Dear Sanda....i am not sure if I see what you mean....There are red (roundish) areas which I would identify as nuclei, and a lot of roundish, "dark lined" structures the center of which are +/- colorless (or with the same colour of "background" which I frankly and honestly at first glance would identify as micro-air-bubbles under the coverglass due to either careless mounting the coverslide or poor dehdyration - humidity left after xylene treatment prior to or at least inherent IN the mounting medium you used). Please specify your prep's and staining mor in detail. Thank you!

Dear Wolfganag,

Thank you for your answers. I will try to clarify more.

Yes, my question is what could be that roundish "dark line" structures. It is not due to staining which was regular Giemsa staining(on 96 well plate-no coverglass used). I attached that picture just because it is easier to see them when cells are fixed and dyed,but I will add one more picture which was done without staining on alive cells and structures are still visible. And we see them every time when we treate cells with this molecule and also size of vacuoles depends on concentration used.  Thank you for your help!

Dear Sanda: will come back later on...... lost the whole text of my reply due to Transmission-problems as I have seen now....

Now that I found again this thread/question without a further e-mail notification (no additional answer) I'll try again / to remember what I'd written/proposed in my first attempt to reply:

Thanks for adding another / the second image (without staining)

First of all:

to have a better imagination or - then in the end - understanding of  the "textures in question" (= roundish "dark line" structures) we need to have details on the "histological - LM- processing" of your  cell culture until imaging and documenting by LM-photography. I understand that you can not / don't like to comment or give utmost specific detail on the culture conditions and / or the 'molecule' you are presenting to or treating the cells (hsFb) with, the knowledge of which (or with / at least the knowledge of some background-facts) could "trigger" sufficient help in characterization of your "empty looking vacuolar structures".

Secondly:  regarding staining: you mentioned Giemsa staining (which one?) as the used staining method ....  AFTER / without? which? fixation, washing(s), etc.,etc. Embedding ? /  direct live imaging after intravital staining?.

Further: have you tried ...(if not, I recommend to try)

i) PAS-stain (just in order to be able to test (also pH-dependent) for carbohydrates / glycoconjugates / mucin(s).... etc.   or

ii)  applying proper stain for demonstration of starch (if you, e. g. use HES(=hydroxy-ethyl-starch") as your "molecule" or,  finally,

iii) apply also (best: try first)  a (generally histological,  or vital stain, if necessary) fat stain / stain for lipids, because the "double rim" of your vacuoles reminds me of refraction properties of [perhaps eluted] lipidic material (depending naturally on the previous processing of your cultured cells - therefore my request on disclosure of all your previous spec-prep-steps). thank you in advance for clarification, best wishes and good luck, WM.

Note added: this post has been edited for some minor grammar and spelling errors three minutes after initial posting

They appear more artefactual as mentioned above by Dr Wolfgang. In addition to what he has discussed, I would add another possibility of endocytotic vesicles containing the 'molecule' that you are using to treat these cultured cells as dermal fibroblasts are known to have phagocytotic properties. Keep us updated. Thanks.