Re: Finding the 458nm laser line of a Coherent innova Spectrum Laser70 on FACSVantageSE

Alex,
What a coincidence ... I just tuned my I90-5 to 458nm yesterday. My 1984
Coherent manual just happens to list the krypton lines (for I90-K) along
with the argon lines (5W laser shown below):

488.0nm: 1500mW  Ar
482.5  :   30	 Kr (3)
476.5  :  600	 Ar
476.2  :   50	 Kr (3)
472.7  :  200	 Ar
465.8  :  150	 Ar
457.9  :  350	 Ar
454.5  :  120	 Ar (3)
406.7-415.4: 150 Kr (3,4)
(3) "Special optics and testing required."
(4) "Powers with high magnetic field."

Try as I might, I cannot lase at 454.5nm with my 457.9-514.5 standard optics
(nor UV, for that matter). Assuming (a) these are the lines you can get, (b)
you're able to tune 476.5 and 476.2 separately, and (c) you're not hitting
the violet lines (406.7-415.4), then yes, I would think your 6th visible
line below 488 would be 458.

If you have a narrow 458/1 bandpass and a light meter, you should be able to
tell for sure.

Somewhat by accident I discovered that BD's DNA QC Kit has a nice particle
for aligning 458 as the second laser. The PI-labeled CEN (or CTN, but they
have higher %CV) excite well there (and UV, too). Obviously though, it is not
specific for 458.

Good luck,
Eric


>Hi FlowCytonauts,
>
>I ran into trouble finding the 458nm laser line of our Innova Spectrum70 
>Laser on our FACSVantage SE for ECFP analysis.
>Actually I can tune to 6 lines below the very strong 488nm line. (UV is 
>probably not visible)
>
>The Innova handbook says:
>70C-Spectrum:
>488.0: 0.25Watt;
>476.5: 0.10
>472.2: ----
>465.8: ----
>457.9: 0.03
>Multiline UV: 0.05
>So which one is the 458 ?. The first visible one ?
>
>Unfortunately I currently have no strong ECFP expressing cell line for 
>fishing in the dark.
>Are there any beads that are somewhat specific for that laserline ?
>I tried Sphero-Yellow bright with not much success...
>I am running the laser as  secondary laser (488 primary, outsplitted UV 
>as laser3).
>I used a 649LP filter and collected the reflected light with a special 
>473/19 filter (as discussed previously in that group).
>
>Any help is greatly appreciated ...
>
>/Alex

Eric Van Buren <eric.vanburen@wayne.edu>
Manager, Flow Cytometry Core Facility
Karmanos Cancer Institute
Detroit, Michigan, USA