An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections.
A number of human brain diseases have been associated with disturbances in the structure and function of cortical synapses. Answering fundamental questions about the synaptic machinery in these disease states requires the ability to image and quantify small synaptic structures in tissue sections and to evaluate protein levels at these major sites of function. We developed a new automated segmentation imaging method specifically to answer such fundamental questions. The method takes advantage of advances in spinning disk confocal microscopy, and combines information from multiple iterations of a fluorescence intensity/morphological segmentation protocol to construct three-dimensional object masks of immunoreactive (IR) puncta. This new methodology is unique in that high- and low-fluorescing IR puncta are equally masked, allowing for quantification of the number of fluorescently-labeled puncta in tissue sections. In addition, the shape of the final object masks highly represents their corresponding original data. Thus, the object masks can be used to extract information about the IR puncta (e.g., average fluorescence intensity of proteins of interest). Importantly, the segmentation method presented can be easily adapted for use with most existing microscopy analysis packages.
Fish
Kenneth N
KN
Department of Psychiatry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA. fishkn@upmc.edu
Sweet
Robert A
RA
Deo
Anthony J
AJ
Lewis
David A
DA
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008252:Macaca fascicularis; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D013569:Synapses / Q000648:ultrastructure*
Nat Protoc. 2006;1(3):1287-96
17406413
J Cell Biol. 1990 Dec;111(6 Pt 2):2815-28
2125300
J Neurosci Res. 1993 Apr 15;34(6):689-706
8315667
J Comp Neurol. 2003 Oct 20;465(3):385-400
12966563
Trends Neurosci. 2004 Jul;27(7):370-7
15219735
Biol Psychiatry. 2008 Apr 15;63(8):759-65
17945195
J Neurosci. 1994 Mar;14(3 Pt 2):1834-55
8126575
J Neurosci Methods. 2006 Apr 15;152(1-2):32-9
16198002
Trends Neurosci. 2001 Aug;24(8):479-86
11476888
Cereb Cortex. 2005 Aug;15(8):1178-86
15590911
J Microsc. 2007 Dec;228(Pt 3):390-405
18045334
Appl Opt. 1994 Feb 1;33(4):603-15
20862055
J Microsc. 1984 May;134(Pt 2):127-36
6737468
J Comp Neurol. 1993 Oct 15;336(3):400-18
8263229
Brain Res Mol Brain Res. 1999 Aug 25;71(2):201-9
10521574
Science. 2002 Oct 25;298(5594):789-91
12399581
J Cell Biol. 1989 Nov;109(5):2105-15
2808521
Brain Res Dev Brain Res. 1996 Oct 23;96(1-2):261-76
8922688
J Microsc. 2005 May;218(Pt 2):148-59
15857376
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2014-9
11830634
J Comp Neurol. 2002 Jun 24;448(2):186-202
12012429
Nat Methods. 2005 Sep;2(9):677-84
16118638
J Neurosci Res. 1995 Dec 15;42(6):742-57
8847736
J Comp Neurol. 1999 May 24;408(1):11-22
10331577