Optimal LMD shapes

[lukaskraller]

Hi,

I would like to ask two questions regarding optimal tissue cutting for laser microdissection. Are there recommendations or best practices for tissue annotation that could improve dissection efficiency?

1. Is there a limitation in tissue size when cutting - How large or small can the dissected region be?

2. Does the shape of the selected area influence dissection efficiency?
   For example, are circular, elongated, or irregular shapes more prone to issues during cutting or capture?

Thank you in advance for your time and support!

[josenimo]

Dear @lukaskraller ,

You are hitting on a very common concern, laser microdissection can be technically challenging and the tissue variability requires case-by-case optimization.

In general:

1. The size limitation is based on the objective used for cutting, commonly for collecting single cells we use the 63X objective, and I recommend having shapes of at least 500umˆ2. On terms of upper limit, I have cut entire TMA cores in 4 shapes, so here the limit is the FOV at 5X I guess.
2. In general, again this varies per tissue, I think that shapes from 20,000 to 50,000 are in that sweet spot where gravity is strong enough to pull it down (single-cells need middle pulse), and small enough that they won't bend while the cutting is underway. Keep in mind, many people have collected 1000s of single cells (<1000umˆ2) with decent >80% collection efficiency.
3. The shape of the collection can influence collection efficiency. in broad terms the more circular a shape is, the better the cutting efficiency. The more elongated or snakey a shape gets the larger the chances of the shape folding while cutting and sticking to the underside of the membrane.

To get a better grasp about this I suggest, if you can, performing some tryouts with as representative tissue as you can. Prepare various shapes and sizes, and see what happens.