Leica Thunder and a comparison between 4.2 cmos versus Hamamatsu ORCA-Fusion

Andrew_Seeber
Sat, 05 Oct 2019 01:28:43 +0000
Hardware

In my quest to build the cheapest high-end spinning disk microscope I ended up demoing a Leica Thunder widefield system in my lab. A few people wrote to me asking for my impressions so I will share them. While I’m under no illusion that a widefield could replace a confocal I was curious what Leica was doing in terms of image clearing/denoising and if this was just a Leica version of deconvolution or something else.

So here is the Thunder setup:
It’s on a fully automated new DMI8. No complaints with the stand at all. There’s a Lumencor LED at the back.

The Leica guys very kindly brought a Hamamatsu Fusion with a Hamamtasu Gemini. The Gemini is an image splitter; you can project two channels on different parts of the camera chip. On the other camera port was a Leica branded 4.2 cmos sensor camera.

One of my favourite parts of this scope was the nifty Leica filter wheel (below). It was switching easily at 25 ms.

Last, here is the Thunder computer. It’s a HP Z4 with a Nvidia 2080 RTX graphics card and a sticker:

This was the first time I’ve used any modern version of Leica microscope and acquisition software. For whatever reason, in every institute I’ve worked, the oldest microscope is always a Leica and I previously had bad impressions of their software. This wasn’t the case now. It’s easy to use and setup. It also had a few nice features such as scanning an area on low mag, creating a map of your sample that you can “click” into at high mag. Convenient.

So what’s up with all this Thunder business? Leica has bundled three different imaging processing techniques in their software: Thunder, deconvolution (I think they call it small volume clearing) and a combination of Thunder and deconvolution (again, I think they call it large volume clearing). The deconvolution does as stated and I won’t really talk about it here. The Thunder processing is definitely interesting though.

Leica sells Thunder as a computational clearing method. Which it is. It works in 2D which is nice and it’s not deconvolution (at least it’s not fitting a PSF). It’s also very fast and can be done live during image acquisition.
Here’s a few examples of what it does on 2D images (this was a 100x):

And a zoomed in version:

In my opinion, Leica’s algorithm doesn’t seem to be making things up. The structures that it makes clearer are visible in the widefield image. However, my biggest complaint, and many people will share my view, is that there is no information about what Thunder is actually doing. It makes it a little hard to trust it. However, I will admit that it certainly does clear the image. I imagine this probably works really nicely for large opaque-ish samples.

This is definitely not a replacement for a confocal system but it’s a useful exercise to compare them. Try and guess which is the spinning disk and which is Thunder in the image below (both taken on a Fusion):

If you guessed the bottom image is Thunder you are correct. One give away is the “banding” around the edge of the nucleus. I’m not entirely sure what causes this but maybe it’s due to the out of focus light on the edge of the nucleus. Since these cells are pretty flat, that could be causing issues. I think Thunder does a pretty good job with the spots. This was the thunder+decon processing method which is why the Thunder images look so smooth.

Overall I think Thunder is promising. I would like to know exactly what it is doing otherwise it’s not really possible to justify using these images in a quantitative way. I think you currently can’t buy the Thunder processing separately from the system as a whole, and I believe that you have to use a PC from Leica as well. The system is a pretty inexpensive widefield with some nice image processing added on. It works well as a whole integrated system and Leica have done a lot to make sure it is very easy to use. It could work well in imaging cores where confocals are booked up all the time and users just need a few nice pictures. I would also be curious to see a head-to-head between Thunder and say Nikon noise.ai.

In the next post I’ll show a comparison between a 4.2 cmos sensor camera and the newish Hamamatsu ORCA Fusion.

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