Re: Intensity of LED excitation for Fura2 or MgFura2

[Debora Keller]

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Hi,

I've been following this LED conversation intently, as we are also trying to ditch the
conventional mercury or Xenon lamp. We also have the issue that our Xenon arc lamp
(Cairn Optosource) gets so hot during overnight experiments that it leads the excitation
filter wheel in front to stall and crash the experiment...

However, some of our users are working with physiology dyes and do ratiometric studies,
that need alternate excitation at low wavelengths such as 340 and 380 nm for fura-2, 340
and 375 for MgFura2... As said, it works well with the Arc lamp except for the fact that it
overheats (and thus crashes the excitation filter wheel just in front).

Does anyone have any experience with LED systems such as spectraX or pE-4000 to
excite so low?

cheers

Debbi
FILM - Facility for Imaging by Light Microscopy
- Facility Manager - Super-Resolution Specialist -

[James Kerin]

***Commercial Response***


Dear Debbi,


Sorry to hear about your heat problems with the Optosource and filter wheel; please email us at [hidden email] and we will help to get this resolved, but we will need to know the make of filter wheel as I suspect it is not ours.  My best guess is that the light source needs to be fitted with a heat filter and perhaps a baffle (we usually use the OptoSource in conjunction with our OptoScan monochromator for Fura 2 measurements - this uses a reflective grating and therefore doesn't need a heat filter).  In our opinion a monochromator is a better device for imaging Fura 2 than a filter wheel as it allows high, low, and isosbestic calcium signals to be wavelength optimised, it has a switching time of <1msec versus > 30msec, and realtime bandwidth selection allows relative optimisation of signal/noise and dynamic range.    The reason why the lamphouse itself does tend to run rather hot is that it's a fully enclosed design.  As well as
avoiding any light leakage, the enclosed design provides a more stable environment around the lamp than ventilation slots would provide, which in turn gives a more stable optical output (minimising convection currents).


We have some experience of using LEDs for Fura 2 measurements.  Isosbestic ratioing works very well with 365 and 385nm LEDs filtered at 360 and 380nm.  These LEDs tend to be temperature sensitive in terms of both intensity and spectral output so some form of closed-loop control is required for most applications.  We incorporate this into our OptoLED system by regulating the light passing through each excitation filter.  We have also had some success in high / low calcium imaging by pushing the short wavelength filter down below 360nm; however the light levels quickly become very dependent on precise peak wavelength of each individual (nominally) 365nm LED.  We would stress that using LEDs for Fura 2 produces superior signal/noise, but the dynamic range is slightly limited by having little or no contribution from the high calcium wavelength. We are currently testing the red-shifted Fura-8 dyes, which we expect to work perfectly with commercially
available LEDs at 365nm and 405nm.  This would still require closed-loop control to avoid temperature-related intensity and spectral shift artefacts. 


Cairn Optoscan Monochromator - http://www.cairn-research.co.uk/catalogue/illumination/optoscan/product/optoscan-monochromator


Cairn Optospin filter wheel - http://www.cairn-research.co.uk/catalogue/detection/optospin/product/optospin-iv


Cairn OptoLED systems - http://www.cairn-research.co.uk/catalogue/illumination/led-light-sources/product/optoled-light-source-0


With our best regards
 J. Graham (on Jim's confocal account)Managing DirectorCairn Research LtdGraveney RoadFavershamKent, ME13 8UPUKwww.cairn-research.co.uk
  filters and mirrors now in stock!


 




On  06/18/2015, 03:45pm, Debora Keller ([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.
 
*****
 

 
Hi,
 

 
I've been following this LED conversation intently, as we are also trying to ditch the
 
conventional mercury or Xenon lamp. We also have the issue that our Xenon arc lamp
 
(Cairn Optosource) gets so hot during overnight experiments that it leads the excitation
 
filter wheel in front to stall and crash the experiment...
 

 
However, some of our users are working with physiology dyes and do ratiometric studies,
 
that need alternate excitation at low wavelengths such as 340 and 380 nm for fura-2, 340
 
and 375 for MgFura2... As said, it works well with the Arc lamp except for the fact that it
 
overheats (and thus crashes the excitation filter wheel just in front).
 

 
Does anyone have any experience with LED systems such as spectraX or pE-4000 to
 
excite so low?
 

 
cheers
 

 
Debbi
 
FILM - Facility for Imaging by Light Microscopy
 
- Facility Manager - Super-Resolution Specialist -