UV (365nm) and visible (546nm) microscopy of a diatom

[colinbm]

Thanks Jonathan

[JMC]

Firstly, Merry Christmas everyone.

 

A single image from an 'Amician test slide' by Watson and Sons (no stacking with this one). Navicula rhomboides, used for testing resolution. Done on my modified Olympus BHB microscope, using 365nm light (Nemo torch). 100x Leitz Pl Apo NA 1.32-0.60 objective, oil immersion. Reichert Neo 1.42/1.18 dark ground condenser, oil immersion. 2.5x Nikon CF PL photoeyepiece. Monochrome converted Nikon d850 camera. Reduced in resolution for sharing here (original image size 6136x5540). Dots are about 360nm apart.

 

[colinbm]

Fascinating Johnathan

[JMC]

Been absent for a while (holiday and away from the microscope as a result) but here's a new image from the microscope taken using 365nm light.

 

 

A Navicula of some type. Olympus BHB microscope using 365nm LED light (Nemo torch). 63x Leitz Pl Apo NA 1.40 objective with oil immersion. Olympus Abbe condenser, oil immersion. 2.5x Nikon CF PL photoeyepiece. Monochrome converted Nikon d850 camera. Single image, no stacking. The slide is a diatom strew by John Dale, where a thin layer of aluminium has been deposited over the diatoms to help with contrast, hence the image looking like a dark field one. The image has been reduced is size for sharing.

 

Going in tighter and cropping the image, shows the areolae with a spacing of 261nm as measured with ImageJ. A hint of the pores in the areolae is also there, but not well resolved.

 

I also did an image of the same diatom with 450nm light (below). Shows the improvement in resolution when using 365nm light.

 

[Andy Perrin]

The FWHM would be 0.51*lambda/NA which is 0.51*365nm/1.4 if I understood your description. That comes out to 133nm. The areolae spacing was about double that — does that mean you have more room for improvement?

[JMC]

31 minutes ago, Andy Perrin said:

The FWHM would be 0.51*lambda/NA which is 0.51*365nm/1.4 if I understood your description. That comes out to 133nm. The areolae spacing was about double that — does that mean you have more room for improvement?

Theoretically there would be room for improvement, but 200-250nm is about my limit with this setup Andy. Keep in mind I am using a homemade microscope with objectives and a photoeyepiece from different manufacturers. Hence theoretical and practical are different.

[JMC]

While I like using 365nm at times for extra resolution, there is an issue in that my decent condenser (Olympus Aplanat Achromat) blocks it, and so do some microscope mountants. To give me some potential improvements over 450nm, I got an Alonefire 395nm torch, and have tried it with a 390nm Thorlabs 10nm FWHM filter. I added a diffuser to the torch to smooth out the light. This was made from a roughened PMMA plate used for sunscreen testing.

The image below was made on my microscope using this new light source, and is of an Actinoptychus heliopelta diatom from Dunkirk, Maryland. Resolution has been dropped to 50% of the original to help with sharing.

 

[colinbm]

These must be pretty amazing in 3D.
What magnification is this please ?

[Andy Perrin]

I love it, JMC! Impressively sharp image, at least at this resolution.

[JMC]

Thanks guys. I used a 63x Leitz Plan Apo NA 1.40 objective, and a 2.5x Nikon photoeyepiece for this one, and the diatom pretty much filled the field of view on the monochrome converted Nikon d850.