Dear Confocalists
In addition to the previous comments made on this
list regarding measurements of calcium with CSLM
I would like to add the following points of view. These
are based on my experience in this area particularly
with tip growing plant cells. In general, I agree with the
flow chart initially proposed here but the exceptions
to the rule are many.
1 - Why use SW (single wavelength) dyes ?
SW dyes can be extremely useful if properly used.
There are lots of excellent papers with this technique
and most significant contributions of Ca2+
imaging for biology and biophysics (waves,
oscillations, annihilation) were done with SW dyes.
SW dyes are easier to use, respond quite rapidly
to changes in Ca2+ and, in general, they are brighter
allowing less dye in the cell and bleach less than
Fura-2 or Indo-1. Among these, Calcium-green 1 seems
to be an excellent dye.
2 - Advantages/disadvantages of SW dyes
There are obviously some major drawbacks: it's
virtually impossible to measure absolute values
of Ca2+ and asymmetric distribution of the dye
can complicate interpretation of the data. Methods
which use Fo/Fmax cannot always be used. For
instance with polarized tip growing cells such as
rhizoids, pollen tubes and fungal hyphae this
method is virtually impossible to achieve. Not only
these cells grow at rates that can reach 4 um/sec
but they also show a cytoplasmic zonation
making 2 consecutive images different.
However these premises can be dismissed if
one s interested in relative changes rather than
absolute values and if one gets a uniform signal
in a resting cell.
Occasionally, absolute values for Ca2+ are needed.
When that s the case, dual-wavelength (DW)
dyes must be used.
But DW dyes also have problems and in certain
cases their use is much more complicated. If one
wants to use caged-probes, fura-2 and indo-1 are
out of question; fura-red can be used but
fluorescence goes down with high Ca2+ and the
signal coming from the released area can be too
low to be imaged resulting in artefacts. To ratio
Ca2+ with 2 different dyes can also result in several
errors, as was pointed here already. 2 dyes bleach,
sequester and leak at different rates making any
calibration virtually impossible, specially in plant
cells with all their problems.
3 - Sequestration/bleaching/leakage
This is a problem common to both SW and DW dyes.
Ratio does indeed solves most of these 3 problems
but does not eliminate them.
Sequestration and leakage is a problem that varies
from system to system. In some systems it s terribly
high (see for instance J. of Microsc 166, 57-86).
In such conditions, imaging with SW dyes probably
needs to be reconsidered. But even in these cases, with
all the apparent sequestration, the few dye in the
cytosol keeps responding to extracellular stimulus.
The question in this case is what kind of response
are we measuring? Cytosolic Ca2+ ? Organelle
Ca2+ ? Ca2+ in general?
Having said that, imaging with DW dyes may
present exactly the same problem. So in this
case, biological material may be problem, and
not the type of dye. The dextran dyes may be
quite useful in such cases because their sequestration
is much more reduced. Still, even dextran dyes can
be taken up by some cells (Protoplasma 175: 126-130).
Bleaching is a problem more frequent with DW
dyes (needing excitation in the UV range) than
with SW dyes. Bleaching of calcium green can
be extremely low if one reduces laser intensity,
scan speed and box size while indo-1 bleach
quite fast. Sure, ratio will take that into account
but our last data presented at the Royal Microsc
Soc (Parton et al, 5th Int Botanical Microsc Meeting)
shows that even the ratio value of a DW dye is
affected by intensity of the signal and imaging
conditions. In this work we shown that if
one uses the wrong imaging setup, gradients
can be easily obtained: fluorescence intensity
above or below a critical level results in non-linear
responses to ion concentration.
4 - Loading conditions
My experience tells me that loading conditions
can strongly influence the quality of dye
distribution. Ester loading of Ca2+ dyes in plant
cells seems to be impossible (either due to heavy
sequestration or no loading), acid loading
never works, permeabilization with detergents
is to be avoided and electroporation does not
allow big molecules inside the cells. So
microinjection seems to be the only solution.
Ionophoresis, if done in bad conditions, can
result in lots of problems. However, if the
process is optimized, can produce very nice
and consistent data. Same is true for pressure
microinjection.
5 - Confocal imaging with SW dyes
Why don t we see the true Ca2+ distribution
with this technique?
Several reasons can account for this. First of all
the dye distribution. A problem of using SW dyes,
even in confocal images free of out-of-focus blur,
is that the amount of dye fluorescence detected is very
dependent on the amount of dye measured in
the cytosol. In tip growing cells, probably due to the
asymmetric distribution of cytosol, it is likely to exist
a tip-to-base gradient in dye which is opposite to the gradient in [Ca2+]c. This is not due to the lower pathlength in the tip since its still observed when we collect very
thin optical sections. Thus, the true distribution of Ca2+ can not be assessed.
In summary, SW dyes can be used if the proper
controls are performed but not if the research
pretends to establish absolute Ca2+ values. If
that s the case, then DW dyes must be used.
DW confocal can solve most of the problems but
again precise controls must be done otherwise
artefacts will outnumber the true data.
Rui
Rui Malhs
__________________________________________
Dept Biologia Vegetal, FCL, Bloco C2
Univ de Lisboa, Campo Grande, 1700 Lisboa,
Portugal. Tel. 01 7500069 Fax. 01 7500048
Email. [log in to unmask]
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