The Atomic Force Microscopy and the MicroElectrode Arrays in the study of mechanoelectrical properti

1
The Atomic Force Microscopy and the
Micro-Electrode Arrays in the study of
mechano-electrical properties of electrogenic
cells

• José Francisco Sáenz Cogollo
• Colombia
• Electronics Engineer, Biomedical Engineering
  Specialist
• Ph.D. Student in Bioengineering
• Università degli Studi di Genova - Italia

2
Contents

• The Atomic Force Microscopy (AFM)
• Principle of operation
• AFM for studying cell properties
• Advantages in cell biology
• The Micro-Electrode Arrays (MEAs)
• Principle of operation
• Uses of (MEAs)
• AFM-MEA combined set-up
• Experiments with dissociated neural networks
• Study of mechanically induced arrhythmias

3
The Atomic Force Microscope (AFM)

• It belongs to the family of the Scanning Probe
  Microscopy (SPM) invented in 1981 by G.Binning
  and H.Rohrer.
• In the SPM a sharp probe is scanned across a
  surface and some probe sample interaction or
  interactions are monitored.
• The AFM senses interatomic forces that occur
  between a probe tip and a substrate.

4
AFM for study cell properties
5
Advantages of using the AFM

• Unlike other techniques, AFM can use samples with
  just minor preparation, over a large range of
  temperatures and in repetitive studies.
• The high resolution allows topographical imaging
  of samples such as DNA molecules, protein
  adsorption or crystal growth, and living cells
  adsorbed on biomaterials, etc.
• In addition to topographical measurements, AFM is
  also capable of complementary techniques that
  provide information on other surface properties,
  e.g. stiffness, hardness, friction, or
  elasticity.
• It can be also used as a tool for controlled
  mechanical nano-stimulation and manipulation

6
Micro-Electrode Arrays (MEAs)

• Are arrangements of several (typically 60) planar
  electrodes in a square recording area of 700um up
  to 5mm.
• An MEA allows the targeting of several sites for
  stimulation and extracellular recording of
  electrical active cells (single cells, neuronal,
  muscle, or cardiac tissue) simultaneously.

7
MEA principle of operation
8
Basic uses of MEAs

• To investigate the interactions between
  electrogenic cells at different locations in the
  same tissue, which may be used to analyze the
  spatio-temporal dynamics of activity or the
  representation of information in neuronal
  networks.
• To reduce the time required for an experiment by
  simultaneously recording at several sites in
  parallel, and thus sample the distribution of
  electrophysiological behavior efficiently, which
  may include comparison of tissue properties at
  different locations.
• To monitor changes of electrical activity over
  periods of time not accessible with individual
  conventional electrodes (e.g., glass capillary or
  tungsten electrodes) in in vitro experiments.

9
AFM/MEA combined set-up

• Little work has been done in order to study the
  simultaneous electrical and mechanical response
  of cells in vitro when applying electrical or
  mechanical stimulus.

Both AFM and MEA techniques are non invasive and
therefore allow to monitor, in real time, in
situ, and at the nanometer scale (for what
relates to AFM) subtle changes in the physiology
of viable cell networks, thus forming together a
novel tool for functional nano-characterization
of electrically active cells.
10
AFM/MEA combined set-up

• We developed an analytical platform based on a
  combined AFM/MEA set-up for deliver/record
  electric currents/potentials and measure minimal
  changes in the morphology/mechanical properties
  of electrically active cell cultures as well as
  to measure the changes in the electric activity
  when single cells are stimulated by means of the
  AFM tip.

A micromanipulator stage together with the motors
of AFM head allow the X-Y-Z positioning of the
AFM scanner over the MEA, while the X-Y movements
of the optical microscope sample stage allow the
whole system positioning over the microscope
objective.
11
Experiments with dissociated neural networks

• Its possible to perform, in situ, both Phase
  Contrast (PC) and Fluorescence Microscopy on the
  cells at the same time while doing the AFM
  scanning and the electrical measurements

12
Study of mechanically induced arrhythmias

• Until now few experiments at the single cell
  level have been performed in order to correlate
  the changes in spontaneous electric activity due
  to a controlled mechanical stimulation
• A study of the real time electrical response of a
  syncytium of cardiac cells to nano-mechanical
  stimuli can represent an accurate model to study
  the mechano-electric behavior of cardiac tissue.

13
The role of fibroblast in cardiac
mechano-electric feedback?

• Fibroblast are the majority of heart cells and
  its number increase with age, infarct and other
  patologies
• Fibroblasts may affect the origin and spread of
  excitation in several ways above and beyond
  formation of passive barriers that obstruct
  electrical conduction
• The inherent mechano-sensitivity of cardiac
  fibroblasts could allow them to play a sensory
  role and to affect cardiac electrophysiology via
  the mechanoelectric feedback phenomenon.

Kohl, P., P. Camelliti, et al. (2005).
"Electrical coupling of fibroblasts and myocytes
relevance for cardiac propagation." Journal of
Electrocardiology 38(4) 45-50.
14
Thanks for your attention

• The Atomic Force Microscopy and the
  Micro-Electrode Arrays in the study of
  mechano-electrical properties of electrogenic
  cells
• Authors
• José Francisco Sáenz Cogollo
• jose.saenz_at_unige.it
• Roberto Raiteri
• Mariateresa Tedesco
• Sergio Martinoia