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February 2014

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From:
Reto Fiolka <[log in to unmask]>
Reply To:
Confocal Microscopy List <[log in to unmask]>
Date:
Mon, 3 Feb 2014 11:39:08 -0600
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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

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