Three-dimensional imaging of living and dying neurons with atomic force microscopy

Abstract

Atomic Force Microscopy (AFM) has been used to image the morphology of developing neurons and their processes. Additionally, AFM can physically interact with the cell under investigation in numerous ways. Here we use the AFM to both three-dimensionally image the neuron and to inflict a nano/micro-puncture to its membrane. Thus, the same instrument used as a tool to precisely penetrate/cut the membrane at the nanoscale level is employed to image the morphological responses to damage. These first high resolution AFM images of living chick dorsal root ganglion cells and cells of sympathetic ganglion and their growing processes provide confirmation of familiar morphologies. The increased resolution of the AFM revealed these structures to be significantly more complex and variable than anticipated. Moreover we describe novel, dynamic, and unreported architectures, particularly large dorsally projecting ridges, spines, and ribbons of cytoplasm that appear and disappear on the order of minutes. In addition, minute (ca. 100 nm) hair-like extensions of membrane along the walls of nerve processes that also shift in shape and density, appearing and disappearing over periods of minutes were seen. We also provide "real time'' images of the death of the neuron cell body after nano/micro scale damage to its membrane. These somas excreted their degraded cytoplasm, revealed as an enlarging pool beneath and around the cell. Conversely, identical injury, even repeated perforations and nanoslices, to the neurite's membrane do not lead to demise of the process. This experimental study not only provides unreported neurobiology and neurotrauma, but also emphasizes the unique versatility of AFM as an instrument that can (1) physically manipulate cells, (2) provide precise quantitative measurements of distance, surface area and volume at the nanoscale if required, (3) derive physiologically significant data such as membrane pressure and compliance, and (4) during the same period of study-provide unexcelled imaging of living samples.

Comparative three-dimensional imaging of living neurons with confocal and atomic force microscopy

“…The fine hairlike structures extending in the plane of growth may actually be axonal spines, although these are generally reported to be much larger in size. The resolution of AFM has allowed us to capture and characterize these vertical and horizontal extensions with much greater detail (see also McNally and Borgens, 2004). The versatility of the AFM will also allow us to investigate the form and function of these projections, providing further explanation for their appearance and/or retraction.…”

“…Others have used AFM technology to image neurons in the fixed state (Tojima et al, 2000;Weissmuller et al, 2000;Melling et al, 2001). However, the overall architecture of living neurons at high resolution has not been thoroughly evaluated with this technology until the first report in this series (McNally and Borgens, 2004). In this work we have utilized AFM and confocal laser scan-ning microscopy (CLSM) to evaluate and confirm novel 3-D architectures of chick dorsal root ganglia (DRG) and sympathetic neurons.…”

“…However, the versatility of AFM can be harnessed for many novel experiments in neurobiology. For example, we have recently used the AFM tip to produce nanoscale injury to the plasmalemma of growth cones and somas of DRG cells, subsequently imaging the responses of the cell to this damage with the same instrument (McNally and Borgens, 2004). AFM allows mechanical interaction with the sample, suggesting future studies into the form and function of localized areas.…”

Chitosan produces potent neuroprotection and physiological recovery following traumatic spinal cord injury

“…The acute compromise of intact cell membranes in response to mechanical forces leads to the deterioration of the barrier function of the plasma membrane and leads to cell and tissue necrosis (Borgens, 1988;Borgens, 2001;Povlishock et al, 1997). Compromise of the damaged membrane can be simply a local increase in membrane permeability to a more extensive open breach in the membrane (McNally and Borgens, 2004). Pathological transport of intracellular and extracellular ionic substances across this region leads to a rapid conduction block in nerve fibers, and eventually cell and tissue death resulting from the persistent absence of cellular resealing and the re-establishment of physiological homeostasis (Borgens, 2003).…”

Neuroprotection from secondary injury by polyethylene glycol requires its internalization

“…The mechanical damage that cell membranes undergo indeed leads to collapse and death of the cell body at variable rates dependent on many factors, including the magnitude of the insult [for real time imaging using atomic force microscopy, see McNally and Borgens (McNally and Borgens, 2004)]. However, cells that survive this initial damage undergo a process of biochemically mediated, progressive collapse and death called 'secondary injury'.…”