Introduction to stereology and sampling strategies in stereology

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1 Introduction to stereology and sampling strategies in stereology Prof. Suleyman KAPLAN Department of Histology-Embryology Medical School Ondokuz Mayıs University, Samsun, Turkey

2 Stereology (1967) a branch of science concerned with inferring the three-dimensional properties of objects or matter ordinarily observed two-dimensionally (WEBSTER S Dictionary)

3 Stereology Number Volume Area Length Density

4 Stereology Particle counting: Glomeruli Cells Organelles Synapses

5

6 1 2 4

7

8

9 4 2

10 ?

11

12

13

14 ?

15 Stereology

16 Stereology

17 Stereology????

18 Stereology Using Numerical Density Unit length (e.g. mm) Unit area (e.g. mm 2 ) Unit volume (e.g. mm 3 )

19 Stereology The Concept of Numerical Density shrinkage swollen

20 Stereology The Concept of Numerical Density Increased volume Decreased volume

21 Stereology The Concept of Numerical Density N V N A N N L

22 Stereology Sampling

23 Stereology - Sampling

24 Stereology - Sampling How many animals in a group? How many sections from an animal? How many areas on a section? How many cells (or object) in an individual??

25 Stereology introduced by Gundersen in 1977

26 Stereology Sahin et al. Image Anal Stereol; 20:9-13, 2001

27 Stereology Kaplan et al. J. Microscopy (Oxford) 203: , 2001

28 Stereology Kaplan et al. J. Neurosci. Methods, 145: , 2005

29 Stereology Kaplan et al. J. Neurosci. Methods, 145: , 2005

30 Stereology

31 Stereology

32 Stereology A simple technique to measure the movements of the microscope stage along the x and y axes for stereological methods Kaplan S, Canan S, Aslan H, Unal B, Sahin B JOURNAL OF MICROSCOPY (OXFORD) 203: , 2001

33 Stereology A stereological investigation on the effects of forebrain ischemia on hippocampal neuron number in chick Canan S, Pakkenberg B, Kaplan S, Aslan H NEUROSCIENCE RESEARCH COMMUNICATIONS 30:27-33, 2002

34 Stereology Estimation of numerical density and mean synaptic height in chick hippocampus 24 and 48 hours after passive avoidance training Unal B, Bradley PM, Sahin B, Canan S, Aslan H, Kaplan S DEVELOPMENTAL BRAIN RESEARCH 136: , 2002

35 Stereology Depression: reduced number of granule cells in the hippocampus of female, but not male, rats due to prenatal restraint stress Schmitz C, Rhodes ME, Bludau M, Kaplan S, Ong P, Ueffing I, Vehoff J, Korr H, Frye CA MOLECULAR PSYCHIATRY 7: , 2002

36 Stereology A stereological estimation of total neuron number and volume of the hippocampus at one and seven day-old chicks Sahin B, Aslan H, Canan S, Kaplan S, Unal B, Odaci E NEUROSCIENCE RESEARCH COMMUNICATIONS 31:29-38, 2002

37 Stereology Therapeutic effects of intracarotid infusion of spermine/nitric oxide complex on cerebral vasospasm Sonmez OF, Unal B, Inaloz S, Sahin B, Yilmaz M, Aydin A, Kaplan S ACTA NEUROCHIRURGICA 144: , 2002

38 Stereology Unbiased estimation of the liver volume by the Cavalieri principle using magnetic resonance images Sahin B, Emirzeoglu M, Uzun A, Incesu L, Bek Y, Bilgic S, Kaplan S EUROPEAN JOURNAL OF RADIOLOGY 47: , 2003

39 Stereology Quantitative features of the nucleus rotundus in the brain of pre and post hatch chicks Con N, Canbilen A, Bradley PM, Kaplan S DEVELOPMENTAL BRAIN RESEARCH 146:71 77, 2003

40 Stereology A NOS inhibitor aminoguanidine reduces zinc-induced neuron loss in rat hippocampus Gokce F, Bagirici F, Kaplan S, Demir S, Ayyildiz M, Marangoz C NEUROSCIENCE RESEARCH COMMUNICATIONS 33:53-62, 2003

41 Stereology Does the sagittal plane kyphosis describe destruction of the affected ıntervertebral disc? (Letter to Editor) Odaci E, Sahin B, Sonmez OF, Kaplan S INJURY 35:208, 2004

42 Stereology Enhancement of nerve regeneration and orientation across a gap by using the nerve graft within the nerve vein conduit graft: A functional, stereological, and electrophysiological study. Keskin M, Akbas H, Uysal A.O, Canan S, Ayyildiz M, Agar E, Kaplan S. PLASTIC AND RECONSTRUCTIVE SURGERY 113: , 2004

43 Stereology Effects of Low-Dose Oxcarbazepine Administration on Developing Cerebellum in Newborn Rat: A Stereological Study Odaci E, Kaplan S, Sahin B, Bas O, Gevrek F, Aygun D, Unal B, Sonmez OF, Colakoglu S, Bilgic S NEUROSCIENCE RESEARCH COMMUNICATIONS 34:28-36, 2004

44 Stereology The effect of haloperidol on the numerical density of neurons and nuclear height in the rat hippocampus (A stereological and histopathological study) Unal B, Özbek ME, Aydın D, Aydın N, Bulucu Z, Vuraler Ö, Odaci E, Sahin B, Kaplan S NEUROSCIENCE RESEARCH COMMUNICATIONS 34:1-9, 2004

45 Stereology No-touch technique for autologous fat harvesting Karacalar A, Orak I, Kaplan S, Yıldırım S AESTHETIC PLASTIC SURGERY 28: , 2004

46 Stereology Estimation of breast prosthesis volume by the Cavalieri principle using magnetic resonance images. Akbas H, Sahin B, Eroglu L, Odaci E, Bilgic S, Kaplan S, Uzun A, Ergur H, Bek Y AESTHETIC PLASTIC SURGERY 28: , 2004

47 Stereology Pinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: gross morphological, histological and stereological analysis. Turgut M, Uyanıkgil Y, Baka M, Tunç AT, Yavaşoğlu A, Yurtseven ME, Kaplan S JOURNAL OF PINEAL RESEARCH 38: , 2005

48 Stereology A new approach for the estimation of intervertebral disc volume using the Cavalieri principle and computed tomography images. Bilgic S, Sahin B, Sonmez OF, Odaci E, Colakoglu S, Kaplan S, Ergur H CLINICAL NEUROLOGY AND NEUROSURGERY 107: , 2005

49 Stereology A simple technique for localizing consecutive fields for disector pairs in light microscopy: application to neuron counting in rabbit spinal cord following spinal cord injury Kaplan S, Gökyar A, Ünal B, Turkkanı-Tunç A, Bahadir A, Aslan H. JOURNAL OF NEUROSCIENCE METHODS 145: , 2005

50 Stereology Morphological alterations produced by zinc-deficiency in rat sciatic nerve: a histological, electron microscopic and stereological study. Unal B, Tan H, Orbak Z, Kiki I, Bilici M, Bilici N, Aslan H, Kaplan S. BRAIN RESEARCH 1048: , 2005

51 Stereology Morphological, stereological and radiological changes in pinealectomized chicken cervical vertebrae. Turgut M, Kaplan S, Turgut AT, Aslan H, Güvenc T, Cullu E, Erdogan S. JOURNAL OF PINEAL RESEARCH 39: , 2005

52 Stereology The effects of section thickness on the estimation of liver volume by the Cavalieri principle using computed tomography images. Emirzeoglu M, Sahin B, Selcuk MB, Kaplan S. EUROPEAN JOURNAL OF RADIOLOGY 356: , 2005

53 Stereology Neonatal pinealectomy induces Purkinje cell loss in the cerebellum of the chick: a stereological study Turkkani Tunc A, Turgut M, Aslan H, Sahin B, Yurtseven ME, Kaplan S, BRAIN RESEARCH 1067:95-102, 2006

54 Stereology Effects of constant lightness, darkness and parachlorophenylalanine treatment on tail regeneration in the lizard ophisops elegans macrodactylus: macroscopic, biochemical and histological changes. Turgut M., Kaplan, S. Metin K., Koca Y. B., Soylu E., Şahin B., Ateşlier Z. B. B., Başaloğlu H.K. Anatomia, Histologia, Embryologia: Journal of Veterinary Medicine Series C 35: , 2006

55 Stereology Cortical anastomotic veins occlusion in the rat including the assessment of cerebral swelling. Cokluk C, Aydin K, Yemisci M, Colakoglu S, Kaplan S. JOURNAL OF NEUROSCIENCE METHODS 56: , 2006

56 Stereology Two distinct events, section compression and loss of particles ("lost caps"), contribute to z- axis distortion and bias in optical disector counting Baryshnikova LM, Von Bohlen Und Halbach O, Kaplan S, Von Bartheld CS. MICROSCOPY RESEARCH AND TECHNIQUE 69: , 2006 (Rank: 1/microscopy)

57 Stereology Effect of high fat diet on quantitative feature of adipocyte in the omentum: an experimental, stereological and ultrastructural study Aslan H, Altunkaynak BZ, Altunkaynak ME, Vuraler Ö, Kaplan S, Ünal B. OBESITY SURGERY 16: , 2006 i.f=3.72

58 Stereology Protection of nigral cell death by bilateral subthalamic nucleus stimulation Temel Y, Visser-Vandewalle V, Kaplan S, Kozan R, Daemen MARC, Blokland A, Schmitz C, Steinbusch HWM BRAIN RESEARCH 1120: , 2007.

59 Stereology Effects of formaldehyde exposure on granule cell number and volume of dentate gyrus: a histopathological and stereological study Aslan H, Songur A, Tunc AT, Ozen OA, Bas O, Yagmurca M, Turgut M, Sarsilmaz M, Kaplan S. BRAIN RESEARCH 1122: , 2007

60 Stereology Effect of pinealectomy on the morphology of the chick cervical spinal cord: A stereological and histopathological study Turgut M, Tunc AT, Aslan H, Yazici, AC, Kaplan, S BRAIN RESEARCH 1129: , 2007

61 Stereology Effect of prenatally exposed to an antiinflammatory drug on neuron number in cornu ammonis and dentate gyrus of the rat hippocampus: a stereological study Gokcimen A, Rağbetli M Ç, Baş O, Tunc AT, Aslan H, Yazici AC, Kaplan S. BRAIN RESEARCH 1127: , 2007

62 Stereology Inhibitory effect of pinealectomy on the development of cerebellar granule cells in the chick: A stereological study Tunc AT, Aslan H, Turgut M, Ekici F, Odaci E, Kaplan, S. BRAIN RESEARCH 1138: , 2007.

63 Stereology Volumetric evaluation of the lung expansion following resection: a stereological study. Basoglu A, Buyukkarabacak Y, Sahin B, Kaplan S. European Journal of Cardio-thoracic Surgery 31: , 2007.

64 Stereology Effects of postnatal formaldehyde exposure on pyramidal cell number, volume of cell layer in hippocampus and hemisphere in the rat: a stereological study. Sarsilmaz M, Kaplan S, Songur A, Colakoglu S, Aslan H, Tunc AT, Ozen OA, Turgut M, Bas O. BRAIN RESEARCH 1145: , 2007.

65 Stereology Effect of high fat diet on the volume of liver and quantitative feature of Kupffer cells in the female rat: stereological and ultrastructural study Kiki İ., Altunkaynak B Z., Altunkaynak M.E., Vuraler Ö., Unal D., Kaplan S. Obesity Surgery 2007; 17:

66 Stereology High frequency stimulation of the subthalamic nucleus improves speed of locomotion but impairs fore limb movement in Parkinsonian rats. Vlamings R, Visser-Vandewalle V, Koopmans G, Joosten E, Kozan R, Kaplan S, Steinbusch H, Temel Y. Neuroscience 2007; 148:

67 Stereology Effect of prenatal exposure to diclofenac sodium on Purkinje cell numbers in rat cerebellum: a stereological study Ragbetli MC, Ozyurt B, Aslan H, Odaci E, Gokcimen A, Sahin B, Kaplan S. Brain Research 2007; 1174:

68 Stereology The effect of autogenous vein grafts on nerve repair with size discrepancy in rats: an electrophysiological and stereological analysis Acar M, Karacalar A, Ayyildiz M, Unal B, Canan S, Agar E, Kaplan S. Brain Research 2008; 1198:

69 Stereology An alternative approach to evaluating lumbar lordosis on direct roentgenograms: projection area per length square Kuru O, Sahin B, Kaplan S. Anatomical Science International 2008; 83:

70 Stereology Chronically given risperidone did not change the number of hepatocytes in rats: a stereological and histopathological study Halici Z, Keles ON, Unal D, Albayrak M, Suleyman H, Cadirci E, Unal B, Kaplan S. Basic & Clinical Pharmacology &Toxicology 2008;102:

71 Stereology Extracorporeal shockwave application to the distal femur of rabbits diminishes the number of neurons immunoreactive for substance P in dorsal root ganglia L5 Hausdorf J., Lemmens M., Kaplan S., Marangoz C., Milz S., Odaci E., Korr H., Maier M., Schmitz C. Brain Research 2008; 1207:

72 Stereology Repairing critical-sized rat calvarial defects with periosteal cell seeded small intestinal submucosal layer. Keskin M., Kelly C.P, Moreira-Gonzalez A., Lobocki C., Yarim M., Kaplan S., Jackson I.T. Plastic and Reconstructive Surgery 2008; 122:

73 Stereology Dependence of computed tomography volume measurements upon section thickness: an application to human dry skulls Sahin B., Mazonakis M., Akan H., Kaplan S., Bek Y. Clinical Anatomy 2008; 21:

74 Stereology Prenatally exposed to a non-steroidal antiinflammatory drug or saline solution impair sciatic nerve morphology: a stereological and histological study. Canan S., Aktas A., Ulkay B., Colakoglu S., Ragbetli MC., Ayyildiz M., Geuna S., Kaplan S. International Journal of Developmental Neuroscience 2008; 26:

75 Stereology An efficient stereological sampling approach for quantitative assessment of nerve regeneration Canan S, Bozkurt H. H, Acar M, Vlamings R, Aktaş A, Sahin B, Temel Y, Kaplan S Neuropathology and Applied Neurobiology 2008; 34:

76 Stereology Effects of prenatal exposure to a 900 megahertz electromagnetic field on the dentate gyrus of rats: a stereological and histopathological study Odaci E., Bas O., Kaplan S. Brain Research 2008; 1238:

77 Stereology Piskin A., Kaplan S., Aktas A., Ayyildiz A., Raimondo S., Alic T. Bozkurt H., Geuna S. Platelet gel does not improve peripheral nerve regeneration: an electrophysiological, stereological and electron microscopic study. Microsurgery 2009; 29:

78 Stereology Bas O., Odaci E., Kaplan S., Acer N., Ucok K., Colakoglu S. 900 megahertz electromagnetic field exposure affects qualitative and quantitative features of hippocampal pyramidal cells in the adult female rat. Brain Research 1265:

79 Stereology Bas O., Odaci E., Mollaoglu H., Ucok K., Kaplan S. Chronic prenatal exposure to the 900 megahertz electromagnetic field induces pyramidal cell loss in the hippocampus of newborn rats. Toxicology and Industrial Health 2009; 25:

80 Stereology Vlamings R., Visser-Vandewalle V., Kozan R., Kaplan S., Steinbusch H.W.M., Temel Y. Bilateral high frequency stimulation of the subthalamic nucleus normalizes COX activity in the substantia nigra of Parkinsonian rats. Brain Research 2009; 1288:

81 Stereology So on

82 Basic concept in unbiased quantitative morphology

83 Terminology Biased Introducing a systematic deviation from the true value Unbiased Approaching the true value after repeated measurements

84 Terminology Values General average Systematic deviation Real Value Number of Experiments This method introduces a systematic deviation from the true value, hence it is a biased method.

85 Terminology Efficiency To get less variable results in a relatively short time.

86 Terminology Efficiency To get less variable results in a relatively short time.

87 Terminology Efficiency To get less variable results in a relatively short time.

88 Terminology Particle Counted/measured unit Synapse Organelle Nucleus Cell Glomerulus Alveoli

89 Terminology Profile Representations of tissue constituents on sections...

90 Terminology Profile? Profile Particle

91 Stereology Basic Problems Microscopy and its Problems Sections and profiles

92 Stereology Basic Problems Problem of dimensional reduction 1-dimensional line 0-dimensional point profile Section plane

93 Stereology Basic Problems Problem of dimensional reduction 1-dimensional line 0-dimensional point profile Section plane

94 Stereology Basic Problems Problem of dimensional reduction 2-dimensional surface Section plane 1-dimensional linear profile

95 Stereology Basic Problems Problem of dimensional reduction 3-dimensional object 2-dimensional area profile Section plane

96 Stereology Basic Problems Problem of dimensional reduction Sectional dimension = Real dimension - 1

97 Terminology Probe A set of geometrical questions asked to the samples

98 use volume probe to count particle number

99 L use area probe to estimate length

100 use line probe to estimate surface area

101

102 use point probe to estimate surface area

103

104 Terminology Types of Probes Point (0-D) Line (1-D) Area (2-D) Volume (3-D)

105 Terminology Probe D Parameter + D probe = 3 + = = 3

106 1

107

108

109 SAMPLING STRATEGY

110 Sampling Strategy Systematic sampling Sampling using a pre-defined sampling interval Random sampling Sampling with a truely random order Systematic-Random sampling Sampling using a pre-defined sampling interval beginning with a random start

111 Sampling Strategy Systematic Random Sampling Every part of the structure has an equal chance of being a member of the final sample set.

112 Sampling Strategy Points to ponder: 1. Sampling bias 2. Systematic bias

113 Sampling Strategy Sampling bias

114 Sampling Strategy

115 Sampling Strategy Systematic Bias

116 Sampling Strategy

117 Sampling Strategy

118 Sampling Strategy

119 Sampling Strategy

120 Sampling Strategy

121 Sampling Strategy Systematic Random Sampling

122

123 =?

124 Sampling Strategy

125 Sampling Strategy

126 Sampling Strategy /4

127 Sampling Strategy /4

128 Thanks to Prof. Bünyamin Şahin Prof. Bünyami Unal Prof. Ersan Odacı Prof. Sinan Canan Prof. Stefano Geuna (Italy) Prof. Christoph von Bartheld (USA) Prof. Yasin Temel (NL) Prof. Abdulmonem Al-Hayani (KSA) Dr. Mohammad Farahna (KSA) Qassim University Ondokuz Mayıs University

129

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