CFP detection under 458 excitation
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Recently we upgraded our Olympus
FV-300 confocal system and purchased filters for CFP/YFP detection for FRET
applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line (multi-line
argon laser) and a heavy interference with direct reflection from the glass.
Chroma claims that it only passes 0.3% of 458nm, and theoretically it is
supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of Medicine,
room 511 e-mail: |
 
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Aryeh Weiss |
Re: CFP detection under 458 excitation
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0.3% may not be a small signal compared to your fluorescence. I would
suggest that you use a filter with a cutoff no higher than 470, and also that you use the HQ ot ET series filters (which have very sharp skirts). --aryeh Inna Slutsky wrote: > > Recently we upgraded our Olympus FV-300 confocal system and purchased > filters for CFP/YFP detection for FRET applications. > > > > The problem is that the 465-495 nm band pass filter (Chroma) showed > direct excitation of the 458 line (multi-line argon laser) and a heavy > interference with direct reflection from the glass. Chroma claims that > it only passes 0.3% of 458nm, and theoretically it is supposed to be > OK for 458 nm excitation. > > > > I wonder if someone has encountered a similar problem and may offer a > better solution for detection of CFP signal (together with YFP under > 514 nm excitation). > > > > Thanks, > > Inna. > > > > > > -------------------------------------------------------- > > Inna Slutsky, PhD > Dept. of Physiology and Pharmacology > > Sackler Faculty of Medicine, room 511 > Tel Aviv University > Ramat Aviv, Tel Aviv 69978, Israel > phone: +972 3 640 6021 > > e-mail: [hidden email] > > > > > > > -- Aryeh Weiss School of Engineering Bar Ilan University Ramat Gan 52900 Israel Ph: 972-3-5317638 FAX: 972-3-7384050 Israel Society for Microscopy 2009 meeting website: http://materials.technion.ac.il/ism/ISM2009.html |
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Monique Vasseur |
Re: CFP detection under 458 excitation
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In reply to this post by islutsky
Which dichroic are you using? Monique Vasseur tél. (514) 343-6111 poste 5148 De : Confocal
Microscopy List [mailto:[hidden email]] De la part de
Inna Slutsky Recently we upgraded our
Olympus FV-300 confocal system and purchased filters for CFP/YFP detection for
FRET applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line
(multi-line argon laser) and a heavy interference with direct reflection from
the glass. Chroma claims that it only passes 0.3% of 458nm, and theoretically
it is supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of Medicine, room 511 e-mail: |
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458 / 514. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of Medicine,
room 511 e-mail: From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Vasseur Monique Which dichroic are
you using? Monique
Vasseur tél. (514) 343-6111
poste 5148 De :
Confocal Microscopy List [mailto:[hidden email]] De la part de Inna Slutsky Recently we upgraded our
Olympus FV-300 confocal system and purchased filters for CFP/YFP detection for
FRET applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line
(multi-line argon laser) and a heavy interference with direct reflection from
the glass. Chroma claims that it only passes 0.3% of 458nm, and theoretically
it is supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of
Medicine, room 511 e-mail: |
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Monique Vasseur |
Re: CFP detection under 458 excitation
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I meant the emission dichroic, the one to split emission between
the PMTs. Monique Vasseur tél. (514) 343-6111 poste 5148 De : Confocal
Microscopy List [mailto:[hidden email]] De la part de
Inna Slutsky 458 / 514. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of Medicine, room 511 e-mail: From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Vasseur Monique Which dichroic are you using? Monique Vasseur tél. (514) 343-6111 poste 5148 De : Confocal
Microscopy List [mailto:[hidden email]] De la part de
Inna Slutsky Recently we upgraded our
Olympus FV-300 confocal system and purchased filters for CFP/YFP detection for
FRET applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line
(multi-line argon laser) and a heavy interference with direct reflection from
the glass. Chroma claims that it only passes 0.3% of 458nm, and theoretically
it is supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky, PhD Sackler Faculty of Medicine, room 511 e-mail: |
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In reply to this post by islutsky
I'm not sure I understand the question. What do you
mean by "direct excitation of the 58nm line?" This is presumably a barrier
filter, for CFP, which you'd expect to excite with the 458nm line. Do
you mean that you are getting a reflection image
back?
Points to consider:
1. Is your dichroic correct?
2. Is your filter installed at its designed
angle?
3. Is your blocking system (AOTF?) for the 488nm laser line
functioning properly? It would only take a tiny leakage there to really
screw things up.
Guy
Optical Imaging Techniques in Cell Biology From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Inna Slutsky Sent: Tuesday, 26 May 2009 3:57 PM To: [hidden email] Subject: CFP detection under 458 excitation Recently we upgraded our
Olympus FV-300 confocal system and purchased filters for CFP/YFP detection for
FRET applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line
(multi-line argon laser) and a heavy interference with direct reflection from
the glass. Chroma claims that it only passes 0.3% of 458nm, and theoretically it
is supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm
excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky,
PhD Sackler Faculty of
Medicine, room 511 e-mail:
Internal Virus Database is out-of-date. Internal Virus Database is out-of-date. |
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Prashant Prabhat |
Re: CFP detection under 458 excitation -"Commercial Response"
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In reply to this post by islutsky
***************"Commercial Response" ******************
Dear Inna,
Ideally it is desired to provide very high degree of blocking (several
orders of magnitude of blocking) of the unwanted laser lines to avoid
interference patterns in the emission channel. Both the excitation filter and
the emission filter contribute to this blocking of unwanted laser lines. For
extremely demanding applications such as imaging of single molecules, it may be
even desirable to provide additional blocking by using a suitable notch or a
long pass filter in the emission channel (http://www.semrock.com/Data/Documents/PerfectingTIRF_0109_Reprint.pdf).
This is generally not true for a dichroic mirror, as they are not specifically
designed for blocking.
For
your application, it may be worthwhile considering from following Semrock filter
for imaging CFP & YFP.
1)
CFP-2423A set (http://www.semrock.com/Catalog/SetDetails.aspx?SetBasePartID=21&CategoryID=16).
Note that the emission filter of this filter set, FF01-483/32-25 is
designed to provide >OD 6 blocking at 458 nm.
2)
YFP-2427A set (http://www.semrock.com/Catalog/SetDetails.aspx?SetBasePartID=25&CategoryID=16).
Note that the emission filter of this filter set, FF01-542/27-25 is
designed to provide >OD 6 blocking at 514 nm.
3)
Alternatively, a steep edge long pass filter in the emission channel, such as
Semrock's LP02-458RS-25 (http://www.semrock.com/Catalog/Detail.aspx?FilterPartID=150&CategoryID=69),
which is designed to provide > OD 6 blocking at 458 nm when used in
conjunction with your existing filters should be able to eliminate interference
patterns in this channel.
Please
contact me offline at [hidden email], if you have
questions.
Regards,
Prashant
Prashant Prabhat, Ph.D. Applications Specialist Semrock A Unit of IDEX Corporation 3625 Buffalo Road, Suite 6 Rochester NY 14624 Email: [hidden email] Phone: 585-594-7064 Toll Free: 866-SEMROCK Fax: 585-594-7095 http://www.semrock.com
The Standard in Optical Filters for Biotech & Analytical Instrumentation Hundreds of Thousands of Ion Beam Sputtered filters delivered - extensive inventory now! The information contained in this message and any attachments may be privileged, confidential, and protected from disclosure. If the reader of this message is not the intended recipient, or any agent responsible for delivering this message to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication may be unlawful and therefore strictly prohibited. If you received this message in error, please reply to the message and delete it.
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Inna Slutsky Sent: Tuesday, May 26, 2009 1:57 AM To: [hidden email] Subject: CFP detection under 458 excitation Recently we upgraded our
Olympus FV-300 confocal system and purchased filters for CFP/YFP detection for
FRET applications. The problem is that the
465-495 nm band pass filter (Chroma) showed direct excitation of the 458 line
(multi-line argon laser) and a heavy interference with direct reflection from
the glass. Chroma claims that it only passes 0.3% of 458nm, and theoretically it
is supposed to be OK for 458 nm excitation. I wonder if someone has
encountered a similar problem and may offer a better solution for detection of
CFP signal (together with YFP under 514 nm
excitation). Thanks, Inna. -------------------------------------------------------- Inna Slutsky,
PhD Sackler Faculty of
Medicine, room 511 e-mail:
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