exorcising spirits from Fluoview

[mmodel]

I apologize if this is a second message to the list, I think the first one didn't go through
 
We are having a bizzare scanning problem. Straight vertical lines in an object become slightly zigzagged with a period of up to 6-7 scan lines, and there also may be some oscillation in the intensity. The period and the magnitude of this periodic noise depends on the scan speed and the scan size. So far we (with the help of an Olympus engineer) have established that:
 
1. It doesn't seem to be the scanner controller or the galvo mechanism
2. It does not seem to be the electric power in the building
3. It is not a mechanical vibration
4. It is not a computer
5. It is not the cables
6. It is not a 60 Hz noise
 
Sometimes connecting the scanner controller to the outlet through a long extension cord seemed to help which may suggest a problem with grounding, but as soon as we concluded that, the trick stopped working. The trouble could not be reproduced at the Olympus testing lab.
 
Has anyone experienced anything similar and successfully resolved the problem?
 
Many thanks in advance!
 
Mike Model

[Guenter Giese]

Mike,

although you mentioned that vibrations are not the case for your imaging
problems: It may be worthwhile to check the whole microscope stand for
vibrations.

I experienced such a problem with our ZEISS Axioscope FS2 Mot with a LSM510
scanner on top. This microscope is mounted on a Luigs and Neumann base, and
this all is placed on a Melles-Griot anti-vibration table.

It turned out that the whole microscope showed a resonance.

The main source of the vibration turned out to be a heavily pulsing stream
of cooling water through silicone tubing on the table connected to our
multiphoton laser. Of course I also tried to minimize this source of
vibration, but could not get rid of it totally.

But I could fix vibrations by mounting two thick type 95 aluminium rails
onto the breadboard (left and right of the scanner, respectively), and from
these posts, two minor rails fix ("squeeze") the scanner housing in between
them (damping material between scanner and side rails).

No problems anymore with vibrations / zigzag lines.

Hope this helps,

Guenter


------------------------------------------
Dr. Guenter Giese
Light Microscopy Facility Manager
Dept. of Biomedical Optics
MPI fuer Medizinische Forschung Jahnstr. 29
D-69120 Heidelberg, Germany
Phone [phone] (Fax: -325)
e-mail: [hidden email]  

> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List
> [mailto:[hidden email]] Im Auftrag von
> MODEL, MICHAEL
> Gesendet: Mittwoch, 7. Oktober 2009 13:28
> An: [hidden email]
> Betreff: exorcising spirits from Fluoview
>
> I apologize if this is a second message to the list, I think
> the first one didn't go through
>  
> We are having a bizzare scanning problem. Straight vertical
> lines in an object become slightly zigzagged with a period of
> up to 6-7 scan lines, and there also may be some oscillation
> in the intensity. The period and the magnitude of this
> periodic noise depends on the scan speed and the scan size.
> So far we (with the help of an Olympus engineer) have
> established that:
>  
> 1. It doesn't seem to be the scanner controller or the galvo
> mechanism 2. It does not seem to be the electric power in the
> building 3. It is not a mechanical vibration 4. It is not a
> computer 5. It is not the cables 6. It is not a 60 Hz noise
>  
> Sometimes connecting the scanner controller to the outlet
> through a long extension cord seemed to help which may
> suggest a problem with grounding, but as soon as we concluded
> that, the trick stopped working. The trouble could not be
> reproduced at the Olympus testing lab.
>  
> Has anyone experienced anything similar and successfully
> resolved the problem?
>  
> Many thanks in advance!
>  
> Mike Model
>

[simon walker (BI)]

Hi Mike,

We have seen a similar problem to this, particularly apparent in DIC transmitted light images.  However, of our two FV1000 systems (located in different rooms), it is only readily apparent on one.  This has led us to believe it is a vibration issue, especially as we can cause a more severe, but similar-looking problem by deliberately introducing a source of vibration near to the microscope.  However, if true, we have yet to isolate the cause of the problem vibration.

While I'm here...has anyone properly investigated the effect of the DIC objective prism in confocal fluorescence imaging?  I had always assumed (rightly or wrongly) that it's presence didn't influence the PSF, but last week I was imaging some subresolution beads and found that, particularly on our IX81-based FV1000 confocals, the DIC objective prism had quite a pronounced effect on the psf.  Specifically the psf was distorted along a diagonal axis and at the point of focus, the bead appeared significantly larger with the prism in place. The implication of this is that for confocal fluorescence imaging, the resolution of the microscope is reduced when the DIC objective prism is in place.  I've also looked on our Zeiss Axiovert 200 and Nikon TE-2000 based systems which employ a slightly different method of DIC and there the effect is much less pronounced although noticeable.

Simon


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: 07 October 2009 12:28
To: [hidden email]
Subject: exorcising spirits from Fluoview

I apologize if this is a second message to the list, I think the first one didn't go through
 
We are having a bizzare scanning problem. Straight vertical lines in an object become slightly zigzagged with a period of up to 6-7 scan lines, and there also may be some oscillation in the intensity. The period and the magnitude of this periodic noise depends on the scan speed and the scan size. So far we (with the help of an Olympus engineer) have established that:
 
1. It doesn't seem to be the scanner controller or the galvo mechanism
2. It does not seem to be the electric power in the building
3. It is not a mechanical vibration
4. It is not a computer
5. It is not the cables
6. It is not a 60 Hz noise
 
Sometimes connecting the scanner controller to the outlet through a long extension cord seemed to help which may suggest a problem with grounding, but as soon as we concluded that, the trick stopped working. The trouble could not be reproduced at the Olympus testing lab.
 
Has anyone experienced anything similar and successfully resolved the problem?
 
Many thanks in advance!
 
Mike Model

[James Pawley]

>I apologize if this is a second message to the list, I think the
>first one didn't go through
>
>We are having a bizzare scanning problem. Straight vertical lines in
>an object become slightly zigzagged with a period of up to 6-7 scan
>lines, and there also may be some oscillation in the intensity. The
>period and the magnitude of this periodic noise depends on the scan
>speed and the scan size. So far we (with the help of an Olympus
>engineer) have established that:
>
>1. It doesn't seem to be the scanner controller or the galvo mechanism
>2. It does not seem to be the electric power in the building
>3. It is not a mechanical vibration
>4. It is not a computer
>5. It is not the cables
>6. It is not a 60 Hz noise
>
>Sometimes connecting the scanner controller to the outlet through a
>long extension cord seemed to help which may suggest a problem with
>grounding, but as soon as we concluded that, the trick stopped
>working. The trouble could not be reproduced at the Olympus testing
>lab.
>
>Has anyone experienced anything similar and successfully resolved the problem?
>
>Many thanks in advance!
>
>Mike Model

Hi Michael,

You say "6-7 scan lines" but do not mention the horizontal scan rate.
Assuming it is about 2 ms, then the problem would seem to have a
period of 12-14 ms (or about 83 to 71 hz), which is close the line
mains frequency. Your experience with the longer extension cord also
suggests ground loops.

Ground loops are present whenever any part of the electrical system
contains conductors that permit current to travel between any two
points in space by more than one path. Ideally, any sensitive wiring
is connected in the form of the branches of a tree, especially any
wiring such as that related to sensing small feedback (such as that
between a mirror position sensor in the scan unit to the scan
amplifier in the electronics?).

  In modern microscopes, these problems are sometimes avoided by
digitizing the analog sense signal before it is returned, and also by
keeping wiring for the "earth" return of power circuits separate from
that of signal (sensing) circuits.

The efficacy of this separation can be defeated by any conductive
path that connects the two systems (such as an added accessory that
is connected to grounded plug in the wall or some concatenation of
metal items connecting the two systems - maybe a pair of scissors
resting between the laser benchtop and some unpainted part of the
microscope.)

They can also be connected inductively. A significant mains-frequency
magnetic field (more than a few mGauss) will be produced by any high
current flowing "in a loop". For instance, if some high current
device nearby, or in an adjacent room, is wired incorrectly so that,
instead of the hot and neutral currents passing through wires that
are in the same power cord (i.e., very small loop with dimensions of
mm, and therefore making a dipole field that decays with the third
power of a distance in mm) the return current may flow through some
metal parts of the device and back through some safety ground strap
(making a loop with dimensions of meters that may not be very large
but that decays a thousand times more slowly.

If such a stray magnetic field is present in the area of your
instrument, there are a number of ways that it can induce an
erroneous alternating mains-frequency currents in sensing circuits
and this will cause the amplifiers that drive the scan mirrors to try
to compensate. As they are compensating for a position error that is
not actually present, the result is to create a position error.

So I think that the most important thing is to try to find out the
frequency a little more accurately (measure the horizontal scan
period and find out the number of lines more accurately by counting
the lines in say 20 "wiggles" and diving the result by 20).  If it
does seem to be mains frequency where you are (50 or 60 hz), then get
a stray field sensor (a coil of wire and a sensitive oscilloscope?)
and see what kinds of fields are present in the area. You do this by
waving the coil around and twisting it every-which-way as you do so
to get the maximum reading. Remember magnetic fields have a direction
as well as a magnitude and you will get the maximum reading only if
the plane of the coil is oriented perpendicular to the direction of
the magnetic field lines (or parallel to the loop making the field.).
See whether this reading changes when nearby equipment is turned on
or off and try to isolate the source and remove it by proper
grounding.

Alternatively, you can do the reverse and MAKE a large field by
passing mains-frequency current through a coil (a coil used for
degaussing the CRTs in old TV sets can work well, but if you make
your own, don't electrocute yourself!). If the field from this coil
creates an artifact similar to that you originally saw, then you know
that you are getting close. By varying the coil's position and
orientation you may even be able to cancel the effect out! But this
is not recommended as a permanent fix.

If the problem is new, then something has changed and It is probably
either a change in the ambient stray field or alternatively that some
connector is not making good contact (wiggle the connectors?) or that
a ground loop has been created by the addition of an accessory or the
happenstance position of some metal parts touching things they should
not touch.

It is also just possibly a mechanical effect: Mains frequency
vibration from some transformer etc. being coupled into the scan unit
and making the mirrors wiggle.

Hope that this helps.  I have a couple of PDFs of articles about
hunting down stray fields and currents in SEMs  and can send them if
you contact me directly.

Cheers,

Jim P.
--
               **********************************************
Prof. James B. Pawley,                          Ph.  608-263-3147
Room 223, Zoology Research Building,              
FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
3D Microscopy of Living Cells Course, June 14-28, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/             Applications due by March 15, 2010
               "If it ain't diffraction, it must be statistics." Anon.

[James Pawley]

>Hi Mike,
>
>We have seen a similar problem to this, particularly apparent in DIC
>transmitted light images.  However, of our two FV1000 systems
>(located in different rooms), it is only readily apparent on one.
>This has led us to believe it is a vibration issue, especially as we
>can cause a more severe, but similar-looking problem by deliberately
>introducing a source of vibration near to the microscope.  However,
>if true, we have yet to isolate the cause of the problem vibration.
>
>While I'm here...has anyone properly investigated the effect of the
>DIC objective prism in confocal fluorescence imaging?  I had always
>assumed (rightly or wrongly) that it's presence didn't influence the
>PSF, but last week I was imaging some subresolution beads and found
>that, particularly on our IX81-based FV1000 confocals, the DIC
>objective prism had quite a pronounced effect on the psf.
>Specifically the psf was distorted along a diagonal axis and at the
>point of focus, the bead appeared significantly larger with the
>prism in place. The implication of this is that for confocal
>fluorescence imaging, the resolution of the microscope is reduced
>when the DIC objective prism is in place.  I've also looked on our
>Zeiss Axiovert 200 and Nikon TE-2000 based systems which employ a
>slightly different method of DIC and there the effect is much less
>pronounced although noticeable.
>
>Simon

Hi Simon,

You raise an important point. As I understand it, it all depends on
the polarization direction of the laser light. If the pol direction
is as it should be for DIC operation (i.e., at 45 deg to the tow axes
of the Wollaston prism) then, yes, the light should form two spots
displaced by an amount that depends on the prism but will be close to
the resolution limit of the optics. The direction of the displacement
should be the same as that of the "shadows" in the DIC image.

However, there are some wrinkles. One is that, because the two beams
have different polarizations, they may not both excite fluorescence
in the same way. In particular, if you were looking at the signal
from a single molecule, it might be lined up to be excited by one
beam but not the other, (in which case the PSF would be round but
perhaps slightly displaced) or it might be somewhat excited by both
(in which case it would be more or less distorted at the DIC angle).

Even on large objects such as beads, where pol isn't important, the
relative ellipticity will depend on the extent to which the pol of
the laser is in fact correctly oriented with the Wollastons. (This
can usually be checked by introducing a polarizer into the microscope
tube.) If the laser sends in light polarized in the only the ordinary
(or only the extraordinary) direction of the prism, then the only one
beam will be produced and the result will again be round.

I don't know why you found that the effect was more apparent on the
Olympus than on the Zeiss, but as you know, the DIC prisms are cut
differently for every objective and each one is always a compromise
between parameters that will produce good contrast versus uniformity
over a large field of view and I assume that this will affect the
"real-space" displacement between the two beams. I suppose that the
manufacturers could tell you the parameters that they use.

Glad that you brought it up.

Cheers,

Jim P.
--
               ****************************************
Prof. James B. Pawley,                 Ph.  608-263-3147
Room 223, Zoology Research Building,                         FAX  [phone]
250 N. Mills St., Madison, WI, 53706  [hidden email]
"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39

[Periasamy, Ammasi (ap3t)]

It appears that this is a random phenomena. With our experience I feel like there may be an electrical disturbance in your building electrical system.
We had this kind of scanning problem during 2003 and we went through all kinds of testing. We did not see any issues by continually monitoring the electrical output from the outlet for a week. But later it showed up again. We just added an appropriate UPS between the wall electrical outlet to the control unit and it never returned with our Biorad Radiance2100 system and it still working. May be we are lucky.


Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010
http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Wednesday, October 07, 2009 9:27 AM
To: [hidden email]
Subject: Re: exorcising spirits from Fluoview

>I apologize if this is a second message to the list, I think the
>first one didn't go through
>
>We are having a bizzare scanning problem. Straight vertical lines in
>an object become slightly zigzagged with a period of up to 6-7 scan
>lines, and there also may be some oscillation in the intensity. The
>period and the magnitude of this periodic noise depends on the scan
>speed and the scan size. So far we (with the help of an Olympus
>engineer) have established that:
>
>1. It doesn't seem to be the scanner controller or the galvo mechanism
>2. It does not seem to be the electric power in the building
>3. It is not a mechanical vibration
>4. It is not a computer
>5. It is not the cables
>6. It is not a 60 Hz noise
>
>Sometimes connecting the scanner controller to the outlet through a
>long extension cord seemed to help which may suggest a problem with
>grounding, but as soon as we concluded that, the trick stopped
>working. The trouble could not be reproduced at the Olympus testing
>lab.
>
>Has anyone experienced anything similar and successfully resolved the problem?
>
>Many thanks in advance!
>
>Mike Model

Hi Michael,

You say "6-7 scan lines" but do not mention the horizontal scan rate.
Assuming it is about 2 ms, then the problem would seem to have a
period of 12-14 ms (or about 83 to 71 hz), which is close the line
mains frequency. Your experience with the longer extension cord also
suggests ground loops.

Ground loops are present whenever any part of the electrical system
contains conductors that permit current to travel between any two
points in space by more than one path. Ideally, any sensitive wiring
is connected in the form of the branches of a tree, especially any
wiring such as that related to sensing small feedback (such as that
between a mirror position sensor in the scan unit to the scan
amplifier in the electronics?).

  In modern microscopes, these problems are sometimes avoided by
digitizing the analog sense signal before it is returned, and also by
keeping wiring for the "earth" return of power circuits separate from
that of signal (sensing) circuits.

The efficacy of this separation can be defeated by any conductive
path that connects the two systems (such as an added accessory that
is connected to grounded plug in the wall or some concatenation of
metal items connecting the two systems - maybe a pair of scissors
resting between the laser benchtop and some unpainted part of the
microscope.)

They can also be connected inductively. A significant mains-frequency
magnetic field (more than a few mGauss) will be produced by any high
current flowing "in a loop". For instance, if some high current
device nearby, or in an adjacent room, is wired incorrectly so that,
instead of the hot and neutral currents passing through wires that
are in the same power cord (i.e., very small loop with dimensions of
mm, and therefore making a dipole field that decays with the third
power of a distance in mm) the return current may flow through some
metal parts of the device and back through some safety ground strap
(making a loop with dimensions of meters that may not be very large
but that decays a thousand times more slowly.

If such a stray magnetic field is present in the area of your
instrument, there are a number of ways that it can induce an
erroneous alternating mains-frequency currents in sensing circuits
and this will cause the amplifiers that drive the scan mirrors to try
to compensate. As they are compensating for a position error that is
not actually present, the result is to create a position error.

So I think that the most important thing is to try to find out the
frequency a little more accurately (measure the horizontal scan
period and find out the number of lines more accurately by counting
the lines in say 20 "wiggles" and diving the result by 20).  If it
does seem to be mains frequency where you are (50 or 60 hz), then get
a stray field sensor (a coil of wire and a sensitive oscilloscope?)
and see what kinds of fields are present in the area. You do this by
waving the coil around and twisting it every-which-way as you do so
to get the maximum reading. Remember magnetic fields have a direction
as well as a magnitude and you will get the maximum reading only if
the plane of the coil is oriented perpendicular to the direction of
the magnetic field lines (or parallel to the loop making the field.).
See whether this reading changes when nearby equipment is turned on
or off and try to isolate the source and remove it by proper
grounding.

Alternatively, you can do the reverse and MAKE a large field by
passing mains-frequency current through a coil (a coil used for
degaussing the CRTs in old TV sets can work well, but if you make
your own, don't electrocute yourself!). If the field from this coil
creates an artifact similar to that you originally saw, then you know
that you are getting close. By varying the coil's position and
orientation you may even be able to cancel the effect out! But this
is not recommended as a permanent fix.

If the problem is new, then something has changed and It is probably
either a change in the ambient stray field or alternatively that some
connector is not making good contact (wiggle the connectors?) or that
a ground loop has been created by the addition of an accessory or the
happenstance position of some metal parts touching things they should
not touch.

It is also just possibly a mechanical effect: Mains frequency
vibration from some transformer etc. being coupled into the scan unit
and making the mirrors wiggle.

Hope that this helps.  I have a couple of PDFs of articles about
hunting down stray fields and currents in SEMs  and can send them if
you contact me directly.

Cheers,

Jim P.
--
               **********************************************
Prof. James B. Pawley,                          Ph.  608-263-3147
Room 223, Zoology Research Building,              
FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
3D Microscopy of Living Cells Course, June 14-28, 2010, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/             Applications due by March 15, 2010
               "If it ain't diffraction, it must be statistics." Anon.

[Glen MacDonald-2]

Dear Simon,
I've found a similar diagonal distortion of the PSF induced by the DIC  
objective prism in our FV-1000/IX-81.   It is also apparent while  
looking at 6 um beads.  Another reason  it should be pulled from the  
light path whenever not collecting DIC, besides dropping the  
fluorescent signal by a small amount.

Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

******************************************************************************
The box said "Requires WindowsXP or better", so I bought a Macintosh.
******************************************************************************

> Hi Mike,
>
> We have seen a similar problem to this, particularly apparent in DIC  
> transmitted light images.  However, of our two FV1000 systems  
> (located in different rooms), it is only readily apparent on one.  
> This has led us to believe it is a vibration issue, especially as we  
> can cause a more severe, but similar-looking problem by deliberately  
> introducing a source of vibration near to the microscope.  However,  
> if true, we have yet to isolate the cause of the problem vibration.
>
> While I'm here...has anyone properly investigated the effect of the  
> DIC objective prism in confocal fluorescence imaging?  I had always  
> assumed (rightly or wrongly) that it's presence didn't influence the  
> PSF, but last week I was imaging some subresolution beads and found  
> that, particularly on our IX81-based FV1000 confocals, the DIC  
> objective prism had quite a pronounced effect on the psf.  
> Specifically the psf was distorted along a diagonal axis and at the  
> point of focus, the bead appeared significantly larger with the  
> prism in place. The implication of this is that for confocal  
> fluorescence imaging, the resolution of the microscope is reduced  
> when the DIC objective prism is in place.  I've also looked on our  
> Zeiss Axiovert 200 and Nikon TE-2000 based systems which employ a  
> slightly different method of DIC and there the effect is much less  
> pronounced although noticeable.
>
> Simon
>
>
> -----Original Message-----
> From: Confocal Microscopy List  
> [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
> Sent: 07 October 2009 12:28
> To: [hidden email]
> Subject: exorcising spirits from Fluoview
>
> I apologize if this is a second message to the list, I think the  
> first one didn't go through
>
> We are having a bizzare scanning problem. Straight vertical lines in  
> an object become slightly zigzagged with a period of up to 6-7 scan  
> lines, and there also may be some oscillation in the intensity. The  
> period and the magnitude of this periodic noise depends on the scan  
> speed and the scan size. So far we (with the help of an Olympus  
> engineer) have established that:
>
> 1. It doesn't seem to be the scanner controller or the galvo mechanism
> 2. It does not seem to be the electric power in the building
> 3. It is not a mechanical vibration
> 4. It is not a computer
> 5. It is not the cables
> 6. It is not a 60 Hz noise
>
> Sometimes connecting the scanner controller to the outlet through a  
> long extension cord seemed to help which may suggest a problem with  
> grounding, but as soon as we concluded that, the trick stopped  
> working. The trouble could not be reproduced at the Olympus testing  
> lab.
>
> Has anyone experienced anything similar and successfully resolved  
> the problem?
>
> Many thanks in advance!
>
> Mike Model

[Michael Weber-4]

Dear list,

I am looking for advice on optical "clearing" of fixed tissue before
staining it and using it for light microscopy. Actually "tissue" is not
the precise term, since I would like to clear whole fly embryos. This
process seems to be well established in histology, i.e. using Xylene. I
also found a commercial product called "Histo-Clear" (National
Diagnostics), which claims to preserve tissue structures rather well,
while being less nasty compared to Xylene. Did you guys ever use something
like that? Any input welcome.

cheers,
Michael

[SUBSCRIBE CONFOCALMICROSCOPY Anonymous]

hello michael,

This publication might be interesting for you:


http://www.ncbi.nlm.nih.gov/pubmed/19397748


thorsten


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List im Auftrag von Michael Weber
Gesendet: Mi 07.10.2009 17:56
An: [hidden email]
Betreff: optical clearing of tissue
 
Dear list,

I am looking for advice on optical "clearing" of fixed tissue before
staining it and using it for light microscopy. Actually "tissue" is not
the precise term, since I would like to clear whole fly embryos. This
process seems to be well established in histology, i.e. using Xylene. I
also found a commercial product called "Histo-Clear" (National
Diagnostics), which claims to preserve tissue structures rather well,
while being less nasty compared to Xylene. Did you guys ever use something
like that? Any input welcome.

cheers,
Michael

[Zucker.Robert]

Mike
This is a review that describes our procedure of clearing mammalian and
insect tissue with BABB. Reprints are available on request

Zucker, R.M.Technical note: Whole insects and Mammalian Embryo Imaging
with Confocal Microscopy: Morphology and Apoptosis. Cytometry 2006 69A:
1143-1152

Best wishes
bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Toxicology Assessment Division
Telephone: [phone]   Fax: [phone]
e-mail: [hidden email]

Mail address: USEPA,ORD,NHEERL,TAD
Developmental Biology Branch ( MD 67)
Research Triangle Park, North Carolina, 27711

Shipping address:
2525 E.NC Highway 54
Durham, NC, 27713



                                                                                                                       
  From:       Michael Weber <[hidden email]>                                                                        
                                                                                                                       
  To:         [hidden email]                                                                        
                                                                                                                       
  Date:       10/07/2009 11:56 AM                                                                                      
                                                                                                                       
  Subject:    optical clearing of tissue                                                                              
                                                                                                                       
  Sent by:    Confocal Microscopy List <[hidden email]>                                              
                                                                                                                       





Dear list,

I am looking for advice on optical "clearing" of fixed tissue before
staining it and using it for light microscopy. Actually "tissue" is not
the precise term, since I would like to clear whole fly embryos. This
process seems to be well established in histology, i.e. using Xylene. I
also found a commercial product called "Histo-Clear" (National
Diagnostics), which claims to preserve tissue structures rather well,
while being less nasty compared to Xylene. Did you guys ever use
something
like that? Any input welcome.

cheers,
Michael

[Boswell, Carl A - (cboswell)]

Dr. Periasamy's comment reflects my first thought; insert a UPS device
between your instrument and the house source.  It not only acts as a battery
backup, but as a "line polisher", preventing voltage fluctuations common in
grid electricity.
c


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Periasamy, Ammasi (ap3t)" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, October 07, 2009 7:39 AM
Subject: Re: exorcising spirits from Fluoview


It appears that this is a random phenomena. With our experience I feel like
there may be an electrical disturbance in your building electrical system.
We had this kind of scanning problem during 2003 and we went through all
kinds of testing. We did not see any issues by continually monitoring the
electrical output from the outlet for a week. But later it showed up again.
We just added an appropriate UPS between the wall electrical outlet to the
control unit and it never returned with our Biorad Radiance2100 system and
it still working. May be we are lucky.


Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Biology, Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22904
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu
************************
Workshop on FRET Microscopy, March 9-13, 2010
http://www.kcci.virginia.edu/workshop/workshop2010/index.php
*************************


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of James Pawley
Sent: Wednesday, October 07, 2009 9:27 AM
To: [hidden email]
Subject: Re: exorcising spirits from Fluoview

>I apologize if this is a second message to the list, I think the
>first one didn't go through
>
>We are having a bizzare scanning problem. Straight vertical lines in
>an object become slightly zigzagged with a period of up to 6-7 scan
>lines, and there also may be some oscillation in the intensity. The
>period and the magnitude of this periodic noise depends on the scan
>speed and the scan size. So far we (with the help of an Olympus
>engineer) have established that:
>
>1. It doesn't seem to be the scanner controller or the galvo mechanism
>2. It does not seem to be the electric power in the building
>3. It is not a mechanical vibration
>4. It is not a computer
>5. It is not the cables
>6. It is not a 60 Hz noise
>
>Sometimes connecting the scanner controller to the outlet through a
>long extension cord seemed to help which may suggest a problem with
>grounding, but as soon as we concluded that, the trick stopped
>working. The trouble could not be reproduced at the Olympus testing
>lab.
>
>Has anyone experienced anything similar and successfully resolved the
>problem?
>
>Many thanks in advance!
>
>Mike Model

Hi Michael,

You say "6-7 scan lines" but do not mention the horizontal scan rate.
Assuming it is about 2 ms, then the problem would seem to have a
period of 12-14 ms (or about 83 to 71 hz), which is close the line
mains frequency. Your experience with the longer extension cord also
suggests ground loops.

Ground loops are present whenever any part of the electrical system
contains conductors that permit current to travel between any two
points in space by more than one path. Ideally, any sensitive wiring
is connected in the form of the branches of a tree, especially any
wiring such as that related to sensing small feedback (such as that
between a mirror position sensor in the scan unit to the scan
amplifier in the electronics?).

  In modern microscopes, these problems are sometimes avoided by
digitizing the analog sense signal before it is returned, and also by
keeping wiring for the "earth" return of power circuits separate from
that of signal (sensing) circuits.

The efficacy of this separation can be defeated by any conductive
path that connects the two systems (such as an added accessory that
is connected to grounded plug in the wall or some concatenation of
metal items connecting the two systems - maybe a pair of scissors
resting between the laser benchtop and some unpainted part of the
microscope.)

They can also be connected inductively. A significant mains-frequency
magnetic field (more than a few mGauss) will be produced by any high
current flowing "in a loop". For instance, if some high current
device nearby, or in an adjacent room, is wired incorrectly so that,
instead of the hot and neutral currents passing through wires that
are in the same power cord (i.e., very small loop with dimensions of
mm, and therefore making a dipole field that decays with the third
power of a distance in mm) the return current may flow through some
metal parts of the device and back through some safety ground strap
(making a loop with dimensions of meters that may not be very large
but that decays a thousand times more slowly.

If such a stray magnetic field is present in the area of your
instrument, there are a number of ways that it can induce an
erroneous alternating mains-frequency currents in sensing circuits
and this will cause the amplifiers that drive the scan mirrors to try
to compensate. As they are compensating for a position error that is
not actually present, the result is to create a position error.

So I think that the most important thing is to try to find out the
frequency a little more accurately (measure the horizontal scan
period and find out the number of lines more accurately by counting
the lines in say 20 "wiggles" and diving the result by 20).  If it
does seem to be mains frequency where you are (50 or 60 hz), then get
a stray field sensor (a coil of wire and a sensitive oscilloscope?)
and see what kinds of fields are present in the area. You do this by
waving the coil around and twisting it every-which-way as you do so
to get the maximum reading. Remember magnetic fields have a direction
as well as a magnitude and you will get the maximum reading only if
the plane of the coil is oriented perpendicular to the direction of
the magnetic field lines (or parallel to the loop making the field.).
See whether this reading changes when nearby equipment is turned on
or off and try to isolate the source and remove it by proper
grounding.

Alternatively, you can do the reverse and MAKE a large field by
passing mains-frequency current through a coil (a coil used for
degaussing the CRTs in old TV sets can work well, but if you make
your own, don't electrocute yourself!). If the field from this coil
creates an artifact similar to that you originally saw, then you know
that you are getting close. By varying the coil's position and
orientation you may even be able to cancel the effect out! But this
is not recommended as a permanent fix.

If the problem is new, then something has changed and It is probably
either a change in the ambient stray field or alternatively that some
connector is not making good contact (wiggle the connectors?) or that
a ground loop has been created by the addition of an accessory or the
happenstance position of some metal parts touching things they should
not touch.

It is also just possibly a mechanical effect: Mains frequency
vibration from some transformer etc. being coupled into the scan unit
and making the mirrors wiggle.

Hope that this helps.  I have a couple of PDFs of articles about
hunting down stray fields and currents in SEMs  and can send them if
you contact me directly.

Cheers,

Jim P.
--
               **********************************************
Prof. James B. Pawley,                           Ph.  608-263-3147
Room 223, Zoology Research Building,
FAX  [phone]
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 14-28, 2010, UBC, Vancouver
Canada
Info: http://www.3dcourse.ubc.ca/      Applications due by March 15, 2010
       "If it ain't diffraction, it must be statistics." Anon.

[Rosemary.White]

Dear Simon,

The DIC prism degrades the image quite substantially on our Leica SP2.  I
understood this to be because the fluorescence is "split", but maybe the
laser excitation is affected also.  It has a quite noticable
fuzzing/doubling effect on the final image.  I always recommend that for DIC
overlays, people take two images - fluorescence first, DIC plus fluorescence
second.  Have not looked at the psf, though.

cheers,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334
 


On 8/10/09 12:25 AM, "simon walker (BI)" <[hidden email]> wrote:

> Hi Mike,
>
> We have seen a similar problem to this, particularly apparent in DIC
> transmitted light images.  However, of our two FV1000 systems (located in
> different rooms), it is only readily apparent on one.  This has led us to
> believe it is a vibration issue, especially as we can cause a more severe, but
> similar-looking problem by deliberately introducing a source of vibration near
> to the microscope.  However, if true, we have yet to isolate the cause of the
> problem vibration.
>
> While I'm here...has anyone properly investigated the effect of the DIC
> objective prism in confocal fluorescence imaging?  I had always assumed
> (rightly or wrongly) that it's presence didn't influence the PSF, but last
> week I was imaging some subresolution beads and found that, particularly on
> our IX81-based FV1000 confocals, the DIC objective prism had quite a
> pronounced effect on the psf.  Specifically the psf was distorted along a
> diagonal axis and at the point of focus, the bead appeared significantly
> larger with the prism in place. The implication of this is that for confocal
> fluorescence imaging, the resolution of the microscope is reduced when the DIC
> objective prism is in place.  I've also looked on our Zeiss Axiovert 200 and
> Nikon TE-2000 based systems which employ a slightly different method of DIC
> and there the effect is much less pronounced although noticeable.
>
> Simon
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of MODEL, MICHAEL
> Sent: 07 October 2009 12:28
> To: [hidden email]
> Subject: exorcising spirits from Fluoview
>
> I apologize if this is a second message to the list, I think the first one
> didn't go through
>  
> We are having a bizzare scanning problem. Straight vertical lines in an object
> become slightly zigzagged with a period of up to 6-7 scan lines, and there
> also may be some oscillation in the intensity. The period and the magnitude of
> this periodic noise depends on the scan speed and the scan size. So far we
> (with the help of an Olympus engineer) have established that:
>  
> 1. It doesn't seem to be the scanner controller or the galvo mechanism
> 2. It does not seem to be the electric power in the building
> 3. It is not a mechanical vibration
> 4. It is not a computer
> 5. It is not the cables
> 6. It is not a 60 Hz noise
>  
> Sometimes connecting the scanner controller to the outlet through a long
> extension cord seemed to help which may suggest a problem with grounding, but
> as soon as we concluded that, the trick stopped working. The trouble could not
> be reproduced at the Olympus testing lab.
>  
> Has anyone experienced anything similar and successfully resolved the problem?
>  
> Many thanks in advance!
>  
> Mike Model

[Rosemary.White]

We had a very similar problem, but it turned out to be the connection
between the fibre-optic cable and the laser - it had come slightly loose.
Since you've checked the cables, I guess it has to be something else.

good luck,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334


On 7/10/09 10:27 PM, "MODEL, MICHAEL" <[hidden email]> wrote:

> I apologize if this is a second message to the list, I think the first one
> didn't go through
>  
> We are having a bizzare scanning problem. Straight vertical lines in an object
> become slightly zigzagged with a period of up to 6-7 scan lines, and there
> also may be some oscillation in the intensity. The period and the magnitude of
> this periodic noise depends on the scan speed and the scan size. So far we
> (with the help of an Olympus engineer) have established that:
>  
> 1. It doesn't seem to be the scanner controller or the galvo mechanism
> 2. It does not seem to be the electric power in the building
> 3. It is not a mechanical vibration
> 4. It is not a computer
> 5. It is not the cables
> 6. It is not a 60 Hz noise
>  
> Sometimes connecting the scanner controller to the outlet through a long
> extension cord seemed to help which may suggest a problem with grounding, but
> as soon as we concluded that, the trick stopped working. The trouble could not
> be reproduced at the Olympus testing lab.
>  
> Has anyone experienced anything similar and successfully resolved the problem?
>  
> Many thanks in advance!
>  
> Mike Model

[mmodel]

This is one thing we haven't checked! Very interesting, thank you!

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Wednesday, October 07, 2009 4:18 PM
To: [hidden email]
Subject: Re: exorcising spirits from Fluoview

We had a very similar problem, but it turned out to be the connection
between the fibre-optic cable and the laser - it had come slightly loose.
Since you've checked the cables, I guess it has to be something else.

good luck,
Rosemary

Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

ph 61 2 6246 5475
fx 61 2 6246 5334


On 7/10/09 10:27 PM, "MODEL, MICHAEL" <[hidden email]> wrote:

> I apologize if this is a second message to the list, I think the first one
> didn't go through
>
> We are having a bizzare scanning problem. Straight vertical lines in an object
> become slightly zigzagged with a period of up to 6-7 scan lines, and there
> also may be some oscillation in the intensity. The period and the magnitude of
> this periodic noise depends on the scan speed and the scan size. So far we
> (with the help of an Olympus engineer) have established that:
>
> 1. It doesn't seem to be the scanner controller or the galvo mechanism
> 2. It does not seem to be the electric power in the building
> 3. It is not a mechanical vibration
> 4. It is not a computer
> 5. It is not the cables
> 6. It is not a 60 Hz noise
>
> Sometimes connecting the scanner controller to the outlet through a long
> extension cord seemed to help which may suggest a problem with grounding, but
> as soon as we concluded that, the trick stopped working. The trouble could not
> be reproduced at the Olympus testing lab.
>
> Has anyone experienced anything similar and successfully resolved the problem?
>
> Many thanks in advance!
>
> Mike Model

[Mario Faretta]

Dear List,
We are testing Chromomycin A3 as nuclear dye in alternative to DAPI in
mouse embryo fibroblasts. We noticed that the obtained staining pattern  
looks quite different from the usual one we get.
Mouse cells usually have bright intense spots corresponding to
heterochromatin foci when stained with DAPI,  but I have the impression
that the number of spots identified by ChromoA3 is less in number and
relative intensity. Do you know if according to the dye specificity we
have to expect this result (due to the relative distribution of AT or GC
bases)? (Cells were fixed (PAF) and permeabilized to reduce
accessibiltiy limitations)
Any suggestion/experience about nuclear dyes excited in the violet blue
range but with greater affinity for heterochromatin (Olivomycin,
Quinacrine Mustard, Syto Molecular Probes Dyes..) and feasible for DNA
quantitation (like cell cycle evaluation)?
Thanks
Mario

--
Mario Faretta
Department of Experimental Oncology
European Institute of Oncology
c/o IFOM-IEO Campus for Oncogenomics
via Adamello 16
20139 Milan
Italy
Phone: ++39-02574303054
email: [hidden email]
http://www.ifom-ieo-campus.it

[Thomas Trusk]

We've had good success using the BABB procedure, described here:

http://www.ncbi.nlm.nih.gov/pubmed/16060974

Thomas C. Trusk, PhD
Josh Spruill Imaging Facility
Regenerative Medicine and Cell Biology
MUSC


-----Original Message-----
From: Michael Weber [mailto:[hidden email]]
Sent: Wednesday, October 07, 2009 11:56 AM
Subject: optical clearing of tissue

Dear list,

I am looking for advice on optical "clearing" of fixed tissue before
staining it and using it for light microscopy. Actually "tissue" is not
the precise term, since I would like to clear whole fly embryos. This
process seems to be well established in histology, i.e. using Xylene. I
also found a commercial product called "Histo-Clear" (National
Diagnostics), which claims to preserve tissue structures rather well,
while being less nasty compared to Xylene. Did you guys ever use something
like that? Any input welcome.

cheers,
Michael

[Stanislav Vitha]

Thorsten,
could you comment on the importance of TDE mountant purity?
Your article (MICROSCOPY RESEARCH AND TECHNIQUE 70:1–9, 2007) used the
Biochemica grade TDE (>99%, Sigma #88559, 250ML for $160.50). I have
been using the Aldrich cat. #166782 (>99%, 500g $27.70) with good success
for conofcal 3D imaging of pollen grains (autofluorescent, no staining needed).
It performed really well for these samples,  but I am uncertain if this lower
grade TDE would work for e.g., immunostained slides.

Also, if anyone is using TDE with an antifade compound, I would appreciate
information what you used, what concentration, and any specific tips on
mixing.  

Thank you!

Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

On Wed, 7 Oct 2009 18:10:56 +0200, Staudt Thorsten <T.Staudt@DKFZ-
HEIDELBERG.DE> wrote:

>hello michael,
>
>This publication might be interesting for you:
>
>
>http://www.ncbi.nlm.nih.gov/pubmed/19397748
>
>
>thorsten
>
>
>-----Ursprüngliche Nachricht-----
>Von: Confocal Microscopy List im Auftrag von Michael Weber
>Gesendet: Mi 07.10.2009 17:56
>An: [hidden email]
>Betreff: optical clearing of tissue
>
>Dear list,
>
>I am looking for advice on optical "clearing" of fixed tissue before
>staining it and using it for light microscopy. Actually "tissue" is not
>the precise term, since I would like to clear whole fly embryos. This
>process seems to be well established in histology, i.e. using Xylene. I
>also found a commercial product called "Histo-Clear" (National
>Diagnostics), which claims to preserve tissue structures rather well,
>while being less nasty compared to Xylene. Did you guys ever use something
>like that? Any input welcome.
>
>cheers,
>Michael

[Stanislav Vitha]

We had a somewhat similar problem with our FV1000 a while ago, where
images would show a striped pattern, with orientation and period depending on
scan size and speed. It turns out that the fiber bringing the laser light from
the combiner/AOTF to the scan box was inserted too deep in the mount (or
something to that effect) and was touching or rubbing against the housing (I
do not remember exactly, it was few years ago). This caused vibrations and
consequently stripes in the images.

Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257  
On Wed, 7 Oct 2009 07:27:47 -0400, MODEL, MICHAEL <[hidden email]>
wrote:

>I apologize if this is a second message to the list, I think the first one didn't
go through
>
>We are having a bizzare scanning problem. Straight vertical lines in an object
become slightly zigzagged with a period of up to 6-7 scan lines, and there also
may be some oscillation in the intensity. The period and the magnitude of this
periodic noise depends on the scan speed and the scan size. So far we (with
the help of an Olympus engineer) have established that:
>
>1. It doesn't seem to be the scanner controller or the galvo mechanism
>2. It does not seem to be the electric power in the building
>3. It is not a mechanical vibration
>4. It is not a computer
>5. It is not the cables
>6. It is not a 60 Hz noise
>
>Sometimes connecting the scanner controller to the outlet through a long
extension cord seemed to help which may suggest a problem with grounding,
but as soon as we concluded that, the trick stopped working. The trouble
could not be reproduced at the Olympus testing lab.
>
>Has anyone experienced anything similar and successfully resolved the
problem?
>
>Many thanks in advance!
>
>Mike Model

[SUBSCRIBE CONFOCALMICROSCOPY Anonymous]

hey stanislav,

it is not mandatory to use tde with the highest purity grade. Sometimes, the lower grade tde is a bit more acidic, but that does not matter, since one has to adjust the ph anyway (if you are lucky, you get a batch with ideal ph). The lower grade TDE is sometimes yellowish coloured, so it might contain uv or blue light absorbing impurities, fluorescing green. But in general, the concentration of absorbing or fluorescent entities in tde is extremly low. Because of that, tde is sometimes used as an immersion oil. There is also no evidence of enhanced photobleaching using lower grade tde. So if it works fine, i would not change the tde to higher grade tde. One reason to use the highest grade tde is that you might have more reliable conditions.
As antifades, you can add a small amount of DABCO (1mM). The best way to do that is to solve dabco in the water you use to adjust the refractive index of tde to 1.517.
By the way, there is a new protocol for adjustin the ph:
 use liquid pH indicators like Phenol red or bromthymolblue.
(phenolred changes from yellow to redviolet at pH 7.3; bromthymolblue changes from green to blue at 7.5)
Make a reference set of different pH values (6.5 to 8 in 0.2 steps)(water, ph adjusted
with naoh or hcl, measured with a ph electrode) and the indicators and compare the colors
with 10ml tde plus indicator. Then, add small amounts of base or acid until you have reached
 the color (by comparing with the reference set) corresponding to the desired pH. Because you are examining a large volume
(and not only a glas surface), the method is fast and reliable.


if you have any problem with the tde, don´t hesitate to contact me.

best wishes,

thorsten



Thorsten,
could you comment on the importance of TDE mountant purity?
Your article (MICROSCOPY RESEARCH AND TECHNIQUE 70:1-9, 2007) used the
Biochemica grade TDE (>99%, Sigma #88559, 250ML for $160.50). I have
been using the Aldrich cat. #166782 (>99%, 500g $27.70) with good success
for conofcal 3D imaging of pollen grains (autofluorescent, no staining needed).
It performed really well for these samples,  but I am uncertain if this lower
grade TDE would work for e.g., immunostained slides.

Also, if anyone is using TDE with an antifade compound, I would appreciate
information what you used, what concentration, and any specific tips on
mixing.  

Thank you!

Stanislav Vitha
Microscopy and Imaging Center
Texas A&M University
BSBW 119
College Station, TX 77843-2257

On Wed, 7 Oct 2009 18:10:56 +0200, Staudt Thorsten <T.Staudt@DKFZ-
HEIDELBERG.DE> wrote:

>hello michael,
>
>This publication might be interesting for you:
>
>
>http://www.ncbi.nlm.nih.gov/pubmed/19397748
>
>
>thorsten
>
>
>-----Ursprüngliche Nachricht-----
>Von: Confocal Microscopy List im Auftrag von Michael Weber
>Gesendet: Mi 07.10.2009 17:56
>An: [hidden email]
>Betreff: optical clearing of tissue
>
>Dear list,
>
>I am looking for advice on optical "clearing" of fixed tissue before
>staining it and using it for light microscopy. Actually "tissue" is not
>the precise term, since I would like to clear whole fly embryos. This
>process seems to be well established in histology, i.e. using Xylene. I
>also found a commercial product called "Histo-Clear" (National
>Diagnostics), which claims to preserve tissue structures rather well,
>while being less nasty compared to Xylene. Did you guys ever use something
>like that? Any input welcome.
>
>cheers,
>Michael

[Steffen Dietzel]

Mario,

while your question is 10 days old by now, I missed it so far and it
seems to be still unanswered.

Your suspicion is correct: DAPI (and the Hoechst dyes) are AT
specific while Chromomycin A3 is GC specific. (Shafer RH et al, Eur.
J Biochem ,173:377, 1988). Mouse chromosomes have enormous AT-rich
pericentromeric and paracentromeric repetitive regions (minor and
major satellite) which cluster in interphase nuclei to chromocenters,
the "bright intense spots" you observed. The degree of clustering is
actually dependent on the cell type. If you want to get the oposite
pattern, you can try Equine cells.

I never did it myself, but I seem to remember that DAPI has been used
for flowcytometer cell cycle studies.

Steffen


At 13:24 08.10.2009, you wrote:

>Dear List,
>We are testing Chromomycin A3 as nuclear dye in alternative to DAPI
>in mouse embryo fibroblasts. We noticed that the obtained staining pattern
>looks quite different from the usual one we get.
>Mouse cells usually have bright intense spots corresponding to
>heterochromatin foci when stained with DAPI,  but I have the
>impression that the number of spots identified by ChromoA3 is less
>in number and relative intensity. Do you know if according to the
>dye specificity we have to expect this result (due to the relative
>distribution of AT or GC bases)? (Cells were fixed (PAF) and
>permeabilized to reduce accessibiltiy limitations)
>Any suggestion/experience about nuclear dyes excited in the violet
>blue range but with greater affinity for heterochromatin
>(Olivomycin, Quinacrine Mustard, Syto Molecular Probes Dyes..) and
>feasible for DNA quantitation (like cell cycle evaluation)?
>Thanks
>Mario
>
>--
>Mario Faretta
>Department of Experimental Oncology
>European Institute of Oncology
>c/o IFOM-IEO Campus for Oncogenomics
>via Adamello 16
>20139 Milan
>Italy
>Phone: ++39-02574303054
>email: [hidden email]
>http://www.ifom-ieo-campus.it