TY - JOUR
T1 - High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument
T2 - Essential sample preparation aspects
AU - Doak, Shareen H.
AU - Rogers, Dale
AU - Jones, Beverley
AU - Francis, Lewis
AU - Conlan, R. Steven
AU - Wright, Chris
PY - 2008/11
Y1 - 2008/11
N2 - The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM-CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM-CLSM images. We demonstrated for the first time that filopodia have a "quilted" surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure-function relationships.
AB - The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM-CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM-CLSM images. We demonstrated for the first time that filopodia have a "quilted" surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure-function relationships.
KW - AFM
KW - Confocal microscopy
KW - Filopodia
KW - Prostate
KW - Quantum dots
UR - http://www.scopus.com/inward/record.url?scp=54049095844&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=54049095844&partnerID=8YFLogxK
U2 - 10.1007/s00418-008-0489-5
DO - 10.1007/s00418-008-0489-5
M3 - Article
C2 - 18719934
AN - SCOPUS:54049095844
VL - 130
SP - 909
EP - 916
JO - Histochemistry and Cell Biology
JF - Histochemistry and Cell Biology
SN - 0948-6143
IS - 5
ER -