Posted by
Reto Fiolka on
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581519p7581522.html
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Dear all
Besides confocal microscopy, SIM very much benefits from high NA on the
illumination side.
The higher the NA, the smaller the line spacing can be. The resolution gain in
SIM is reciprocal to the line spacing.
To give numbers, with 488nm excitation, I have achieved 120nm lateral
resolution with SIM with a NA 1.2 objective, whereas with a NA 1.45 TIRF
objective, 80nm lateral resolution is possible.
For the latter case, the corresponding line spacings, as low as 170nm, only
exist in the near field at 488nm.
This is of importance (also for confocal imaging) when imaging watery samples.
An NA above ~1.33 will not improve resolution in the far field, as anything
above is lost in the nearfield.
I have seen many papers where people employed high NA oil objectives for
confocal imaging in biological samples. Besides not being indexed matched, they
will not provide more resolution, as any illumination above the critical angle is
lost in the near field (critical angle for n=1.33 is reached at NA 1.33).
Best,
Reto