Comparison of methods for the estimation of the thickness of ultrathin tissue sections

Abstract

For a number of quantitative electron microscopical techniques it is relevant to obtain an estimate of the thickness t' of the section which, in general, will differ from the actual distance t between the two cuts that generate the section. To estimate t' of ultrathin sections, several techniques have been adopted in the past, both with and without the aid of the electron microscope and additional equipment, which are summarized in an appendix. In the present study five methods have been evaluated experimentally using sections of ten different interference colours: (a) the 'small-fold' technique, (b) the 'electron scattering' method, (c) interference microscopy with (A) the Vicker's M86@ scanning microinterferometer and (B) the Jenoptik Amplival Interphako@ interference microscope and (d) the 're-embedding' method. Reliable, reproducible and comparable results were obtained with the small-fold technique, with the Vickers M86@ scanning microinterferometer and with the electron scattering method. For the last method, standard test lines for the different settings of the electron microscope were developed. The results obtained with the Jenoptik Amplival InterphakoB interference microscope are reproducible, but show a constant difference, i.e. a factor of 1-36, in thickness compared with the other three techniques. The possible cause of this 'systematic error' is discussed. The re-embedding method proved to be more laborious and slightly less reliable than the other techniques.The variation in t' between sections of a particular interference colour (inter-section variation) was found to be larger than the variation in t' within a section (intra-section variation).

Orexin axons in the rat ventral tegmental area synapse infrequently onto dopamine and γ‐aminobutyric acid neurons

“…where D ϭ mean diameter of varicosities, ls ϭ mean length of synapses, and t ϭ mean thickness of ultrathin sections, in this case 0.057 m, as determined using the small fold method (De Groot, 1988). Synaptic incidence was estimated from all Orximmunoreactive varicosities sampled in tissue singly labeled by immunogold-silver for Orx or dually labeled by immunoperoxidase for Orx and gold-silver for TH or GABA.…”

Termination of the geniculocortical projection in the striate cortex of macaque monkey: A quantitative immunoelectron microscopic study

“…For determination of the distance between the reference and the look-up sections, the section thickness was measured by using Small's method of minimal folds (Small, 1968) as described by Weibel (1979) and Royet (1991). This method provides accurate estimates of section thickness compared with other techniques (Calverley et al, 1988;DeGroot, 1988;Hunter and Stewart, 1989). Minimal folds lie perpendicular to the section plane and have a width that equals twice that of the section thickness.…”

Unbiased stereological estimation of the total number of synapses in a brain region

“…The thickness of ultrathin sections was estimated with Small's method of minimal folds (Small, 1968) as described by Weibel (1979) and Royet (1991) because this method provides accurate estimates of section thickness compared with other techniques (Calverley et al, 1988;De Groot, 1988;Hunter & Stewart, 1989). Minimal folds lie perpendicular to the section plane and have a width that equals twice that of the section thickness.…”