Question on Water Immersion Objective compatibility with pgFocus

[Brandon Lee]

Hello,

We need a drift correction system for a few microscopy applications in single molecule localization microscopy as well as long term light sheet imaging.

We currently have an Nikon TE-2000-E but have a few spare PI 721 PIFOCs for piezo scanning.

We want to ideally build our own drift correction system and have been heavily looking into the pgFocus.  The main issue that we are seeing:  there seems to be a pretty standard requirement of using an objective with an NA of 1.4 or higher (since pgFocus was primarily designed for TIRF).  Our single molecule localization microscopy application and our long term light sheet imaging application necessitate the following 2 objectives:

One of them is a Nikon 60x Water Immersion with 1.27 NA and the other is a Nikon 60x Water Immersion with 1.2 NA.

Is it safe to assume that neither of these objectives will be compatible with the pgFocus?

If not, we will have unfortunately have to sell our TE-2000-E system to make way for a newer Nikon system with Perfect Focus.  

Thanks in advance for your help.

 

[kyle.m....@gmail.com]

Hi Brandon,

Is it safe to assume that neither of these objectives will be compatible with the pgFocus?

 

That's correct so long as your sample's minimum refractive index is greater than the NA of either objective. pgFocus uses total internal reflection (TIR) of the laser beam from a coverslip. The NA of an objective has to be at least equal to the refractive index of the sample to do TIR. Since your objective's NAs are 1.2 and 1.27 but the refractive index of water is 1.33, this criterion is not satisified. See the paragraph below eq. 16 here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285862/

If you imaged in air, though, then that's another story ;)

Kyle

[Brandon Lee]

Thanks so much for your help and for the quick response, Kyle.  We will keep this in mind and switch to focusing on the Nikon system/PFS.

Thanks again.

[Karl Bellve]

Kyle is right. At the moment, high NA objective is required for pgFocus. 

One day, I might revise the light path of pgFocus, and do it the same way Perfect Focus (and others) maintain focus. The electronics wouldn't have to change, just the firmware, and the optics. It is a much more universal approach.

Cheers Karl

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Cheers,

Karl Bellvé