[Cytometry] Red laser "cleanup" filters

Howard Shapiro hms at shapirolab.com
Mon Mar 21 22:04:44 EDT 2011


Bob Hoffman wrote:

> A detail note on clean up filters for diode lasers. The filter is not actually needed to suppress the the main laser line, but suppresses light (known as amplified SPONTANEOUS emission, ASE) that is emitted along with the laser (amplified STIMULATED emission) light. The emission spectrum from red diode lasers includes ASE as "tails" on the low and high wavelength sides of the laser wavelength. ASE for red diode lasers I measured was less than 1% of the main laser line, but was significant enough that scattered ASE light caused a large increase in the signal from unstained cells in the APC channel when a 660/20 APC filter was used with a 635 nm laser diode. Although the 660/20 filter completely blocked 635 nm light, it passed enough of the ASE long wavelength tail that a fraction of ASE (not 635 nm light) was getting through the 660/20 filter. Adding a band pass that cut off ASE before the APC filter cut on eliminated the ASE scatter and made the autofluorescence in the APC channel essentially zero.
> 
> Basically the laser filter and APC filter have to be a matched set- just as the excitation and emission filters for a fluorescence microscope. But since ASE is much lower intensity than the laser emission, the filter on the laser is less critical than the excitation filter on a microscope that has white light excitation.
> 
> ASE filters are critical for Raman scatter measurements. See for an example www.ondax.com/products/noiseblock™-ase-suppression-filter. For an example of an ASE spectrum, click on the link for the NoiseBlock data sheet.
> 
> The filter is installed on the laser at an angle to prevent light reflected by the filter going directly back into the laser diode and causing instability.
> 
> Other comments are correct, that if you are only using the laser for stream viewing, you don't need a filter on the laser.
> 
> Bob Hoffman
> P.S. My first message to the list since retiring from BD over a year ago.
> 

Welcome back to the list, Bob!

Another issue with diode lasers, not mentioned above, has to do with the variability of the output wavelength. A red He-Ne laser emits at 632.8 nm, period.
The actual wavelength of the laser (stimulated) emission from a red "635 nm" diode may vary from the low 630s to (more likely) the low 640s. A 660/20 filter used on a detector will pass significantly more light at 642 nm than at 635 nm, meaning that background due to scattered laser light getting through the detector filter will be higher if the laser wavelength is 642 nm than if it is 635 nm. This can't be fixed with a cleanup filter, but may be reduced by substituting a detector filter with a longer center wavelength for the 660/20, provided the material to be detected does not emit significantly less at the longer wavelength. There are also wavelength variability issues with violet diode lasers; one that emits above 410 nm is significantly less effective for excitation of Hoechst 33258 or 33342 than is one that emits at or below 405 nm. You should be able to get the precise wavelength of any of the lasers in your instrument from the manufacturer or whoever else installed the laser; if not, borrow an Ocean Optics or similar spectrophotometer.

-Howard




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