Dear Carola,

first of all: the images you show for comparison are not the same magnification. Therefore it might be that we are "impressed" by a 'wrong feeling of blackening / dark vs light structures'  via the overall  "contrast" .

Secondly: Regarding the comparability:

Have you managed to ask the former institution about their

i) type of EM,

ii) their staining method for the ultrathin sections (as compared to your staining method) and at least

iii) the type of image acquisition (digital, analog?)?.

Assuming you are using a digital camera it might be also useful [to know which system you use AND ) to assess the hardware used by the former external EM facility (or if they used analog technique, i. e. negative film, physical development and B/W paper printing).

Good ol' AMT. I agree mostly with Wolfgang. Especially the third point. Differences in imaging devices can have a HUGE impact. Our facility upgraded our camera and my images are sharper than ever (and also far more sensitive to astigmatism). AMT is something I've used a lot and their software aims for ultrahigh contrast when set to automatic. You've played around with it, though, so that's one thing down (and unfortunately the easiest).

Your mags seem similar enough on first glance. It looks like it's something with your embedding.

Apologize for lengthiness....

To be honest:  calculation of image magnification from my monitor display:

old-EM.tif:  500 nm bar = 10 mm,    therefore follows: 1 µm = 20 mm

new_EM.tif    2 µm bar = 35 mm,     therefore follows: 1 µm = 17.5 mm

which means image 'old' has an original magnification of approx.  x20,000,

                 and image 'new'has an original magnification of approx. x17,500.

@Colin: thank you for mentioning that fact as well as the possibility of differences in staining characteristics due to other embedding parameters (i.e. chemical as wll as physical:  as the tissue was sent as "perfused fixed tissue" it can be assumed that unequal tissue processing chemicals&steps, different embedding and staining parameters (e. g. resin type, resin mixture including hardness and catalyst concentration, infiltration, polymerization; make and properties as well as application of staining to ultrathins) may end up with different staining characteristics.

One could try the following:

*) since uranyl ions strongly react with phosphate and amino groups, thus staining DNA and some proteins (but not staining well organelles composed of membranes)*) 

**)Lead citrateenhances the contrast by interacting with proteins and glycogens [enhances to a weaker extent also the effects of UA-Ac-stain)  and has binding / staining effects for /on a wide range of cellular structures such as ribosomes, lipid membranes, cytoskleleton and other compartments of the cytoplasm. The enhancement of the contrasting effect depends on the interaction with reduced osmium, since it allows the attachment of lead ions to the polar groups of molecules*).   

Not knowing about your (?)UO2Ac(?) - lead citrate staining recipes (composition / staining time) it might be possible to increase the contrast of unmyelinated nerves / axons (i.e. their membranes) by decreasing UO2-ac incubation  and/or  increasing leadcitrate incubation time. Perhaps you could try also a triple "contrast" staining procedure, consisting of first incubation (pretreating) of ultrathin section in/with filtered hydrous 0.05% tannic acid solution (either at RT say for 5-10 min or even slightly elevated temps up to 40° for some min.),  followed - after jet washing the sections with A.bidest. and short drying - by the usual double-staining sequence UO2Acetate (hydrous,alcoholic) and lead citrate (e.g. Venable&Coggeshall, or Reynolds).

Note: *) in part taken/cited from:    "EM Sample Preparation Contrasting"

https://www.leica-microsystems.com/fileadmin/academy/2013/Contrasting_final.pdf

Disclaimer:

no affiliation to LEICA Company, no financial interest, unfortunately also not having had the opportunity to run a Leica Ultrostainer during my active profession in the EM-Lab. I will not comment on the use of "contrasting"(German root") instead of 'staining' (English "term"), as I don't comment on the wrong citation for the modification of lead citrate by "The alkaline Wenable & Goggeshalls "  lead solution (p. 8.) which correctly should read:

VENABLE J.H, Coggeshall R. (1965): A Simplified Lead Citrate Stain for Use in Electron Microscopy; J. Cell Biol.(N.Y.) 25, 407-408.

 Thank you for your reply Colin and the very detailed reply Wolfgang.

Meanwhile, I did find out the external facility, we previously sent our samples to, was using 1% uranyl acetate (UA) prior to polimerisation. However, at our current facility we can't use UA due to strict H&S regulations. Instead the TEM technician suggested adding gadolinium acetate to the staining protocol (Gadolinium acetate (first stain) -> Wash -> Blot dry -> Lead citrate (second stain) -> Wash -> Dry).

I found a recent paper testing a (what they call) modified UA replacement method which they found to produce better quality images. However, the staining was tested on kidney rather than brain tissue. 

http://clinmedjournals.org/articles/ijmnr/international-journal-of-medical-nano-research-ijmnr-3-013.pdf

We already embedded all the CC samples we are planning to use. So we are currently sectioning gold / silver ribbons. The only process I can modify for the current batch of samples is the staining stage (and of course try a few more settings of intensity / focus on the TEM), which I am planning to try on a few grids tomorrow. 

In terms of the TEM, the model I am using now is the Tecnai Spirit one. Not sure how /if the camera system is different from the one used before.

Apologize for lengthiness [but I am going into detail as usual, therefore anybody for free to downvote]

Dear Carol, great !

Thank you for detailed info on your processing....

Since your tissue at the former processing institution has been "UO2Ac-block-stained"

(which also means "fixation of tissue matrix substrate -which perhaps would be eluted otherwise - already prior to embedding"

it might be worth a try only to stain ultrathins with lead citrate or a 'double' combination

It is now clear for me  that most of the e-dense "stain" = structures seen by contrast forming may be attributable to the digital imaging system you use. There is some information [ *) ]     concerning the Sm-Gd-stain (not knowing for now about improvement of "dye"= the UAR/Uranylacetate replacement substances which are sold via the special market). Therefore: thank you for mentioning the paper testing the substance  (UAR-MUAR) = 

----------------------------------------------------------------------------

Santhana RL, Paramsavaran S, Koay BT, Izan SA, Siti AN (2016) Modification of

the Uranyl Acetate Replacement Staining Protocol for Transmission Electron Microscopy.

Int J Med Nano Res 3:013 [**)]   [note: they have 90 nm 'thick' ultrathin sections which naturally - combined with a small objective aperture and 100kV beam acceleration voltage- will produce huge contrast especially using digital camera system the authors of the paper unfortunately didn't reveal to the reader ! Additionally:  within the text on p. 2  'EM (FEI Tecnai G2) at 100 kW correctly should read kV )   which I did not know about yet. I wonder a bit about the effectivity / efficacy of their UAR solution but it may be that they found out the right dilution of the UAR(=Sm-Gd)-stock solution as commercially available)

----------------------------------------------------------------------------

It would be interesting to know more in detail (i.e. parameters) about your practical staining on your ultrathins with the UAR/MUAR-Pb-sequence....

Other possibilities for successful ultrathin section staining  IMHO would be ('oldies' but perhaps goldies in your case):  use or at least try Bi-(=wismuth-)  instead of UO2Ac - staining (cf: Ainsworth & Karnovsky (1972) J.Histochem. Cytochem. 20:995), thorium, perhaps also Indium, lanthanum or last but not least, PTA / phosphotungstic acid staining could be used.

MY best wishes and good luck, WM.

cf. e.g. 

[ *) ] Staining ultrathin sections with Uranylacetate replacements?

 https://www.researchgate.net/post/Staining_ultrathin_sections_with_Uranylacetate_replacements

and:

https://www.researchgate.net/publication/269709034_DATASET_for_RG_suppl_for_POSTER_%27UO2Acas_superb_staining_reagent_used_in_%27MUSS_WH14-12-19docx

as well as: 

 https://www.researchgate.net/publication/264527770_Uranylacetate_as_superb_staining_reagent_used_in_diagnostic_as_well_as_scientific_Transmission_Electron_Microscopy_really_at_risk_or_mandatory_to_do_the_job_properly

 [**)] Int J Med Nano Res (and publisher ClinMedLibrary) is listed in: https://scholarlyoa.com/publishers/   (2015 list)

Conference Paper Uranylacetate as superb staining reagent used in diagnostic ...

Data DATASET for RG suppl. for POSTER 'UO2Ac.as superb staining r...