Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Better resolution at shorter shorter wavelengths, but when working with thicker tissue slices, results are better at longer wavelengths because of dispersion.
Cheers,
Jeff
________________________________
From: Marc Thibault [mailto:[log in to unmask]]
Sent: Tue 2/5/2008 3:59 PM
To: [log in to unmask]
Subject: Re: Polarized light in a Zeiss LSM510 Meta
---------------------- Information from the mail header -----------------------
Sender: Confocal Microscopy List <[log in to unmask]>
Poster: Marc Thibault <[log in to unmask]>
Subject: Re: Polarized light in a Zeiss LSM510 Meta
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
HI, I have nothing to add but maybe another question: Is there a =
preffered
laser line for transmitted light DIC images of cells (with no concerns =
with
photoxicity) ?
I also have weird intensity variations with channel D (transmitted) on =
the
LSM.
=20
Thanks
=20
Marc
=20
------------------------------------------------
Marc Thibault, Ph.D.
Postdoctoral Fellow
NSERC/Biosynthech Chair in Hybrid Biomaterials
for Innovative Regenerative Technologies
Department of Chemical Engineering
Ecole Polytechnique
Montreal, Qc, Canada
514 340 4711 (4781)
------------------------------------------------
=20
_____ =20
De : Carol Norris [mailto:[log in to unmask]]=20
Envoy=E9 : 5 f=E9vrier 2008 10:18
=C0 : [log in to unmask]
Objet : Re: Polarized light in a Zeiss LSM510 Meta
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal We had a =
similar
problem on our Leica SP2, except that the cycle occurred over a couple =
of
hours. With the 488 line, background intensity in the DIC channel =
varied
from 10 to about 250 and back down; there were similar changes with the =
458
line off the same laser. Transmitted light images using the 543 laser =
over
the same time period showed a steady decrease from 200 down to 20. All =
this
happened while fluorescent images showed essentially no variation. Our
tech person fixed it by adjusting the polarization of the lasers. =20
Carol Norris
On Feb 5, 2008, at 3:25 AM, Carlos Sanchez wrote:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal=20
Hello everybody.
=20
We would like to know your expert opinion about a problem =
with
polarized transmitted light which we have been observing in our Zeiss =
LSM510
Meta confocal system since Summer-2007.
=20
Please, download examples from:
http://bacterio.cbm.uam.es/confocal/Temp/Polarized_light_CBM_Madrid_2008.=
zip
=20
The Zeiss database include three =93*.lsm=94 files with some timelapse =
images
taken with the transmission detector and using the 488nm laser line in
channel 1, the 543nm one in channel two and 633nm in the third one:
1- METAwithpolarizer.lsm- taken in the LSM510 Meta system with just the
polarizer (never the analyzer since lasers are already polarized) =
inserted
in the condenser,
2- withoutpolarizer.lsm- exactly the same as before but without
inserting the polarizer,
3- Withpolarizerinnewconfocal0.lsm- the same, inserting the =
polarizer,
but in another newer Zeiss LSM510 confocal system.
=20
As you will see the signal is rather stable in samples 2 and =
3
with all laser lines. However, when the polarizer is inserted in the
condenser of the Zeiss LSM510 Meta system we get fluctuations with slow
cycles of around two minutes. Zeiss technicians changed the optical =
fiber
and aligned lasers with the fiber several times. In fact they were able =
to
solve some additional strange line pattern got with the 543nm laser line =
but
slow light fluctuations still persist.
We would be very pleased if you could send us your ideas, impressions,
similar experiences.
Thank you very much in advance for your help and time.
Regards,
=20
Carlos S=E1nchez Mart=EDn
Servicio de Microscop=EDa Optica y Confocal (Lab.310)
Centro de Biolog=EDa Molecular "Severo Ochoa" (CSIC-UAM)
C/Nicol=E1s Cabrera,1
Universidad Aut=F3noma de Madrid. Cantoblanco.
28049. Madrid
Tlfs. 91 196 4613/4643
Fax. 91 196 4420
E-mail: [log in to unmask]
Web: http://www.cbm.uam.es/confocal
=20
Carol E. Norris, Ph.D
Facility Scientist
Flow Cytometry/Confocal Microscopy Facility
Biotechnology/Bioservices Center
University of Connecticut Unit 3149
91 N. Eagleville Rd
Storrs, CT 06269-3149
Phone (860) 486-3080
Fax (860) 486-5005
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<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
<HTML><HEAD>
<META http-equiv=3DContent-Type content=3D"text/html; =
charset=3Diso-8859-1">
<META content=3D"MSHTML 6.00.6000.16544" name=3DGENERATOR></HEAD>
<BODY=20
style=3D"WORD-WRAP: break-word; -webkit-nbsp-mode: space; =
-webkit-line-break: after-white-space">
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial=20
size=3D2>HI, I have nothing to add but maybe another question: Is there =
a=20
preffered laser line for transmitted light DIC images of cells (with no =
concerns=20
with photoxicity) ?</FONT></SPAN></DIV>
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial size=3D2>I=20
also have weird intensity variations with channel D (transmitted) on the =
LSM.</FONT></SPAN></DIV>
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial=20
size=3D2></FONT></SPAN> </DIV>
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial=20
size=3D2>Thanks</FONT></SPAN></DIV>
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial=20
size=3D2></FONT></SPAN> </DIV>
<DIV dir=3Dltr align=3Dleft><SPAN class=3D451365420-05022008><FONT =
face=3DArial=20
size=3D2>Marc</FONT></SPAN></DIV>
<DIV> </DIV><!-- Converted from text/plain format -->
<P><FONT =
size=3D2>------------------------------------------------<BR>Marc=20
Thibault, Ph.D.<BR><BR>Postdoctoral Fellow<BR>NSERC/Biosynthech Chair in =
Hybrid=20
Biomaterials<BR>for Innovative Regenerative Technologies<BR>Department =
of=20
Chemical Engineering<BR>Ecole Polytechnique<BR>Montreal, Qc, =
Canada<BR>514 340=20
4711=20
(4781)<BR>------------------------------------------------<BR><BR><BR></F=
ONT></P>
<DIV> </DIV><BR>
<DIV class=3DOutlookMessageHeader lang=3Dfr dir=3Dltr align=3Dleft>
<HR tabIndex=3D-1>
<FONT face=3DTahoma size=3D2><B>De :</B> Carol Norris=20
[mailto:[log in to unmask]] <BR><B>Envoy=E9 :</B> 5 f=E9vrier =
2008=20
10:18<BR><B>=C0 :</B> =
[log in to unmask]<BR><B>Objet :</B>=20
Re: Polarized light in a Zeiss LSM510 Meta<BR></FONT><BR></DIV>
<DIV></DIV>Search the CONFOCAL archive at=20
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal We had a =
similar problem=20
on our Leica SP2, except that the cycle occurred over a couple of hours. =
With the 488 line, background intensity in the DIC channel varied =
from 10=20
to about 250 and back down; there were similar changes with the 458 line =
off the=20
same laser. Transmitted light images using the 543 laser over the same =
time=20
period showed a steady decrease from 200 down to 20. All this =
happened=20
while fluorescent images showed essentially no variation. Our =
tech person=20
fixed it by adjusting the polarization of the lasers.
<DIV><BR class=3Dwebkit-block-placeholder></DIV>
<DIV>Carol Norris<BR>
<DIV><BR class=3Dwebkit-block-placeholder></DIV>
<DIV><BR>
<DIV>
<DIV>On Feb 5, 2008, at 3:25 AM, Carlos Sanchez wrote:</DIV><BR=20
class=3DApple-interchange-newline>
<BLOCKQUOTE type=3D"cite"><SPAN class=3DApple-style-span=20
style=3D"WORD-SPACING: 0px; FONT: 12px Helvetica; TEXT-TRANSFORM: =
none; COLOR: rgb(0,0,0); TEXT-INDENT: 0px; WHITE-SPACE: normal; =
LETTER-SPACING: normal; BORDER-COLLAPSE: separate; orphans: 2; widows: =
2; -webkit-border-horizontal-spacing: 0px; =
-webkit-border-vertical-spacing: 0px; =
-webkit-text-decorations-in-effect: none; -webkit-text-size-adjust: =
auto; -webkit-text-stroke-width: 0">Search=20
the CONFOCAL archive at <A=20
=
href=3D"http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal">http:/=
/listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal</A>
<DIV class=3DSection1>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">Hello=20
everybody.</SPAN></FONT></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 12pt"></SPAN></FONT> </P>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: =
12pt"> =
We would like to know your expert opinion about a problem with =
polarized=20
transmitted light which we have been observing in our Zeiss LSM510 =
Meta=20
confocal system since Summer-2007.</SPAN></FONT></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 12pt"></SPAN></FONT> </P>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">Please,=20
download examples from:</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Courier New" size=3D2><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 10pt; FONT-FAMILY: 'Courier New'"><A=20
style=3D"COLOR: blue; TEXT-DECORATION: underline"=20
=
href=3D"http://bacterio.cbm.uam.es/confocal/Temp/Polarized_light_CBM_Madr=
id_2008.zip">http://bacterio.cbm.uam.es/confocal/Temp/Polarized_light_CBM=
_Madrid_2008.zip</A></SPAN></FONT></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 12pt"></SPAN></FONT> </P>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 35.4pt; =
FONT-FAMILY: 'Times New Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">The=20
Zeiss database include three =93*.lsm=94 files with some timelapse =
images taken=20
with the transmission detector and using the 488nm laser line in =
channel 1,=20
the 543nm one in channel two and 633nm in the third =
one:</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">1-=20
METAwithpolarizer.lsm- taken in the LSM510 Meta system with just the =
polarizer=20
(never the analyzer since lasers are already polarized) inserted in =
the=20
condenser,</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; TEXT-INDENT: -18pt; =
FONT-FAMILY: 'Times New Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">2-<FONT=20
face=3D"Times New Roman" size=3D1><SPAN=20
style=3D"FONT: 7pt 'Times New Roman'"> <SPAN=20
=
class=3DApple-converted-space> </SPAN></SPAN></FONT></SPAN></FONT><S=
PAN=20
lang=3DEN-GB>withoutpolarizer.lsm- exactly the same as before but =
without=20
inserting the polarizer,</SPAN></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; TEXT-INDENT: -18pt; =
FONT-FAMILY: 'Times New Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">3-<FONT=20
face=3D"Times New Roman" size=3D1><SPAN=20
style=3D"FONT: 7pt 'Times New Roman'"> <SPAN=20
=
class=3DApple-converted-space> </SPAN></SPAN></FONT></SPAN></FONT><S=
PAN=20
lang=3DEN-GB>Withpolarizerinnewconfocal0.lsm- the same, inserting the =
polarizer,=20
but in another newer Zeiss LSM510 confocal system.</SPAN></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 12pt"></SPAN></FONT> </P>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: =
12pt"> =
As you will see the signal is rather stable in samples 2 and 3 with =
all laser=20
lines. However, when the polarizer is inserted in the condenser of the =
Zeiss=20
LSM510 Meta system we get fluctuations with slow cycles of around two =
minutes.=20
Zeiss technicians changed the optical fiber and aligned lasers with =
the fiber=20
several times. In fact they were able to solve some additional strange =
line=20
pattern got with the 543nm laser line but slow light fluctuations =
still=20
persist.</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; TEXT-INDENT: 35.4pt; =
FONT-FAMILY: 'Times New Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">We=20
would be very pleased if you could send us your ideas, impressions, =
similar=20
experiences.</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB =
style=3D"FONT-SIZE: 12pt">Thank=20
you very much in advance for your help and time.</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN=20
style=3D"FONT-SIZE: 12pt">Regards,</SPAN></FONT></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN=20
style=3D"FONT-SIZE: 12pt"></SPAN></FONT> </P>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">Carlos S=E1nchez=20
Mart=EDn</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">Servicio de=20
Microscop=EDa Optica y Confocal (Lab.310)</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">Centro de Biolog=EDa=20
Molecular "Severo Ochoa" (CSIC-UAM)</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">C/Nicol=E1s=20
Cabrera,1</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">Universidad=20
Aut=F3noma de Madrid. Cantoblanco.</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">28049.=20
Madrid</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">Tlfs. 91 196=20
4613/4643</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: 12pt">Fax. =
91 196=20
4420</SPAN></FONT></DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN style=3D"FONT-SIZE: =
12pt">E-mail:<SPAN=20
class=3DApple-converted-space> </SPAN><A=20
style=3D"COLOR: blue; TEXT-DECORATION: underline"=20
=
href=3D"mailto:[log in to unmask]">[log in to unmask]</A></SPAN></FONT>=
</DIV>
<DIV=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: 12pt">Web:<SPAN =
class=3DApple-converted-space> </SPAN><A=20
style=3D"COLOR: blue; TEXT-DECORATION: underline"=20
=
href=3D"http://www.cbm.uam.es/confocal">http://www.cbm.uam.es/confocal</A=
></SPAN></FONT></DIV>
<P class=3DMsoNormal=20
style=3D"FONT-SIZE: 12pt; MARGIN: 0cm 0cm 0pt; FONT-FAMILY: 'Times New =
Roman'"><FONT=20
face=3D"Times New Roman" size=3D3><SPAN lang=3DEN-GB=20
style=3D"FONT-SIZE: =
12pt"></SPAN></FONT> </P></DIV></SPAN></BLOCKQUOTE></DIV><BR></DIV><=
/DIV><BR><BR>
<DIV>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Carol E. Norris, Ph.D</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Facility Scientist</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Flow Cytometry/Confocal Microscopy Facility</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Biotechnology/Bioservices Center</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>University of Connecticut Unit 3149</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>91 N. Eagleville Rd</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Storrs, CT 06269-3149</FONT></P>
<P style=3D"MIN-HEIGHT: 14px; MARGIN: 0px; FONT: 12px =
Helvetica"><BR></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Phone (860) 486-3080</FONT></P>
<P style=3D"MARGIN: 0px"><FONT style=3D"FONT: 12px Helvetica" =
face=3DHelvetica=20
size=3D3>Fax (860) 486-5005</FONT></P></DIV><BR></BODY></HTML>
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