MIFE Key Features
There
are are several major features that, taken together, provide a
significant advantage of the MIFE approach over other techniques for flux
measurements. These include:
1.
Non-destructiveness. In contrast to many other methods, the MIFE system allows in situ measurements of net ion
fluxes, in physiologically realistic conditions.
2.
High spatial resolution. The electrode tip is typically 2-3
µm in diameter, which makes it possible to measure net ion fluxes from
single cells (Babourina et al. 2000; Shabala et al. 2001b) or
protoplasts derived from plant cells (Shabala et al. 1998; Tyerman et
al. 2001). Moreover, for some ionophores with high signal-to-noise
ratio (such as H+), the electrode tip diameter can be further reduced
to 0.8-1.0 µm. As a
result, the cell surface can be "mapped" (Shabala et al. 1998; Tegg et
al. 2005), providing information about spatial distribution and
functional expression of specific ion transporters.
3.
High temporal resolution. The "default" MIFE settings assume
electrode movement with 10 sec period. This could be further reduced
without difficulty to 2 or 3 sec in some cases. Such high temporal
resolution is crucial in studying rapid signaling events at plant
membranes. Most other non-invasive techniques operate on a time scale
at least one order of magnitude slower. This gives the MIFE technique a
unique opportunity to provide insights into very early (and fast)
events associated with plant responses to environmental changes.
4.
Duration of measurements. There is essentially no limitation on
how long fluxes could be measured from the cell or tissue. The
technique is non-invasive, and its application is practically limited
only by the lifetime of the ion-selective electrode (typically 15 to 20
h). Moreover, electrodes may be replaced easily,
and measurements resumed after only a few minutes' break. None of the
other techniques of the same time resolution (e.g. patch-clamp or
fluorescence imaging) provide this opportunity. Due to dye bleaching,
fluorescence measurements are usually restricted to a limited number of
images being taken. Maintaining a 'gigaseal' for several hours is also
a big problem in every patch-clamp study. As for internal ion-selective
microelectrodes, their application is limited by the likelihood of
electrode being clogged by the dense cytosol after a certain period of
time.
5.
Measurement of fluxes of neutral molecules can also be made if
the appropriate micro-sensor is available. Pang et al. (2006) have used MIFE for this and other systems have also been used in Porterfield's lab (McLamore et al. 2011).
6. Simultaneous measurements of several fluxes. The
possibility of measuring kinetics of fluxes of several ions
or neutrals simultaneously, and essentially at the same spot, is important in
understanding the underlying ionic mechanisms of cell adaptive
responses. By assessing stoichiometry ratios between various ions,
valuable information about the membrane transporters involved can be
gained. MIFE applications: summarised below:
Ø
plant physiology (stress; adaptation; mineral
nutrition; photosynthesis; long-distance transport; growth & development; water relations;
osmoregulation; hormonal physiology, stomatal physiology, plant movements)
Ø
cell biology (signaling; perception; elicitors)
Ø
ecophysiology (plant responses to abiotic and
biotic factors)
Ø
biophysics (properties of ion channels and
transporters)
Ø
developmental biology (morpho- and embrio-genesis;
polarity)
Ø
functional genomics (in planta studies of
specific gene functions; heterologous expression systems)
Ø
agronomy and plant
breeding (plant
screening for environmental fitness)
Ø
soil science (soil-root interface; heavy metal
toxicity; remediation)
Ø
marine biology (algae; phytoplankton; marine
biofilms and mats; sediments)
Ø
bryology (physiology and development)
Ø
mycology (factors controlling growth and
development)
Ø
food microbiology (effect of food-related treatments
on bacteria; food preservation studies; interrelation of pathogenic and
probiotic bacteria; biofilms)
Ø
medical microbiology (pathogenic bacteria; bacterial
physiology an genetics; host-pathogen interactions)
Ø
environmental microbiology (functional genomics;
bioremediation; environmental physiology)
Ø
medical research (screening of new drugs;
physiology; pathology)
Ø
human and animal
physiology
(receptors; signaling; homeostasis)
Ø
toxicology (receptors; selectivity and action
spectrum; molecular targets)
.
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