Tapping Mode Atomic Force Microscopy allows the in situ Imaging of Fragile Membrane Structures and of Intact Cells Surface at High Resolution

Abstract

Because of its potential applications in the understanding of membrane function, high resolution in situ imaging by AFM of the surface of cells in their aqueous medium remains an important goal. Based on two examples, the in situ imaging of detergent-resistant plasma membrane domains and of the lateral membrane of outer hair cells from the inner ear, we show that low-force tapping mode AFM allows the access to the topography of very fragile membrane structures and that details of a cell can be acquired with a lateral resolution of about 5 nm.

Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study

“…The adsorption of melanosomes on the surface of such aggregates caused a disruption in the melanosome distribution within the cell. As a matter of fact, the formation of protein clusters after chemical fixation has already been observed through confocal laser microscopy ( 5 – 7 ) and AFM ( 8 – 11 ). However, while confocal microscopy required protein labeling, AFM allowed only the investigation of the external morphology of the cell membrane, leaving proteins organization in the cytoplasm unexplored.…”

“…The presence of abnormal protein clusters at the cell membrane of fixed cells has been proven through confocal laser microscopy ( 5 – 7 ) and atomic force microscopy (AFM) ( 8 – 11 ). However, the former requires fluorescent labeling of the proteins and allows observing only large objects (>0.3 µm), while the latter provides information about the morphology of very restricted areas of the cell membrane, losing track of organization inside the cell and at a wider spatial range.…”

Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study

“…The adsorption of melanosomes on the surface of such aggregates caused a disruption in the melanosome distribution within the cell. As a matter of fact, the formation of protein clusters after chemical fixation has already been observed through confocal laser microscopy ( 5 – 7 ) and AFM ( 8 – 11 ). However, while confocal microscopy required protein labeling, AFM allowed only the investigation of the external morphology of the cell membrane, leaving proteins organization in the cytoplasm unexplored.…”

“…The presence of abnormal protein clusters at the cell membrane of fixed cells has been proven through confocal laser microscopy ( 5 – 7 ) and atomic force microscopy (AFM) ( 8 – 11 ). However, the former requires fluorescent labeling of the proteins and allows observing only large objects (>0.3 µm), while the latter provides information about the morphology of very restricted areas of the cell membrane, losing track of organization inside the cell and at a wider spatial range.…”

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins

“…This has a possibility to cause artefacts by creating pseudo-clusters. Previous studies reported that fixatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns 12 – 25 . However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers because it is still difficult to observe living cell surfaces at the scale of a few nanometers.…”

Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

“…Previous studies reported that xatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns. However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers 12,13,14,15,16,17,18,19,20,21,22,23,24,25 , because it is still di cult to observe living cell surface at several nanometer scales.…”

Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study

“…The adsorption of melanosomes on the surface of such aggregates caused a disruption in the melanosome distribution within the cell. As a matter of fact, the formation of protein clusters after chemical fixation has already been observed through confocal laser microscopy ( 5 – 7 ) and AFM ( 8 – 11 ). However, while confocal microscopy required protein labeling, AFM allowed only the investigation of the external morphology of the cell membrane, leaving proteins organization in the cytoplasm unexplored.…”

“…The presence of abnormal protein clusters at the cell membrane of fixed cells has been proven through confocal laser microscopy ( 5 – 7 ) and atomic force microscopy (AFM) ( 8 – 11 ). However, the former requires fluorescent labeling of the proteins and allows observing only large objects (>0.3 µm), while the latter provides information about the morphology of very restricted areas of the cell membrane, losing track of organization inside the cell and at a wider spatial range.…”

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins

“…This has a possibility to cause artefacts by creating pseudo-clusters. Previous studies reported that fixatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns 12 – 25 . However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers because it is still difficult to observe living cell surfaces at the scale of a few nanometers.…”

Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

“…Previous studies reported that xatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns. However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers 12,13,14,15,16,17,18,19,20,21,22,23,24,25 , because it is still di cult to observe living cell surface at several nanometer scales.…”

Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study

“…The adsorption of melanosomes on the surface of such aggregates caused a disruption in the melanosome distribution within the cell. As a matter of fact, the formation of protein clusters after chemical fixation has already been observed through confocal laser microscopy ( 5 – 7 ) and AFM ( 8 – 11 ). However, while confocal microscopy required protein labeling, AFM allowed only the investigation of the external morphology of the cell membrane, leaving proteins organization in the cytoplasm unexplored.…”

“…The presence of abnormal protein clusters at the cell membrane of fixed cells has been proven through confocal laser microscopy ( 5 – 7 ) and atomic force microscopy (AFM) ( 8 – 11 ). However, the former requires fluorescent labeling of the proteins and allows observing only large objects (>0.3 µm), while the latter provides information about the morphology of very restricted areas of the cell membrane, losing track of organization inside the cell and at a wider spatial range.…”

Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins

“…This has a possibility to cause artefacts by creating pseudo-clusters. Previous studies reported that fixatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns 12 – 25 . However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers because it is still difficult to observe living cell surfaces at the scale of a few nanometers.…”

Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

“…Previous studies reported that xatives can change a part of the cell surface structures at scales of hundreds of nanometers to microns. However, no reports investigated surface structures on mammalian cells with a resolution of several nanometers 12,13,14,15,16,17,18,19,20,21,22,23,24,25 , because it is still di cult to observe living cell surface at several nanometer scales.…”