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Dear Ms. Schmaus,
I can tell you something about Fura-2 from my own experiences. I have
been onvolved in Calcium ratio imaging for cardiology studies in a
pharmaceutical company.
People have been using ultrafast camera's for Calcium ratio imaging,
which puzzles me a bit. From what I remember, at 20 deg. Celsius, the
Calcium sensitive probe Fura-2 needs 5-10 ms to reach equilibrium in a
solution of about 140 mM Calcium. We used regular PAL video cameras (25
fps, 40 msec./frame) with a frame splitter device, which enabled us to
monitor intracellular Calcium changes and relocation at 1/4 of the frame
rate, at 10 msec in individual cells. Taking the Nyquist sampling
theorem into account, this is sufficient to monitor most phenomena we
were interested in.
We used Fura-2, as the use of Calcium ratio imaging which has some
advances compared to non-ratio Calcium measurement (i.e. concentration
changes of the probe). For Calcium ratio measurements, a
Mercury arc lamp provides the best result I guess, with its high
emission of light in the near-UV range (I do not like Mercury arcs for
other fluorescent work, due to their "spiky" spectrum, but prefer Xenon
arcs instead, as their emission is more evenly spread through the
visible spectrum).
For the optics we used Fluorite lenses, as they transmit better in the
near-UV range, than glass objectives. What is your opinion of the optics
for Calcium ratio measurements ?
We used mechanical filter changers for switching between the excitation
wavelengths, what about electronic devices ? From what I remember, their
transmission efficiency is much lower than "traditional" filters ?
Some data I remeber from my own work:
The concentration of intracellular Calcium lies between 10 nM and 10 uM
or even higher I believe. The excitation wavelength optimum changes when
Fura-2 binds Calcium. Fura-2 has a Kd of about 145 nM and is about
saturated above 1 uM Calcium (this may change depending on the
conditions ?).
When Fura-2 binds Calcium, the excitation optimum shifts between 300 and
400 nm, 363 (ion free) and 335 nm, in practice 340 nm and 380 nm are
taken for the two excitation wavelengths (Mercury arc peaks). The more
Calcium is present, the higher the absorption shift towards 340 nm. One
can detect this shift by monitoring the emission at 510 nm (~green
light), the change in emission intensity will reflect the absorption
shift between 340 and 380 nm. The Kd (dissociation constant) for Calcium
of Fura-2 is ~135 nM in Mg2+ free Calcium buffers and ~224 nM in the
presence of 1 mM Mg2+. Although Fura-2 may accurately indicate peaks of
Calcium up to 500 nM in cells, Fura-2 exhibits limited sensitivity above
500 nM Calcium.
There is no Calcium dependent change in absorption at about 360 nm.,
which is called the isosbestic point of Fura-2. Measurement of the 510
nm emission when exciting at 360 nm can be used as a measure for
fotobleaching of Fura-2.
Best regards,
Peter Van Osta
Union Biometrica N.V./S.A.
European Scientific Operations (ESO)
Cipalstraat 3
B-2440 Geel
Belgium
Tel.: +32 (0)14 570 619
Fax.: +32 (0)14 570 621
http://www.unionbio.com/
http://ourworld.compuserve.com/homepages/pvosta/cvwww.htm
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Date: Tue, 8 Apr 2003 20:18:50 +0200
From: Klughammer - Anneliese Schmaus <[log in to unmask]>
Subject: Fura + CCD Camera
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Hi,
I am looking for a CCD camera which allows me to capture
Fura-2 stained objects. A CCD camera what kind of technical
specification should it have? What is important?
mfg / regards
Anneliese Schmaus
klughammer gmbh
Strassbach 9
85229 Markt Indersdorf
Germany
Tel. +49 08136 6011
Fax +49 08136 7098
[log in to unmask]
www.klughammer.de
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