Super resolution using imageJ

[Knecht, David]

I am trying to get advice on how to convert our existing microscope system to super resolution capabilities. We have a Nikon inverted stand with an Andor spinning disk confocal on one port and wide field on the other port.  Coming in the back is a motorized TIRF arm and the laser excitation from the Andor can be diverted either the spinning disk or the TIRM arm.  I looked at Henriques et al (Nature Methods) and the implication is that we can use this system with Quickpalm to acquire data, but I am unclear on the excitation pathway for doing this. It sounds like we would come in the TIRF arm, but how would we fill the BFP of the objective?  Also, there is mention of a “clever” way to insert a cylindrical lens in a holder under the objective, but I could not find more details.  Are there more detailed instructions somewhere or alternative approaches? Any advice welcome as we are new to super resolution.  Thanks- Dave

Dr. David Knecht
Professor of Molecular and Cell Biology
Core Microscopy Facility Director
University of Connecticut
Storrs, CT 06269
860-486-2200

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[John Oreopoulos]

David,

Many specifics about Ricardo's setup can be found at his Youtube channel:

https://www.youtube.com/user/paxcal/videos

He uses a home-built TIRF system to deliver laser light into the microscope system at an oblique angle (TIRF or quasi-TIRF). The light is focused at the back-focal plane, not filled. This condition is needed to get a collimated beam impinging on the sample. Expanding the beam at the back of the objective concentrates the light at the sample plane (which is sometimes useful for PALM/STORM - higher power densities), and some setups use a drop-in lens to do that, but the penetration depth is less-defined in that case (which doesn't really matter for this application. In general, people are using TIRF illuminators to get laser light into a widefield microscope setup, but it doesn't have to be strictly TIRF illumination.

You might also consider Nico Stuurmann's localization microscopy plugin for MicroManager which is also very good:

https://micro-manager.org/wiki/Localization_Microscopy

There are a number of other free-ware localization microscopy applications floating out there on the web, but I've lost track of them because there are so many now.


Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2014-11-22, at 9:49 AM, Knecht, David wrote:

> I am trying to get advice on how to convert our existing microscope system to super resolution capabilities. We have a Nikon inverted stand with an Andor spinning disk confocal on one port and wide field on the other port.  Coming in the back is a motorized TIRF arm and the laser excitation from the Andor can be diverted either the spinning disk or the TIRM arm.  I looked at Henriques et al (Nature Methods) and the implication is that we can use this system with Quickpalm to acquire data, but I am unclear on the excitation pathway for doing this. It sounds like we would come in the TIRF arm, but how would we fill the BFP of the objective?  Also, there is mention of a “clever” way to insert a cylindrical lens in a holder under the objective, but I could not find more details.  Are there more detailed instructions somewhere or alternative approaches? Any advice welcome as we are new to super resolution.  Thanks- Dave
>
> Dr. David Knecht
> Professor of Molecular and Cell Biology
> Core Microscopy Facility Director
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

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ImageJ mailing list: http://imagej.nih.gov/ij/list.html