comprehensive introduction to microscopy and stereology, including graduate students who need to use modern microscopy or stereology in their projects, and investigators new to histological analysis who need a rapid and complete introduction to these techniques.

For more information and to register, please visit: http://www.neurorenew.com

P.S. Please note that MBF Mindset only recommends courses taught by capable and qualified instructors. It does not promote any course taught by commercially-affiliated instructors.

Woods Hole NeuroStereology Workshop

Interested in developing your design-based stereology skills? Dr. Mark West’s “NeuroStereology Workshop: An Introduction to Stereology for Neuroscientists” offers an excellent opportunity for scientists to refine their techniques.

The six-day workshop, held at the Marine Biological Laboratory in Woods Hole, Massachusetts, takes place April 17 – 22, 2010. A small group of graduate students, postdocs, and senior scientists will have the opportunity to design, supervise, and critically evaluate stereological studies of the nervous system, under the expert tutelage of Dr. West, neurobiology professor in the Anatomical Institute of Aarhus University in Denmark.

Learn more, and register for the workshop at Neurostereology.info.

See the full list of this year’s Stereology courses presented by MBF Bioscience

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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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Introduction to Stereology for Neuroscientists

There are still a few open spaces in Dr. Mark West’s “Introduction to Stereology for Neuroscientists” course on October 15-16, at the Silversmith Hotel in Chicago. Register for the two-day course, held immediately prior to this year’s meeting of the Society for Neuroscience, and learn how to design, supervise, and critically evaluate stereological studies of the nervous system.

Stereology is a methodology that provides meaningful quantitative descriptions of the geometry of three-dimensional structures from measurements that are made on two-dimensional images sampled from a structure of interest. The workshop will emphasize modern ”design based” stereology, which is characterized by the preparation and probing of tissue in such a manner that no assumptions about the size, shape, and orientation of the structures being quantified are necessary.

Stem Cell Transplants Aid in Spinal Recovery

SCI-Figure-9

by Dan Peruzzi, PhD.

Thousands of people in the United States have spinal cord injuries (SCIs), with associated loss of movement and sensation below the site of the injury. Neural and glial cell transplants into research animals after SCI have correlated with recovery of function. The improvement may be caused by the transplanted cells; it’s thought that remyelination by the transplanted glial cells is the main reason for the improvement. Also, if adult neural stem cells are transplanted, there is evidence they form new neurons. In “Analysis of Host-Mediated Repair Mechanisms after Human CNS-Stem Cell Transplantation for Spinal Cord Injury: Correlation of Engraftment with Recovery” (2009, Hooshmand MJ, Sontag CJ, Uchida N, Tamaki S, Anderson AJ, Cummings BJ, PLoS One) the authors use Stereo Investigator’s powerful quantitative tools to determine whether changes in the host environment may also be correlated with improved function.

Using Stereo Investigator, specific aspects of the host milieu were compared between spinal cord injured animals (Non-Obese-
Diabetic-severe combined immunodeficient mice) that received transplants of human central nervous system-stem cell neurospheres (hCNS-SCns) and those that did not. The sampling parameters such as section interval, grid size, and counting frame size, were determined by checking the coefficient of error to make sure it was low. In some cases, additional post-hoc power analysis of data from previous publications was used to demonstrate that the parameters were appropriate for the required precision. Serotonergic fiber length was estimated using the Isotropic Virtual Planes probe with a 60X objective. Blood vessel length was estimated using the Space Balls probe with a 40X objective. The areas and volumes of lesions, spared tissue, and astrogliosis, were estimated using the Cavalieri probe with a 20X objective. Stereological results were complimented by biochemical protein analysis. In addition, the Optical Fractionator probe was used to estimate a non-host parameter, the number of neurons that live and proliferate from the hCNS-SCns transplant.

There were no differences found in the host characteristics between hCNS-SCns transplant animals and control animals. For example, there was no difference in the length of blood vessels. Platelet/ endothelial cell adhesion molecule immunohistochemistry was used to identify blood vessels. Some treatments following CNS trauma may promote behavioral recovery associated with vascular remodeling. Blood vessel length was estimated at the injury center, one mm rostral, and one mm caudal to the injury. There was no statistical difference between controls and hCNS-SCns transplanted animals (see figure, controls are vehicle and human fibroblasts (hFb)). Regarding the non-host characteristic of how many transplanted cells lived, multiplied, and migrated, the Optical Fractionator estimate showed that the transplanted cell number increased 194 percent after transplantation and migrated from the injection site. Ablation of some transplanted cells with Diptheria toxin correlated with a loss of locomotor recovery. This study shows that the direct consequences of the transplanted cells such as proliferation -
correlate with improved function – while the transplant does not have an effect on host characteristics such as lesion volume,
spared tissue, fiber sprouting, and angiogenesis, ruling out any correlation of an indirect effect of the transplanted stem cells with recovery.

Dan Peruzzi is a staff scientist at MBF Bioscience.

First published in The Scope, fall 2009.

MBF Unveils Neurolucida and Stereo Investigator 9

Neurolucida 9

Image courtesy of Juan Carlos Tapia, PhD, Harvard University

Now available, release 9 of our flagship Neurolucida and Stereo Investigator systems represent one of the largest changes in the history of these products. With many new features and an improved interface, we’re listening to you, our loyal customers, and delivering products with innovation, power, and speed.

Stereo Investigator and Neurolucida New Features

Image Handling: We’ve been busy when it comes to handling and visualizing your valuable image data. High bit depth, Nchannel (up to 16 bits per channel) image files are now accessible via an intuitive user interface. Acquire up to 6 independent channels of high bit depth data per image from any of our cameras. You can even load dozens of image stacks at once. Our new image handling lets you work with the latest image file formats from the major confocal microscope manufacturers. We’ve enhanced your ability to work with virtual slides to include 3D virtual slide stacks and allow you to save portions of these large montage images (and stacks) to separate files. All of this is controlled by a new image display control feature providing precise histogram adjustments.

Automatic Contouring: Automatically trace contours while you watch the software identify the tissue boundaries from images, image stacks, virtual slices, and even off the live camera feed. If you need to move the stage, the software does it automatically and continues tracing the outline of your region.

Automatic Object Detection: Now with just a few clicks you can let the software find cells, synapses, grains, or similar objects of interest in your images. Mark and count the objects or ask our software to automatically trace around the outside of an object. JPEG2000 Image File

Support: We now offer state-of-the-art image file storage with JPEG2000 support, which dramatically reduces disk space usage for all your image data. For instance, you’ll see image stacks that were 100 MB shrink to about 5 MB, depending on the image data and compression ratio you choose. Create very large virtual slices that exceed the computer’s physical memory by using JPEG2000 support, and store these files
using manageable file sizes. An example, we recently captured a large virtual slice (556,800 x 520,000) that compressed to only 40 GB; with earlier technology, it saved as an 800 GB image file.

Multiple User Setup: If you have a system that serves many users, you’re life just got easier. Use one login with all users or give each user a private login with their own customized settings. If you have a system with multiple hardware setups, this also makes it easy to keep the settings separate and switch between them.

Support for Aperio and Hamamatsu Nanozoomer Files: Now you can load Aperio SVS and Nanozoomer virtual slide files into Neurolucida or Stereo Investigator for analysis.

Improved 3D Visualization: A fully redesigned 3D visualization window makes it easier to view, manipulate, and understand image stacks and tracings. We’ve included cut planes in the X, Y, and Z axis from any view angle. Manipulate the image transparency to get the look you want from your image and tracing data. Look for the new automatic rotation tool that lets you adjust the angle of rotation
and speed.

Hardware: We’ve added support for monochrome and color cameras under 64-bit Vista and Windows 7. Our streamlined camera interface gives you the image you desire easily and quickly. Now, swap between multiple cameras without exiting the application to easily utilize the multiple camera ports on your microscope. Zeiss AxioCam, QImaging, and Baumer cameras are part of our line as well, along with tight integration for the Zeiss ApoTome and support for the Qioptic Optigrid structured illumination offerings.

Simplified Menus and Hot Keys: Streamlined workflows with more intuitive menus and dialog, along with new navigation keys help you maneuver around with less effort.

Drag and Drop: Drag and drop your images, image stacks, and data files for easier loading and display.

Tracing with Transparency: Ever want to look at or trace something where the tracing was in the way? Now simply adjust the transparency of the tracing to see the underlying image while tracing.

Serial Sections: Do you trace your sections separately and combine them later? We’ve made it much easier and intuitive to combine files together into a complete data file with fewer steps. Use the improved Serial Section Manager to define many sections in one step and view multiple sections.

These are just some of the new enhancements found in version 9.

More Neurolucida 9 Additions

Neurolucida has added new analysis tools for tracing and classifying dendritic spines, and improved editing features to correct your AutoNeuron tracings.

And More In Stereo Investigator 9

Stereo Investigator has a number of other new features including the Connectivity Assay (Pulmonary Edition). New enhancements for the Optical Fractionator include faster counting of multiple cell populations, new displays for the results, and improved Excel export for easier and more comprehensive reports. The Area Fraction Fractionator is also faster and more streamlined. An important
new tool, the Oversample and Resample allows you to methodologically pick the most efficient sampling parameters for your material.

First published in The Scope, fall 2009.

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New, Easy Setup of Multiple-User Accounts

by Julie Keefe, M.S.

With version 9.0 of our software, you can now easily configure a Neurolucida or Stereo Investigator system to support multiple users, each with their own personalized settings.

There is no need to worry if the program is pointing to the right directory or accessing the correct configuration files for each user.

The new multiple-user setup only takes about 30 seconds for each user. It includes a Group option for different laboratories using the same system, and it also allows individual Profile log-ins within each group.

You can create different groups for your lab, or you can organize by experiment type, with shared parameters, to assure consistency among users.

To create a new profile, all you need to do is import the configuration file from an existing profile. In seconds, the settings and calibrations are completed, and you can begin working with the software. This feature is ideal for core facilities, since it has the added benefit of reducing the administrative overhead for the manager.

You can simply adjust your settings and be assured that those settings will be remembered whenever you return to work at that computer.

If you are an existing customer, this new setup uses the same configuration files you are already using. There is no need to perform another setup, and it automatically applies any needed updates.

Julie Keefe is the technical support manager at MBF Bioscience.

First published in The Scope, fall 2008.

Need Help With Stereology? Consider an MBF Labs Pilot Study

by Susan Hendricks, Ph.D.

Stereology is a powerful tool that can reduce your workload and provide accurate, unbiased quantification. Unfortunately, stereology is often not the most intuitive method for the uninitiated. If it isn’t done properly, the results you attain may not be accurate. Here’s where we can help. Not only do we make Stereo Investigator, the most cited software for stereology, but now we’re offering to do your work for you. Send us your slides, and we’ll complete a comprehensive pilot study for you; providing an objective analysis of your tissue thickness and staining penetration, outlining sampling parameters and counting procedures, and providing you with the knowledge and confidence you need to collect the remainder of the data yourself. Our staff includes experienced Ph.D. research scientists who are experts with stereology methodology, our software, and who have demonstrated experience in neuroscience research applications.

Using a technique created at MBF and adapted from Slomianka and West (2005), we will calculate an adequate sampling fraction based on the section thickness, section interval, and the frequency of the objects to be counted within your tissue. We can also empirically determine the appropriate guard zones for your tissue. This data will be provided to you with the return of your slides in a report detailing how the parameters were chosen. Additional support as you begin your work is also available; we can walk you through your first counting procedure or even audit your work to ensure that data collection is proceeding as expected. Confidence in the technique will translate to confidence in the results.

Consider using our pilot study service as a way to optimize and streamline the quantification for all the animals in your study. And by using MBF to acquire the data of your pilot study, you are saved the time and labor involved in oversampling, to free you and your staff to complete other important experiments. Our research staff scientists and stereology technicians will work with you to outline the histology requirements for your experiment. Based on your hypothesized result, the number of animals needed for the pilot study will be determined. Do you expect to see a large, robust difference between control and experimental groups? Provide a subject from each group to ensure that the sampling is as efficient as possible. Need to remain blind to condition? The stereology pilot study should be performed, at a minimum, on the group in which you expect the fewest number of objects. This will guarantee that enough data is collected for the remainder of the subjects in the study.

A properly designed stereology pilot study will reduce time, effort, and costs for the overall experiment by optimizing the sampling parameters to identify how many sections are needed (and therefore generated and stained) to obtain a result with adequate precision.

Of course, if you want to outsource your complete study, we would be glad to help you with that as well.

For more information, visit mbflabs.com.

Citation: Slomianka L and West MJ (2005) Estimators of the precision of stereological estimates: an example based on the CA1 pyramidal cell layer. Neuroscience. 136(3): 757.

Susan Hendricks is a staff scientist at MBF Bioscience.

First published in The Scope, summer 2008.

Stereology: Avoiding Bias Using the Optical Fractionator

Optical Fractionator Stereo Investigator 8

by Dan Peruzzi, Ph.D.

Stereology allows us to estimate the amount and size of biological features that are impossible or prohibitive to measure exhaustively. If the stereological probe is designed well, and we can manage to
follow all the rules, bias due to sampling design and estimation methods will be eliminated, resulting in a more accurate estimate. For instance, when using the Optical Fractionator to estimate the number of cells in a particular region, we follow rules to make sure a given cell cannot be counted more than once. The reminders listed here will help you avoid some of the common sources of bias.

Section the Entire Region of Interest

Sampling should be systematic and random. Use the cryostat, vibratome, or microtome to take sections of the whole anatomical area; not just at a supposedly representative section or sections. Pick a section interval and randomly choose the starting section for each animal. This gives every section throughout
the entire region an equal chance of being picked for sampling.

Choose a “Point” on the Cell

If you apply the counting rules to the whole cell, large cells, and those oriented perpendicular to the plane of sectioning, will have a greater chance of being counted than smaller cells or cells with parallel orientation. To avoid size and orientation bias, do not use the whole volume of the cell as the basis of your counting decisions. Instead, dwindle the volume down to a point as much as possible. The finer the resolution in the optical plane (focus), the easier this is to do. The ‘point’ you choose must be uniquely identifiable on the particle. Some suggestions include the cell ‘top’ (i.e., the first part of the cell to come into focus) or the cell nucleolus, if there is one and only one per cell.

Use Thicker Sections

An Optical Fractionator probe typically requires approximately ten to fifteen optical planes through the z-axis of the disector. This is easier to achieve with better z-resolution and with thicker sections. Having many optical planes, instead of just a few, allows for a finer visual discrimination. The advantage is being able to distinguish more easily where the “point” on the cell is in order to determine if it falls inside the disector and should be counted. With fewer optical planes, observer bias can result and begin to reach significant levels.

Eliminating bias is desirable, but we must also do the proper amount of sampling, so the estimate will be precise. This makes it more likely that any given estimate will be closer to the mean of all possible estimates. In other words, eliminating bias will not be enough to efficiently get a good estimate. You must also do enough work for your required level of precision, but not so much that you are wasting time and valuable resources.

Learn more about using the Optical Fractionator at mbfbioscience.com.

Dan Peruzzi is a staff scientist at MBF Bioscience.

First published in The Scope, summer 2008.