The lines have seemed to hold up quite well. I have not seen any
noticeable difference over four years on either the argon or HeNe laser.
The argon laser is a 25 mW argon Ion laser and the HeHe lasers are
(according to one fo the Zeiss manuals) 0.5 mW for the 543 nm laser and
5 mW for the 633 nm. The power is adjustable on the argon laser but I
almost always have left it running at 4 amps on the current meter. The
HeNe lasers are not adjustable.
While the power differences are substantial, I have not found this to be
a problem in either bleaching (fluorescein specimens) or difference in
signal intensity from red or green emitting fluorochromes.
As to the question of overlap or crossover of signal: if the signal
from each of the fluorochromes is about the same intensity, and one is
not extremely brighter than the other, fluorescein and rhodamine signals
do seem to be clean enough for double label experiments. However, one
should always do the proper controls by doing single labels and checking
the opposite channel (i.e., checking single labeled fluorescein
specimens with the HeNe excitation and rhodamine filters and vice
versa).
Another way around this is to use Cy2 or Texas Red as the red
fluorochrome. These have a higher excitation maximum (i.e., ~590 nm)
and there is no overlap between fluorescein and these fluorochromes.
The downside is that you are not exciting these fluorchromes maximally
with a 543 nm line. In my experience however, these fluorochromes are
excited enough to give a very good signal with the 543 HeNe laser and
work well in the couble label experiments with my laser configuration.
Hope this helps.
Matthew J. Schibler Ph.D.
UCLA Brain Research Institute
73-384 CHS 951761
Los Angeles, CA 90095-1761
(310) 825-9783
FAX (310) 206-5855
E-mail: [log in to unmask]
> -----Original Message-----
> From: Anthony G Moss [SMTP:[log in to unmask]]
> Sent: Wednesday, January 21, 1998 6:05 AM
> To: Schibler, Matthew
> Cc: [log in to unmask]
> Subject: Re: how long should a laser last?
>
> How do the lines hold up? Do they vary much in power? Thanks
>
> **********************************************************************
> ***
> *
> *
> * Anthony Moss voice (334)844-9257
> *
> * 101 Cary Hall fax (334)844-4065
> *
> * Zoology and Wildlife Science email [log in to unmask]
> *
> * Auburn University
> *
> * Auburn, AL 36849
> *
> *
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> **********************************************************************
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>
>
> On Tue, 20 Jan 1998, Schibler, Matthew wrote:
>
> > I have been running a 488/514 argon laser and a 543 Helium neon
> > combination for five years in one institution on a Zeiss LSM 410 and
> a
> > second set (same combination) for one year at another institution
> (on a
> > Zeiss LSM 310) after it was used for two years by another group. So
> far
> > I have not had any problems.
> >
> > I have not used a 633 nm laser.
> >
> > My understanding about these lasers is that an argon laser can last
> from
> > 2000-4000 hrs with proper care and that the helium-neon lasers
> (which
> > are comparatively low power) can last up to 10,000 hrs.
> >
> > Hope this helps.
> >
> > Matthew J. Schibler Ph.D.
> > UCLA Brain Research Institute
> > 73-384 CHS 951761
> > Los Angeles, CA 90095-1761
> >
> > (310) 825-9783
> > FAX (310) 206-5855
> > E-mail: [log in to unmask]
> >
> > > -----Original Message-----
> > > From: Anthony G Moss [SMTP:[log in to unmask]]
> > > Sent: Tuesday, January 20, 1998 4:31 PM
> > > To: [log in to unmask]
> > > Subject: Re: how long should a laser last?
> > >
> > > I remain mystified by the confocal manufacturer's approach to
> this.
> > > Would
> > > it not ALWAYS be better to have long laser life (don't answer
> > > obviously
> > > rhetorical) so isn't there a better way than having the
> short-lived
> > > Kr-Ar
> > > laser, even if it is nifty to use? So much wasted time and effort
> > > with the KrAr combo ..... using 2 lasers seems better to me, the
> > > long-lived Ar ion for 488/512 and a second long-lived laser (HeNe
> ?)
> > > for
> > > the 543, 633 lines. At least I'm given to believe they are both
> > > long-lived.
> > >
> > > Even though there could be some difficulty with getting the two
> lasers
> > > aligned, and perhaps with colocalization, it should be a
> reasonable
> > > and solvable problem for all confocal manufacturers, I would
> think.
> > > Zeiss did this with one of their scopes; I don't know the success
> > > situation with their scope; I noticed their name mentioned in the
> last
> > > discussion. Any comment from Zeiss confocalists withthis
> combination?
> > >
> > >
> **********************************************************************
> > > ***
> > > *
> > > *
> > > * Anthony Moss voice (334)844-9257
> > > *
> > > * 101 Cary Hall fax (334)844-4065
> > > *
> > > * Zoology and Wildlife Science email
> [log in to unmask]
> > > *
> > > * Auburn University
> > > *
> > > * Auburn, AL 36849
> > > *
> > > *
> > > *
> > >
> **********************************************************************
> > > ***
> >
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