Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>To reduce the reflections due to the laser light on the coverglass using the
>Olympus FV300 we typically would put an ND filter in place to reduce the
>light to between 5 and 20% of the incident power. Then we would use the AOTF
>control to choose our power. Typically for live cell work we use 0.1% of the
>488 nm line of a 40 mW Ar laser. So attenuating to 20% gives us 5x more
>sensitivity with the AOTF now controlling the power from 0% to 20% rather
>than 100%. It turns out AOTFs are good attenuators but not great blockers of
>laser light so this will block reflections from both the 488 nm and the 514
>nm lines (as well as weaker lines). I'm not sure other confocal microscopes
>still have ND filters in the light path but this is certainly a
>cheap solution.
>
>Claire Brown
It might be worth pointing out that, in the event that you don't need
to collect a fluorescent signal in its presence. (i.e., you are
interested in structures farther into the specimen), the reflection
from the coverslip interface can be a useful marker for the location
of the this interface.
And in relation to the subject of the original post, using an NA 1.4
objective will make it worse, at least it will as long as the laser
light fills the full input pupil (practically, that the laser beam
diameter is larger than that of the "glass" as the back of the
objective). This is because the fraction of light reflected by the
glass/medium interface depends not only on the RI's of these two
layers but also on the angle. As the angle of incidence increases
beyond that corresponding to NA 1.2, the fraction reflected increases
markedly, reaching 100% at NA larger than about 1.33.
To emphasize what Guy and others have said: unless the structure of
interest is essentially touching the glass, there is NO advantage to
using "the larger NA". a) because it really isn't larger when you
account for all the light lost to reflection at the interface and b)
because SA decreases the resolution so much that the peak brightness
of a point object is lower.
Some folk persist in thinking that this isn't so, perhaps because a)
large objects (i.e, those not near enough to the resolution limit to
be blurred by SA) will appear a little brighter, b) they have not
taken the time to adjust the collar on their water objective for the
coverslip thickness (not so important for an oil lens as the oil and
the coverslip have about the same RI.) and consequently, the image
from the water lens is aberrated and hence dim or 3) they have read
somewhere that the brightness in widefield fluorescence varies with
the fourth power of the NA. The idea is that both the illumination
and the collection of light vary with the square of the NA. This is
true for the illumination as long as the image of the arc in the BFP
actually fills the pupil, that the "high-NA light" is not lost by
reflection at the coveslip interface and you don't use a very small
field diaphragm aperture. It is not true for the collection if the
larger NA produces SA.
If you are working with living cells, get the water lens and learn
how to adjust it.
This topic is so important that it rates an entire chapter in the new
handbook as well as being discussed in many other chapters. There is
also a long discussion of the construction and features of notch
filters, particularly whose fabricated using the new "hard" coatings.
Cheers,
Jim P.
--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-262-9083
250 N. Mills St., Madison, WI, 53706 [log in to unmask]
"A scientist is not one who can answer questions but one who can
question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
"He who can get you to believe absurdities, can get you to commit atrocities."
Voltaire.
|
