Suggestion for setting up Calcium measurement system

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

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Dear listers,

We want to set up a calcium measurement system based on our old Nikon
microscpe (Eclipse TE300). It has a very nice environmental chamber and
equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter wheel.

We don't want something very fancy (like very high resolution or very high
speed acquisition) and really want to make a full use of what we have now
since we have a very limited budget.
Based on my research, we might need to buy a filter cube suitable for calcium
measurement (eg. Fura-2) and acquisition software with ratio imaging
application. Anything else do we need?

I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice
system to do calcium measurement. However, it is quite pricy and sold as a
whole package.

I would really appreciate if anyone here with experience of setting up a
calcium measurement system gives me some suggestion?

Thank you,

Yi

[Johannes Helm]

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Good afternoon,

an important decision - which most probably will be made by your budget -
is whether you want to go for

1)
a system that will be suitable for quantitative ratiometric measurements
at minimum time delay between the measurements of the two channels,

2)
a system that will be suitable for quantitative ratiometric measurements
at which the time delay between the measurements of the two channels can
be "long" - i.e. at least some seconds,

3)
a system for qualitative measurements, only (at which there are some
tricks to make even qualitative measurements a kind of semi-quantitative).

I write this since you explicitely mentioned the dye substance "Fura-2" in
your mail, which is an excitation ratiometric dye substance.

Some comments:
a)
Before you consider ratiometric measurements at all, estimate your photon
statistics. You will need quite a large number of photons to produce fully
quantitative [Ca2+](x, y, z, t) images that are not drowned in noise,
since the ratio imaging algorithm is strongly noise enhancing. Also,
during your data analysis you should avoid using 8 bit or 12 bit values
since truncation errors will have a terrible effect.
Ref. for an analysis of that problem:
Helm, Patwardhan, Manders, 1997, "A study of the precision of confocal,
ratiometric, Fura-2-based [Ca2+]measurements", Cell Calcium 22(4):287-298


b)
If you want to do ratiometric measurements, it will be cheaper to avoid
excitation ratiometric dyes and go for an emission ratiometric dye, if
possible, e.g. one of the Indoes.


c)
Whether excitation or emission ratiometric, you will - dependent on your
measurement system - need either two filter cubes or one filter cube with
two different filters for excitation or emission, which then will not be
mounted in the cubes but somewhere else in the system (either on the
illumination side for the excitation ratiometric system or on the emission
side for the emission ratiometric system).

d)
Your budget might allow you to purchase one of these:
http://www.photometrics.com/products/multichannel/dv2.php
which will allow you to use your single camera as a two channel detector
suitable even for fast- provided your rate of photons is large enough -
emission ratiometric measurements.

e)
If ratiometry is beyond your financial horizon then you will most probably
need to only purchase a suitable filtercube for your dye substance of
choice.

f)
You may write your own data analysis software or check, e.g., ImageJ for
Macros for your data analysis. Alternatively, you might inspect the market
and find commercial data analysis software (possibly even combined with a
suitable acquisition software). Since you have a Hamamatsu Camera, it
might be a good idea to talk to your local Hamamatsu dealer about that.

g)
Definitely worth the investment even at a poor budget situation will be an
electro mechanical shutter (e.g. the "Uniblitz" type by Vincent
Associates, ref. www.uniblitz.com, or by JML, ref.
http://www.jmloptical.com/pages/electronic-shutter-systems.aspx ) that can
be triggered from your camera data acquisition software in order to
minimize bleaching.

Best wishes,

Johannes


--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

> *****
> To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear listers,
>
> We want to set up a calcium measurement system based on our old Nikon
microscpe (Eclipse TE300). It has a very nice environmental chamber and
equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter
wheel.
>
> We don't want something very fancy (like very high resolution or very
high
> speed acquisition) and really want to make a full use of what we have
now
> since we have a very limited budget.
> Based on my research, we might need to buy a filter cube suitable for
calcium
> measurement (eg. Fura-2) and acquisition software with ratio imaging
application. Anything else do we need?
>
> I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice
system to do calcium measurement. However, it is quite pricy and sold as
a
> whole package.
>
> I would really appreciate if anyone here with experience of setting up a
calcium measurement system gives me some suggestion?
>
> Thank you,
>
> Yi
>

[Boswell, Carl A - (cboswell)]

Hi Sara,
An additional item not mentioned in Johannes' excellent reply is the transmission limits of most lenses to UV wavelengths.  If you are set on using Fura2, seriously consider a quartz or other high-UV transmission objective.  Because so little 340nm light gets to the sample using "normal" lenses, the fluorescent signal received from the 340 excitation remains essentially unchanged with changes in intracellular calcium, rendering the sensitivity of your measurements very low.

Good luck,
C

Carl A. Boswell
520-954-7053

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY Sara Smith
Sent: Friday, October 21, 2011 8:12 AM
To: [hidden email]
Subject: Suggestion for setting up Calcium measurement system

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Dear listers,

We want to set up a calcium measurement system based on our old Nikon microscpe (Eclipse TE300). It has a very nice environmental chamber and equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter wheel.

We don't want something very fancy (like very high resolution or very high speed acquisition) and really want to make a full use of what we have now since we have a very limited budget.
Based on my research, we might need to buy a filter cube suitable for calcium measurement (eg. Fura-2) and acquisition software with ratio imaging application. Anything else do we need?

I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice system to do calcium measurement. However, it is quite pricy and sold as a whole package.

I would really appreciate if anyone here with experience of setting up a calcium measurement system gives me some suggestion?

Thank you,

Yi

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

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Dear Johannes,



Thank you for all your inputs. I really appreciate your time and help.

It is very helpful for us and all the information is valuable.



For the measurement system, we want to have a least semi-quantitative
feature.

We are planning to measure the calcium concentration by using ratio metric
dye.

So the change of ratio of the intensity of two channels will present the
change of calcium concentration. At this stage, we might not need to measure
the exactly calcium concentration in uM. Although is semi-quantitative, we
still want the time delay between the measurements of the two channels to be
as short as possible.



The reason I listed Fura-2 as an example is I used to use it and the
microscope is equipped with a filter wheel, however we are open to any
dye that can fulfill this mission. Our microscope is a wide-field
fluorescent microscope not confocal. Is it still cheaper to go with an
emission ratio metric dye? If this is the case, we can reconsider to use
indoes instead of Fura-2.



As for the system set up, I was so clueless and your email helps me a lot.

It is very informative and guides me in the right direction.

Thank you so much.

I will read your paper and do more research about it.

Thanks again!



Yi


On Fri, Oct 21, 2011 at 11:50 AM, P. Johannes Helm
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good afternoon,
>
> an important decision - which most probably will be made by your budget -
> is whether you want to go for
>
> 1)
> a system that will be suitable for quantitative ratiometric measurements
> at minimum time delay between the measurements of the two channels,
>
> 2)
> a system that will be suitable for quantitative ratiometric measurements
> at which the time delay between the measurements of the two channels can
> be "long" - i.e. at least some seconds,
>
> 3)
> a system for qualitative measurements, only (at which there are some
> tricks to make even qualitative measurements a kind of semi-quantitative).
>
> I write this since you explicitely mentioned the dye substance "Fura-2" in
> your mail, which is an excitation ratiometric dye substance.
>
> Some comments:
> a)
> Before you consider ratiometric measurements at all, estimate your photon
> statistics. You will need quite a large number of photons to produce fully
> quantitative [Ca2+](x, y, z, t) images that are not drowned in noise,
> since the ratio imaging algorithm is strongly noise enhancing. Also,
> during your data analysis you should avoid using 8 bit or 12 bit values
> since truncation errors will have a terrible effect.
> Ref. for an analysis of that problem:
> Helm, Patwardhan, Manders, 1997, "A study of the precision of confocal,
> ratiometric, Fura-2-based [Ca2+]measurements", Cell Calcium 22(4):287-298
>
>
> b)
> If you want to do ratiometric measurements, it will be cheaper to avoid
> excitation ratiometric dyes and go for an emission ratiometric dye, if
> possible, e.g. one of the Indoes.
>
>
> c)
> Whether excitation or emission ratiometric, you will - dependent on your
> measurement system - need either two filter cubes or one filter cube with
> two different filters for excitation or emission, which then will not be
> mounted in the cubes but somewhere else in the system (either on the
> illumination side for the excitation ratiometric system or on the emission
> side for the emission ratiometric system).
>
> d)
> Your budget might allow you to purchase one of these:
> http://www.photometrics.com/products/multichannel/dv2.php
> which will allow you to use your single camera as a two channel detector
> suitable even for fast- provided your rate of photons is large enough -
> emission ratiometric measurements.
>
> e)
> If ratiometry is beyond your financial horizon then you will most probably
> need to only purchase a suitable filtercube for your dye substance of
> choice.
>
> f)
> You may write your own data analysis software or check, e.g., ImageJ for
> Macros for your data analysis. Alternatively, you might inspect the market
> and find commercial data analysis software (possibly even combined with a
> suitable acquisition software). Since you have a Hamamatsu Camera, it
> might be a good idea to talk to your local Hamamatsu dealer about that.
>
> g)
> Definitely worth the investment even at a poor budget situation will be an
> electro mechanical shutter (e.g. the "Uniblitz" type by Vincent
> Associates, ref. www.uniblitz.com, or by JML, ref.
> http://www.jmloptical.com/pages/electronic-shutter-systems.aspx ) that can
> be triggered from your camera data acquisition software in order to
> minimize bleaching.
>
> Best wishes,
>
> Johannes
>
>
> --
> P. Johannes Helm, M.Sc. PhD
> Seniorengineer
> CMBN
> University of Oslo
> Institute of Basic Medical Science
> Department of Anatomy
> Postboks 1105 - Blindern
> NO-0317 Oslo
>
> Voice:  [phone]
> Fax:    [phone]
>
> WWW:    folk.uio.no/jhelm
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
>  > Dear listers,
> >
> > We want to set up a calcium measurement system based on our old Nikon
> microscpe (Eclipse TE300). It has a very nice environmental chamber and
> equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter
> wheel.
> >
> > We don't want something very fancy (like very high resolution or very
> high
> > speed acquisition) and really want to make a full use of what we have
> now
> > since we have a very limited budget.
> > Based on my research, we might need to buy a filter cube suitable for
> calcium
> > measurement (eg. Fura-2) and acquisition software with ratio imaging
> application. Anything else do we need?
> >
> > I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice
> system to do calcium measurement. However, it is quite pricy and sold as
> a
> > whole package.
> >
> > I would really appreciate if anyone here with experience of setting up a
> calcium measurement system gives me some suggestion?
> >
> > Thank you,
> >
> > Yi
> >
>

[SUBSCRIBE CONFOCALMICROSCOPY Sara Smith]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear Carl,

Thank you for your email.
It is very good point. I will double check all of our objectives.
Thanks again!

Yi
On Fri, Oct 21, 2011 at 12:55 PM, Boswell, Carl A - (cboswell) <
[hidden email]> wrote:

> Hi Sara,
> An additional item not mentioned in Johannes' excellent reply is the
> transmission limits of most lenses to UV wavelengths.  If you are set on
> using Fura2, seriously consider a quartz or other high-UV transmission
> objective.  Because so little 340nm light gets to the sample using "normal"
> lenses, the fluorescent signal received from the 340 excitation remains
> essentially unchanged with changes in intracellular calcium, rendering the
> sensitivity of your measurements very low.
>
> Good luck,
> C
>
> Carl A. Boswell
> 520-954-7053
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of SUBSCRIBE CONFOCALMICROSCOPY Sara Smith
> Sent: Friday, October 21, 2011 8:12 AM
> To: [hidden email]
> Subject: Suggestion for setting up Calcium measurement system
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear listers,
>
> We want to set up a calcium measurement system based on our old Nikon
> microscpe (Eclipse TE300). It has a very nice environmental chamber and
> equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter wheel.
>
> We don't want something very fancy (like very high resolution or very high
> speed acquisition) and really want to make a full use of what we have now
> since we have a very limited budget.
> Based on my research, we might need to buy a filter cube suitable for
> calcium measurement (eg. Fura-2) and acquisition software with ratio imaging
> application. Anything else do we need?
>
> I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice
> system to do calcium measurement. However, it is quite pricy and sold as a
> whole package.
>
> I would really appreciate if anyone here with experience of setting up a
> calcium measurement system gives me some suggestion?
>
> Thank you,
>
> Yi
>
>

[Johannes Helm]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Yi Zheng,


>
> For the measurement system, we want to have a least semi-quantitative
> feature.
>
> We are planning to measure the calcium concentration by using ratio
> metric dye.

Ok.

>
> So the change of ratio of the intensity of two channels will present the
> change of calcium concentration. At this stage, we might not need to
> measure
> the exactly calcium concentration in uM. Although is semi-quantitative,
> we still want the time delay between the measurements of the two channels
> to be as short as possible.
>
>
>
> The reason I listed Fura-2 as an example is I used to use it and the
> microscope is equipped with a filter wheel, however we are open to any dye
> that can fulfill this mission. Our microscope is a wide-field fluorescent
> microscope not confocal. Is it still cheaper to go with an emission ratio
> metric dye? If this is the case, we can reconsider to use indoes instead of
> Fura-2.

Since you already have a filter wheel attached to your microscope, you
could use an excitation ratiometric dye.
However, unless you would use an IMS style CLSM setup - you write that you
are NOT planning to do this - excitation ratiometric measurements will
un-avoidably result in time delays between the recordings of the
fluorecenses of the Ca2+ bound and free dye molecules. Unless, of course,
there is a technique avoiding this, which I am not aware of.

For an emission ratiometric dye, you can buy equipment, which even in the
wide field case will make it possible for you to avoid these time delays.
Since you would like to attain a good time resolution, an emission
ratiometric dye might, hence, be preferrable (provided that you, sooner or
later, will have the budget to either buy a DV2 or possibly even a second
camera and a DC2). An example for an emission ratiometric dye is Indo-1,
although this dye has to be excited in the UV (excitation max. at approx.
350nm), what, as Carl Boswell has written, will be a problem on many
microscope objectives.


The subject is getting a little bit special and other group members
possibly regard it as bothering. For the technical details, we might
continue our discussion without Cc-ing to the confocal list (unless there
are participants who still are interested, of course).

Best wishes,
Johannes

>
>
>
> As for the system set up, I was so clueless and your email helps me a
> lot.
>
> It is very informative and guides me in the right direction.
>
> Thank you so much.
>
> I will read your paper and do more research about it.
>
> Thanks again!
>
>
>
> Yi
>
>
> On Fri, Oct 21, 2011 at 11:50 AM, P. Johannes Helm
> <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:

http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> Good afternoon,
>> an important decision - which most probably will be made by your budget -
>> is whether you want to go for
>> 1)
>> a system that will be suitable for quantitative ratiometric
measurements
>> at minimum time delay between the measurements of the two channels, 2)
>> a system that will be suitable for quantitative ratiometric
measurements
>> at which the time delay between the measurements of the two channels
can
>> be "long" - i.e. at least some seconds,
>> 3)
>> a system for qualitative measurements, only (at which there are some
tricks to make even qualitative measurements a kind of
>> semi-quantitative).
>> I write this since you explicitely mentioned the dye substance "Fura-2" in
>> your mail, which is an excitation ratiometric dye substance.
>> Some comments:
>> a)
>> Before you consider ratiometric measurements at all, estimate your photon
>> statistics. You will need quite a large number of photons to produce fully
>> quantitative [Ca2+](x, y, z, t) images that are not drowned in noise,
since the ratio imaging algorithm is strongly noise enhancing. Also,
during your data analysis you should avoid using 8 bit or 12 bit values
since truncation errors will have a terrible effect.
>> Ref. for an analysis of that problem:
>> Helm, Patwardhan, Manders, 1997, "A study of the precision of confocal,
ratiometric, Fura-2-based [Ca2+]measurements", Cell Calcium
>> 22(4):287-298
>> b)
>> If you want to do ratiometric measurements, it will be cheaper to avoid
excitation ratiometric dyes and go for an emission ratiometric dye, if
possible, e.g. one of the Indoes.
>> c)
>> Whether excitation or emission ratiometric, you will - dependent on
your
>> measurement system - need either two filter cubes or one filter cube with
>> two different filters for excitation or emission, which then will not
be
>> mounted in the cubes but somewhere else in the system (either on the
illumination side for the excitation ratiometric system or on the
emission
>> side for the emission ratiometric system).
>> d)
>> Your budget might allow you to purchase one of these:
>> http://www.photometrics.com/products/multichannel/dv2.php
>> which will allow you to use your single camera as a two channel
detector
>> suitable even for fast- provided your rate of photons is large enough -
emission ratiometric measurements.
>> e)
>> If ratiometry is beyond your financial horizon then you will most probably
>> need to only purchase a suitable filtercube for your dye substance of
choice.
>> f)
>> You may write your own data analysis software or check, e.g., ImageJ
for
>> Macros for your data analysis. Alternatively, you might inspect the market
>> and find commercial data analysis software (possibly even combined with a
>> suitable acquisition software). Since you have a Hamamatsu Camera, it
might be a good idea to talk to your local Hamamatsu dealer about that.
g)
>> Definitely worth the investment even at a poor budget situation will be an
>> electro mechanical shutter (e.g. the "Uniblitz" type by Vincent
Associates, ref. www.uniblitz.com, or by JML, ref.
>> http://www.jmloptical.com/pages/electronic-shutter-systems.aspx ) that can
>> be triggered from your camera data acquisition software in order to
minimize bleaching.

>> Best wishes,
>> Johannes
>> --
>> P. Johannes Helm, M.Sc. PhD
>> Seniorengineer
>> CMBN
>> University of Oslo
>> Institute of Basic Medical Science
>> Department of Anatomy
>> Postboks 1105 - Blindern
>> NO-0317 Oslo
>> Voice:  [phone]
>> Fax:    [phone]
>> WWW:    folk.uio.no/jhelm
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>>  > Dear listers,
>> >
>> > We want to set up a calcium measurement system based on our old Nikon
>> microscpe (Eclipse TE300). It has a very nice environmental chamber and

equipped with Hamamatsu Digital Camera (C4742-95) and BioPoint filter
wheel.

>> >
>> > We don't want something very fancy (like very high resolution or very
>> high
>> > speed acquisition) and really want to make a full use of what we have
>> now
>> > since we have a very limited budget.
>> > Based on my research, we might need to buy a filter cube suitable for
>> calcium
>> > measurement (eg. Fura-2) and acquisition software with ratio imaging
>> application. Anything else do we need?
>> >
>> > I was looking at Hamamatsu AQUACOSMOS/Basic System. It is a very nice
>> system to do calcium measurement. However, it is quite pricy and sold

as
>> a
>> > whole package.
>> >
>> > I would really appreciate if anyone here with experience of setting
up
>> a
>> calcium measurement system gives me some suggestion?
>> >
>> > Thank you,
>> >
>> > Yi
>> >
>


--
P. Johannes Helm, Dipl. Pys., tekn.lic., tekn.dr.
Senioringeniør
CMBN
IMB
Anatomi
Postboks 1105 - Blindern

kontor: Gaustad, Domus Medica, rom 0347
labor: Gaustad, Domus Medica, rom 0356
telefon: 51159
telefaks: 51499

[B. Prabhakar Pandian]

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Hello Everybody:
                                  It may be an off confocal question,
but I was wondering if someone can point me to where I can get uniform
sizes of carbon particles (5-20µm sizes).

Thanks,

-Prabhakar

[Wolfgang Staroske]

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Hi Prabhakar,

you can try Goodfellow. I ordered carbon particles long time ago there,
but I don't know, if they have your special sizes.

www.goodfellow.com

- No commercial interest -

Bye Wolfgang

Am 20:59, schrieb B. Prabhakar Pandian:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Everybody:
> It may be an off confocal question,
> but I was wondering if someone can point me to where I can get uniform
> sizes of carbon particles (5-20µm sizes).
>
> Thanks,
>
> -Prabhakar

--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: [phone]
Fax.: [phone]
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de

[umsinghbiotech]

Dear Sir,
I am using Fura2 dye for calcium staining with fluorescent microscope (Nikon). The filter which are required for this dye should have absorbance range (340-380nm) and emmission range (504-512nm). But due to unavailability of filter having both absorbance and emmission in any single filter,I used DAPI and FITC filter ,
but the result which we obtained have no any difference between Fura2 (dye) treated sample and control
sample. Please suggest me how I solve this problem.

Thanks


Regards,
Ums