Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review.
Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations.
Mayhew
Terry M
TM
Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen's Medical Centre, E Floor, University of Nottingham, Nottingham, NG7 2UH, UK. terry.mayhew@nottingham.ac.uk
Lucocq
John M
JM
D000818:Animals; D003713:Dendritic Cells / Q000648:ultrastructure; D017979:Gold Colloid / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron / Q000379:methods*; D008867:Microtomy / Q000379:methods*; D013194:Staining and Labeling
D017979:Gold Colloid