FLIM Decay curve reflection

[Alison Dun]

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We have a Leica SP5 integrated with a pico quant TCSPC system. We have been seeing some kind of reflection in our curve, usually around the same place and with varying degrees depending on the lens used (water or oil) and also by the excitation wavelength (significantly smaller if you move away from 488 with WLL). We see this with 40 MHz and 80 MHz.

I wondered if anyone has seen this before in their own system? We have been told it is normal but I worry about whether it will affect the decay curve fitting. It can be very small (say 1% of the peak, but we have seen it at the same height as the peak!).
We have a gated-STED and WLL integrated into the system also.

Any advice would be very welcome! If you do have experience of this and could give me some advice please email me directly,
 
Thanks
Ali

Dr. Alison Dun
Technology and Facility Manager
Edinburgh Super Resolution Imaging Consortium (ESRIC)
Heriot-Watt University
Edinburgh
EH14 4AS

Office: G.40 David Brewster Building
Lab: 3.17 William Perkin Building
Lab Phone: [phone]
Mobile: [phone]
@AliDun_esric

[Vladimir Ghukasyan-2]

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Hi Alison,

You can tell the approximate location of the reflection signal by
calculating it from the length of the cable. We had a similar problem
before and got rid of it by increasing the TAC threshold (this was a
Becker&Hickl system, not sure if PicoQuant allows to do that).

Kind regards,
Vladimir



On Thu, May 22, 2014 at 11:13 AM, Alison Dun <[hidden email]> wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> We have a Leica SP5 integrated with a pico quant TCSPC system. We have
> been seeing some kind of reflection in our curve, usually around the same
> place and with varying degrees depending on the lens used (water or oil)
> and also by the excitation wavelength (significantly smaller if you move
> away from 488 with WLL). We see this with 40 MHz and 80 MHz.
>
> I wondered if anyone has seen this before in their own system? We have
> been told it is normal but I worry about whether it will affect the decay
> curve fitting. It can be very small (say 1% of the peak, but we have seen
> it at the same height as the peak!).
> We have a gated-STED and WLL integrated into the system also.
>
> Any advice would be very welcome! If you do have experience of this and
> could give me some advice please email me directly,
>
> Thanks
> Ali
>
> Dr. Alison Dun
> Technology and Facility Manager
> Edinburgh Super Resolution Imaging Consortium (ESRIC)
> Heriot-Watt University
> Edinburgh
> EH14 4AS
>
> Office: G.40 David Brewster Building
> Lab: 3.17 William Perkin Building
> Lab Phone: [phone]
> Mobile: [phone]
> @AliDun_esric
>

[Unruh, Jay]

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I would try to eliminate it if possible.  If your instrument response function (IRF) doesn't contain the exact same amplitude peak, it can skew your results significantly.  Of course, most FLIM applications are measuring pixel-to-pixel variation and this would likely give a consistent bias towards longer lifetime.  Given the wavelength and spatial distribution of the emission vs. excitation, it would not be surprising if the IRF contains a different amount of scatter.  What is the delay time from the main peak?  You can usually figure out where it is based on that (1 ns = 30 cm).  On many systems, the culprit is the condenser.  Is it dependent on the size of the pinhole?  If not, it is probably in the excitation path--perhaps in the WLL itself.  The fact that it differs with objective argues against this though.  A reflection in the WLL would always be the same amplitude.  Perhaps you can tilt the condenser backwards or put a black felt cloth in front of it?

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alison Dun
Sent: Thursday, May 22, 2014 10:13 AM
To: [hidden email]
Subject: FLIM Decay curve reflection

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We have a Leica SP5 integrated with a pico quant TCSPC system. We have been seeing some kind of reflection in our curve, usually around the same place and with varying degrees depending on the lens used (water or oil) and also by the excitation wavelength (significantly smaller if you move away from 488 with WLL). We see this with 40 MHz and 80 MHz.

I wondered if anyone has seen this before in their own system? We have been told it is normal but I worry about whether it will affect the decay curve fitting. It can be very small (say 1% of the peak, but we have seen it at the same height as the peak!).
We have a gated-STED and WLL integrated into the system also.

Any advice would be very welcome! If you do have experience of this and could give me some advice please email me directly,
 
Thanks
Ali

Dr. Alison Dun
Technology and Facility Manager
Edinburgh Super Resolution Imaging Consortium (ESRIC) Heriot-Watt University
Edinburgh
EH14 4AS

Office: G.40 David Brewster Building
Lab: 3.17 William Perkin Building
Lab Phone: [phone]
Mobile: [phone]
@AliDun_esric

[Pamela Young]

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Good call Jay!  When I was at LOCI, we used to always move the condenser
out of the way (by tipping the top half of the microscope back to avoid
reflections.  I¹m looping Brian Burkel in on this conversation, as I know
he has spent a lot of time thinking about this.

Dr Pamela A. Young
 | Light and Optical Microscopist
Australian Centre for Microscopy & Microanalysis

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On 23/05/2014 1:25 am, "Unruh, Jay" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>I would try to eliminate it if possible.  If your instrument response
>function (IRF) doesn't contain the exact same amplitude peak, it can skew
>your results significantly.  Of course, most FLIM applications are
>measuring pixel-to-pixel variation and this would likely give a
>consistent bias towards longer lifetime.  Given the wavelength and
>spatial distribution of the emission vs. excitation, it would not be
>surprising if the IRF contains a different amount of scatter.  What is
>the delay time from the main peak?  You can usually figure out where it
>is based on that (1 ns = 30 cm).  On many systems, the culprit is the
>condenser.  Is it dependent on the size of the pinhole?  If not, it is
>probably in the excitation path--perhaps in the WLL itself.  The fact
>that it differs with objective argues against this though.  A reflection
>in the WLL would always be the same amplitude.  Perhaps you can tilt the
>condenser backwards or put a black felt cloth in front of it?
>
>Jay
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Alison Dun
>Sent: Thursday, May 22, 2014 10:13 AM
>To: [hidden email]
>Subject: FLIM Decay curve reflection
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>We have a Leica SP5 integrated with a pico quant TCSPC system. We have
>been seeing some kind of reflection in our curve, usually around the same
>place and with varying degrees depending on the lens used (water or oil)
>and also by the excitation wavelength (significantly smaller if you move
>away from 488 with WLL). We see this with 40 MHz and 80 MHz.
>
>I wondered if anyone has seen this before in their own system? We have
>been told it is normal but I worry about whether it will affect the decay
>curve fitting. It can be very small (say 1% of the peak, but we have seen
>it at the same height as the peak!).
>We have a gated-STED and WLL integrated into the system also.
>
>Any advice would be very welcome! If you do have experience of this and
>could give me some advice please email me directly,
>
>Thanks
>Ali
>
>Dr. Alison Dun
>Technology and Facility Manager
>Edinburgh Super Resolution Imaging Consortium (ESRIC) Heriot-Watt
>University
>Edinburgh
>EH14 4AS
>
>Office: G.40 David Brewster Building
>Lab: 3.17 William Perkin Building
>Lab Phone: [phone]
>Mobile: [phone]
>@AliDun_esric