A strategy for correlative microscopy of large skin samples: towards a holistic view of axillary skin complexity

Abstract

Knowledge about the structural elements of skin and its appendices is an essential prerequisite for understanding their complex functions and interactions. The hence necessary morphological description across several orders of scale not only requires the investigation at the light microscopic level but also ultrastructural investigation, ideally on the identical sample. For a correlative and multimodal observation one unique preparation protocol is mandatory. As a compromise between sample sizes of >500 lm in diameter on the one hand and optimal preservation of antigenicity and morphology on the other, we developed a new preparation protocol that allows (i) 3D reconstruction of the resin-embedded sample by confocal light microscopy prior to (ii) direct immunolocalization of target proteins within selected sample planes by light and fluorescence microscopy or transmission electron microscopy. Alternatively, (iii) serial cryosections of the frozen sample can be taken for characterizing the sample in toto. With this unique approach we were able to fully demonstrate the structural complexity of axillary skin samples, increasing the structural resolution from 3D reconstruction of the whole gland up to ultrastructural investigations at the subcellular level. We could demonstrate that axillary sweat glands are not separately distributed, as has been assumed to date; instead, they seem to be intricately twisted into one another. This promotes the concept of a complex axillary sweat gland organ instead of single sweat gland entities.Key words: axillary skin -correlative microscopy -multimodal imaging -sweat gland Please cite this paper as: A strategy for correlative microscopy of large skin samples: towards a holistic view of axillary skin complexity. Experimental Dermatology 2008; 17: 73-80.

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

“…The numerous attempts of correlative microscopy in different systems provide many successful examples of bridging between dynamic LM with the static but resolving electron microscopy (Mironov et al, 2000; van Rijnsoever et al, 2008; Wilke et al, 2008; Nixon et al, 2009). While the work on CLEM for cultured cells has shown major progress in recent years (Verkade, 2008), its use on multicellular organisms lags significantly behind.…”

Correlative microscopy: a potent tool for the study of rare or unique cellular and tissue events

“…Simultaneously, researchers were introducing the use of HPF in CLEM experiments (Biel et al ., 2003; Pfeiffer et al ., 2003). The fluorescent staining of resin‐embedded skin tissue by adding fluorescent dyes, such as acridine orange and safranin O, to the substitution medium during freeze substitution (Biel et al ., 2003) and the uniform procedure based on quick freezing for CLEM analysis (including cryo‐SEM) of unique biopsies were developed (Richter et al ., 2007; Wilke et al ., 2008), and recently automation of the CLEM procedure has been initiated (Nickell et al ., 2007).…”

Correlative microscopy

“…2). 137 Often, however, it is desired to observe the fluorescent target in 138 the living cells before fixation to catch a special event [41][42][43][44][45][46][47] or to 139 prepare a whole sample to find the place of interest, indicated by 140 the fluorescence label, in a three-dimensional environment 141 [38,[48][49][50]. In the following we describe different approaches that 142 allow the identification of fluorescently labelled proteins in whole 143 cells by light and electron microscopy.…”

“…The time resolution is dependent on the speed of penetration and fixation when chemical fixatives 48 are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to 49 identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either trans- 50 fections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence 51 labels in cells on a large section to reduce searching time in the electron microscope.…”