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Hi Gabriel,
I think you have to consider the refractive index (RI) mismatch. In ideal
condition, you want to have a constant RI trough the light pathway, ie:
If you specimen is in aqueous solution (1.338 of RI) you better use a
water immerion objectif. If you specimen is in glycerol-base mounting
media, it is better to use an oil or glycerol immersion objectif.
Imagine yourself immerged water and you want to look outside (in the air)
, the RI mismatch willmake you see very distorted images.
By keeping the RI constant trough the light pathway, you will reduce the
light lost and contrast:
Ex. Microcope objective lenses (glass RI 1.5), oil (RI 1.5), coverslip
(glass RI 1.5), specimen in glycerol base mounting media (RI 1.5).
Hope this help!!!!
Louis
Louis Villeneuve
Research Associate Confocal Microscopy
Montreal Heart Institute- Research Center
5000 East Belanger
Montreal (Qc), Canada
H1T 1C8
514-376-3330 ext 3511
514-376-1355 (Fax)
[hidden email]
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Hi,
I have a user who insist that using a 1,27NA water immersion objective is
brighter and would give better images than using a 1,4NA oil immersion. I
understand that deeper into the media that would be true. But, in that
particular case, we are talking about imaging GFP at less than 100 micron
away with a spinning disk.
So, I was wondering at which distance from the coverslips do we start
seeing benefits of using a water immersion objective over an oil objective
in aqueous media.
Thanks for your help.
Sincerely
*Gabriel Lapointe, M.Sc.*
Lab Manager / Microscopy Specialist
Concordia University, Biology Department
7141 Sherbrooke St. West SP 534
Montréal QC H4B 1R6 Canada
[hidden email]
cmac.concordia.ca
http://gabriellapointe.ca
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