## Just for you: FREE 60-day trial to the world’s largest digital library.

The SlideShare family just got bigger. Enjoy access to millions of ebooks, audiobooks, magazines, and more from Scribd.

Cancel anytime.Free with a 14 day trial from Scribd

- 1. Stereology Theory and Experimental Design Julie Korich, Ph.D. Staff Scientist/Research Liaison
- 2. Demystifying Stereology
- 3. mbfbioscience.com • What are you quantifying? • How will you quantify it? • How do you validate results? Basic Questions Artwork by Sidney Harris
- 4. mbfbioscience.com What are you quantifying? • Need to quantify 3D structures in various brain regions • How do you quantify volume of the cortex? • How do you quantify motor neuron number in the spinal cord? • How do you quantify sprouting in the cerebellum? Whole brain image courtesy of http://science.nationalgeographic.com/science/photos/brain/ Motorneurons image courtesy of MBF Bioscience Cerebellum image courtesy of Dr. Tamily A. Weissman.
- 5. mbfbioscience.com Sampling from Tissue Sections • Measure 3D parameters (number, area, volume, length)on tissue sections http://www.boneclones.com/KO-515.htm
- 6. mbfbioscience.com The How… Non-stereological Methods • Profile counts • Exhaustive Sampling-sample every event in every section through the region of interest • Pitfalls: • Inefficient • Laborious • Biases • One representative section • Pitfalls: Introducing Bias www.PHDComics.com
- 7. Profile Counting: Size and Orientation Bias mbfbioscience.comC. Schmitz and P. R. Hof. Neuroscience 130 (2005) 813–831
- 8. Profile Counting: Double Counts mbfbioscience.com 3 4 4 3 3 3 Profile Counting • Counter would report 20 cells. However, there are only 8 cells. • Also, if using exhaustive sampling, it is necessary to count EVERY cell in EVERY section.
- 9. mbfbioscience.com ‘One Representative Section’ Counts within a single field-of-view (white box) would lead to the false impression that Animal 1 has fewer cells than Animal 2 in the entire region of interest. A B Animal 1 Animal 2
- 10. mbfbioscience.com In Summary: Non-Stereological Methods • Non-stereological sampling can be biased in addition to laborious • With stereological techniques sampling bias is avoided – every event has an equal opportunity of being sampled • Stereology does not make assumptions regarding size, shape, orientation or distribution • Therefore, stereology is considered the gold standard for quantification in neuroscience
- 11. Design-Based Stereology • The sampling is performed on a sub-fraction of the entire region • Within each section, only a subsample is evaluated • Systematic sampling is highly efficient and provides sampling consistency across and within sections • A randomized offset ensures unbiased measures
- 12. mbfbioscience.com What is Stereology • The process of obtaining unbiased, meaningful, quantitative measurements of three dimensional objectives from two dimensional information • The geometrical properties of features in 3-D space can be quantified by „throwing‟ random geometrical probes into the space and recording the way in which they intersect with the structures of interest. • Unbiased Stereology, Second Edition, 2005, Howard, C.V. and Reed, M.G., QTP Publications, Liverpool, page 8.
- 13. Geometric Probes • Geometric probes used for the sampling • Points for volume • Lines for surface area • Planes for lengths • Volume for numbers • Geometric probes are required to report 3D data mbfbioscience.comHoward CV, Reed MG: Unbiased Stereology. 2nd ed., Bios, Oxford, 2005
- 14. mbfbioscience.com Design-Based Stereology •Used to avoid sampling bias and error • Sample whole region using systemic random sampling •Requires isotropy to prevent bias • Ensures that all positions in the structure have the same likelihood of being sampled • How do you achieve isotropy • Object Orientation • Tissue Preparation • Probe
- 15. Achieving Sampling Isotropy Object Orientation • Some objects are isotropic while others have a preferential orientation • If your object population of interest is anisotropic… AnisotropicIsotropic Wikimedia.org
- 16. Achieving Sampling Isotropy • Isotropic tissue sections • All three planes are randomized (3D spin) in the tissue before sectioning • Vertical tissue sections • Two planes are randomized (2D spin) in the tissue before sectioning • Preferential tissue sections (e.g., coronal) • Because the orientation of the tissue is specified, the object or the probe must be isotropic Tissue Preparation Allen Brain Atlas, http://www.brain-map.org/
- 17. Achieving Sampling Isotropy • Using isotropic probes frees you from having to either prove that your objects are isotropic or make your tissue isotropic Stereology Probe
- 18. Stereology Probes Feature • Cell Population • Regional Volume • Area Fraction (fraction of cortex occupied by plaques) • Fiber Length Isotropic Probes • Physical/Optical Fractionator • Cavalieri • Area Fraction Fractionator • SpaceBalls mbfbioscience.com • Cell Size • Nucleator Feature Anisotropic Probes
- 19. mbfbioscience.com Physical Disector • View 2 adjacent thin sections. Sections need to be thinner than the cells being counted • Count cells that appear in one section (green inclusion plane) but not the other (red exclusion plane) • Ideal for very small (e.g EM) or very large structures (e.g. kidney glomeruli)
- 20. mbfbioscience.com Optical Disector • Why not use thick sections and focus through (optical sections) rather than using two thin adjacent sections? • As focus through the tissue, count cells as they appear following specific counting rules • Sections need to be thick…
- 21. mbfbioscience.com Optical Disector • Isotropy is ensured by identifying and marking a unique point • The counting frame combined with the fractionator improves sampling efficiency • Typically it is not required to sample every cell within a section
- 22. mbfbioscience.com The Optical Fractionator • Sampling is done following systematic random sampling (SRS) • The counting frame is laid down on a systematic grid that is randomly placed on the anatomical area of interest
- 23. mbfbioscience.com The Fractionator Overview: A: Entire ROI B: The region of interest has been sectioned with an interval of 2 - every other section will be sampled C: Within each section, a fraction of the tissue will be sampled using the optical fractionator D: 3D view of the optical fractionator and disector Anderson and Gundersen. Journal of Microscopy, Vol. 196, Pt 1, Oct1999, pp. 69±73.
- 24. Formula for the Optical Fractionator The cell population is determined by sampling a subset or subfraction of tissue within the region of interest. Population estimate, N, is equal to: Reciprocal of Volume Fraction X Sum of Counts = N∑Q-1 Volume Fraction X mbfbioscience.com
- 25. Three components constitute the volume fraction: 1. Height sampling fraction (hsf): How much of the tissue (thickness) was sampled (e.g., 80%) 2. Section sampling fraction (ssf): How many sections you examine (e.g., every 4th) 3. Area sampling fraction (asf): How much of each section‟s area was sampled (e.g., 25%) Calculating the Volume Fraction mbfbioscience.com
- 26. mbfbioscience.com Height Fraction:hsf • Disector Height is the thickness of the tissue sampled • Average Mounted Section Thickness is the thickness of the tissue after processing • The disector height ≠ average mounted thickness • The cut surfaces of the tissue can be disturbed to the point that counting is inaccurate. Therefore, only a portion of the tissue is used for counting - disector height
- 27. mbfbioscience.com Guard Zones “Plucked Cell” “Lost Cap” Section Top Section Bottom Side View Disector Height Top Guard Zone Bottom Guard Zone Disector Height
- 28. mbfbioscience.com • Thickness should be measured at every sampling site • Assumptions pertaining to the post-processing thickness can lead to sampling bias and error • Processing of tissue results in shrinkage • With some techniques, tissue can shrink 80% • Avoid assuming shrinkage is homogenous across ages, groups, etc. • Processing can also result in uneven shrinkage – wavy tissue Section Thickness
- 29. mbfbioscience.com Section Sampling Fraction: Lateral View Dorsal View In your experiments you will sample a subset of sections through the region of interest = section interval
- 30. mbfbioscience.com Section Sampling Fraction: ssf | A | A | A | A | A | B | B | B | B | B | C | C | C | C | C | D | D | D | D | D | E | E | E | E | E 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 15 | 14 | 16 | 17 | 18 | 19 | 21 | 20 | 22 | 23 | 24 | 25 | • The interval is systematic (e.g. every 5th section is sampled) • The starting section needs to be random
- 31. mbfbioscience.com • The counting frame ( ) ensures that objects are counted once and only once • The grid ensures that a fraction of the tissue is sampled in a systematic and random manner • Once defined, the grid spacing and counting frame size cannot be changed • Placement of the grid on the ROI is random (via Stereo Investigator) Area Sampling Fraction: asf
- 32. mbfbioscience.com Area Sampling Fraction: asf
- 33. mbfbioscience.com = N∑Q-1 Volume Fraction X Optical Fractionator: Recap • Report the total cell population within the region of interest independent of volume • Important to understand the volume fraction and its components: hsf, asf and ssf • Stereology is not magic its math!
- 34. mbfbioscience.com • What are you quantifying? • Global measures – cell numbers, volumes, area, lengths • How will you quantify it? • Non-stereological methods • Stereology • How do you validate results? • Accuracy vs. Precision • Experimental Design • CE • Pilot Study Basic Questions Artwork by Sidney Harris
- 35. • With sampling, a given estimate of a population will vary from the true number • The goal of stereology is to ensure that the individual sampling error does not overshadow the difference due to experimental manipulation • High Precision, Low Accuracy • High Accuracy, Low Precision • High Precision, High Accuracy mbfbioscience.com True number Accuracy vs. Precision
- 36. Know Your Question • Shape of the region of interest • Uniform in shape: fewer sections • Non-uniform shape: more sections mbfbioscience.comahappyvalentine.blogspot.com geradandlauracoles.com
- 37. Know Your Question • Are the objects normally distributed in your region? • Evenly distributed in structure: fewer sections • Unevenly distributed in structure: more sections mbfbioscience.com
- 38. Know Your Question • How frequent are your objects? • Dense population (more spots on the pup): fewer sections • Sparse populations (fewer spots on the pup): more sections mbfbioscience.commbfbioscience.comhttp://dalmatian-dog-lovers.blogspot.com/
- 39. Know Your Question • Are the objects normally distributed within a section? • Evenly distributed in section: fewer disectors • Unevenly distributed in section: more disectors mbfbioscience.comImages courtesy of MBF Bioscience
- 40. Designing Your Study • How do you plan to visualize the tissue? • Brightfield • Fluorescence • Tissue collection • Collect tissue through the entire ROI • Different series can be used to label different biological features • Cut the tissue at the proper thickness for the probe being used • Same sections can be used for multiple probes • Staining must penetrate entire thickness • „Garbage in, Garbage out‟ 1. www.randform.org; 2.www. brainmuseum.org; 3. Courtesy of Dr. Daniel Peterson 1. 2. 3. mbfbioscience.com
- 41. Tissue Considerations mbfbioscience.comDorph-Petersen,, K.A, Nyengaard, J.R., Gundersen, H.J. G... Journal of Microscopy, Vol. 204, Pt 3, December 2001, pp. 232±246.
- 42. Tissue Considerations Dorph-Petersen,, K.A, Nyengaard, J.R., Gundersen, H.J. G... Journal of Microscopy, Vol. 204, Pt 3, December 2001, pp. 232±246.
- 43. Microscope Considerations • High resolution and a thin depth of field are required to discriminate between objects on top of each other • Necessary for the Optical Fractionator Objective Approx. Depth of Field 40 x (NA 0.65) 1.84 m 40 x (NA 0.95) 0.98 m 60 x (NA 1.0) 0.68 m 100 x (NA 1.4) 0.58 m Image courtesy of Chandra Avinash, http://photography.learnhub.com/lesson/page/41-understanding-depth-of-field
- 44. Source of Methodological Errors mbfbioscience.com • Observer • Defining the ROI • Properly counting cells using the counting rules • This is always present • Sampling • Sampling within sections (noise) and across sections • Number of animals • Number of sections • If enough sampling is performed, the error introduced by your methods will be reduced Modified from Mark West NeuroStereology Workshop 2010
- 45. • Coefficient of Error (CE) is an estimate of the precision of the population size estimate • Reported per animal • A lower CE indicates less chance for sampling error and greater chance for an accurate estimate mbfbioscience.com Coefficient of Error OCV2 = CV2 OCE2+ Observed Group Variance Biological Variabiliy Methodologically Introduced Variance Common CE equations: Gundersen (m=1),Schmitz-Hof
- 46. Why is the CE Important? mbfbioscience.com • If the results are not significant (no difference between groups), could increasing the precision achieve the desired result? • Increase precision (decrease the CE) by sampling more • Helps other researchers evaluate the validity of the results • Important for optimizing your study Modified from Mark West NeuroStereology Workshop 2010 Figure: Simpson, J. et. Devel Neurobio. 2013 Jan;73(1):45-59.
- 47. • Perform a Pilot Study and check the CE • Understand the cellular distribution • Even distribution and/or high density: visit fewer sites per section • Uneven distribution and/or low density: visit more sites per section • It is more efficient to visit more sites per section than increase the number of sections From Theory to Practice mbfbioscience.com
- 48. The pilot study is designed to select sampling parameters that obtain accurate data with low sampling error and the greatest amount of efficiency. It takes into account: • Probe choice • Region of interest • Section thickness & histology • Object distribution The Pilot Study mbfbioscience.com
- 49. Interpreting the Pilot Study • Oversample one animal • Recalculate the estimations using MBF‟s resampling, oversample • Look for the „sweet spot‟ • If visit fewer sites per section, what happens to the estimation • If visit few sections, what happens to the estimations • Optimize the section interval and SRS grid dimensions for remaining study mbfbioscience.com 20000 30000 40000 50000 60000 70000 80000 0 1 2 3 4 5 6 Section Interval CellEstimation 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 SRS Site Interval CellEstimation Figures courtesy of MBF Bioscience
- 50. Pilot Study for Thin Tissue • Tissue is thinner than recommended • Sample pilot study animal without guard zones • Measure the section thickness at every site • Count cells through the entire thickness • Run the data file though MBF‟s resample disector mbfbioscience.com 0 5000 10000 15000 20000 25000 1 2 3 4 5 6 7 Guard Zone Height ( m) CellEstimation Figure courtesy of MBF Bioscience
- 51. mbfbioscience.com Other Probes Cavalieri Area Fraction Fractionator Spaceballs Nucleator
- 52. mbfbioscience.com Area and Volume Estimation: Cavalieri Point Counting • Area of an object is estimated by point counting • Volume of the object is estimated by summing the areas and multiplying by the slice thickness • Used for volume measurements of anatomical regions • Done at low magnification on a single plane Howard CV, Reed MG: Unbiased Stereology. 2nd ed., Bios, Oxford, 2005
- 53. mbfbioscience.com Cavalieri Point Counting Figures courtesy of MBF Bioscience
- 54. mbfbioscience.com Planimetry • Planimetric data is given to users along with Optical Fractionator Results • The volume is correct provided that the user defined the ROI accurately • Can be used to generate density measures • Not Stereology, it can be considered biased Figure courtesy of MBF Bioscience
- 55. mbfbioscience.commbfbioscience.com Estimating Area/Volume Fraction Area Fraction Fractionator • Cavalieri estimate of area performed on a systematically selected fraction of tissue • Sampling is done at low magnification and on one plane • Place marker for subregion (e.g. lesion, non-parenchyma) • Place marker for area within the contour (e.g. lung) area fraction = area of subregion area of total region Figure courtesy of MBF Bioscience
- 56. mbfbioscience.com Estimation of Length: Spaceballs • Report total length of all the processes in the ROI • Uses a SRS sampling • Instead of a counting frame, a sphere is placed at the sampling sites • Mark processes that intersect the sphere as focus through the tissue • To maximize the diameter of the spherical probe, use hemispheres • Length = 2 (∑Q) x x 1 ssf v a Mouton PR, Gokhale AM, Ward NL, West MJ. Journal of Microscopy. 2002 Apr;206(Pt 1):54-64
- 57. Spaceballs
- 58. mbfbioscience.com Area and Volume Estimation: Nucleator • Use in conjunction with the Optical Fractionator • Measure cell size (area & volume) and number • Uses one optical plane • Cells and/or sections need to be isotropic • If the cells and sections have a preferred orientation, Nucleator can only be used to report cross sectional area, not volume (e.g., nerve profiles in ventral root) * X X X X X X X X
- 59. In Conclusion • Today we discussed stereology theory and discussed the importance of using geometric probes to quantify 3D events • We discussed some rules for achieving unbiased estimates • SRS sampling • Isotropy • Discussed experimental design and sampling strategies to ensure efficiency, precision and accuracy • We also introduced the Optical Fractionator for counting cells and briefly discussed other probes mbfbioscience.com
- 60. Learn More • Visit www.stereology.info • View practical demonstration webinars www.mbfbioscience.com/webinars • Email Julie at julie@mbfbioscience.com