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We have found differences in our DeltaVision parfocality depending upon
the immersion oil used. If we want red and green co-localized, we use DF
oil. If we want blue and green co-localized, we use the DV immersion oil
kit. All of this is verified by 100nm TetraSpec beads. Still, the color
that is "off" is only off by 0.5 microns (one pixel). Pretty happy with
the system. Olympus IX70, 100x 1.35.
Kathy
________________________________
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Julio Vazquez
Sent: Tuesday, June 19, 2007 9:13 AM
To: [log in to unmask]
Subject: Re: Objectives
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Glen,
A quick test with Mol Probes 0.5 micron beads, in fluorescence mode on a
Deltavision microscope, gave these results (I am using the green
channel as a reference, and give the offset in z for the other
channels):
Zeiss alpha-plan Fluar 100/1.45:
528nm: 0
457 nm: -0.8 microns
617 nm: -0.1 microns
685 nm: -0.6 microns
Zeiss Plan_Apo 100/1.4:
528 nm: 0
457 nm: -0.4 microns
617 nm: + 0.1 microns
685 nm: + 0.4 microns
green and red channel are parfocal on both, within experimental error;
DAPI and CY5 are off on both, but a bit more so on the Fluar; the range
of variation across all four channels is 0.8 microns for both
objectives, but the PlanApo has a flatter curve. I suspect that some of
the variation could be due to RI mismatch between oil/glass/mounting
medium, making the objectives look a bit worse than they are.
The Zeiss alpha-plan fluar, 100x/1.45, the 63x/1.4 PlanApo and the
PlanApo 40/1.2 Water (all Zeiss) all require the most extreme
corrections when used with two photon excitation, but here again the
corrections are in opposite direction for the 100/1.45 and the other two
lenses. As a comparison, the multi-immersion 25x/0.8, used with oil,
needs no correction at 800 nm excitation.
If you Google alpha Plan-Fluar, you get to a Zeiss site with some specs
for the objective. Peak transmission (~80%) is between 400-700 nm. They
do not provide chromatic correction curves.
--
Julio Vazquez,
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
On Jun 15, 2007, at 10:48 PM, Glen H MacDonald wrote:
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Has anyone checked the parfocality of this lens across the laser
lines, say from 405 nm to 633nm, by reflection or multi-color beads?
Asked with due regard for variability between individual lenses.
Regards,
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923
(206) 616-4156
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