What kind of techniques are you thinking of Christopher? Are you up for transmission electron microscopy? Questions of this sort were [once upon a time] answered using TEM + stereology. In particular I recollect one study when a salivary cell in secreting, the question being: what happens to the membrane which surrounds the secretary vesicle, after it fuses with the plasma membrane? The investigators were Cope and Williams and ??? I think. Probably in the 1980s or even 1970s.

Thank you for the reply Richard. Essentially all the work I do at present is live with primary cells. With the techniques I am using I can essentially keep the cells alive and provide varying stimuli to 'get them going' as such. So for example I can apply pHrodo bacteria (pH sensitive dyed bacterial particles) and then watch live up take into the cell and then further digestion. From this example I am able to follow phagosomes from creation to degredation. But what i would like to do is some how mark the exterior membrane of the phagocytosing cell, possibly with the same pH sensitive dye used for the bacteria. Then i would look at varying ratios of flourescence over a live time course. But this is all theory, nothing has been put in practice yet.

Its interesting you mention TEM. I am planning on making a move to using TEM for characterisation of various structures within the cells I use. And it would be very interesting to take this approach to the question. So how would you use TEM to analyse the recycling of the membrane? I can imagine you would need a specific marker for the secretory vesicle membrane and then one for the target membrane that the vesicle would fuse with, over a detailed time course? Thank you again Richard, I will certainly look up the papers and the use of TEM.

Dear Christopher,

(dear Richard), I am too quite sure some special EM-techniques (TEM, but SEM as well, I guess) , especially Cryo-Techniques combined with diverse labeling methods (ultrarapid freezing, Freeze fracture, Freeze substitution, etc.) could help you in answering your scientific questions. Just only as an "entry" to specific (T)EM technology (which of course is only one out of many ways of possible specific preparation methods, a lot of which have been published within the last 10-20 years)

I would like to point you to a brand new article by

Constantin Craciun, Lucian Barbu-Tudoran

Identification of new structural elements within ‘porosomes’ of the exocrine pancreas: A detailed study using high-resolution electron microscopy

Micron Vol 44, No. 01 pp. 137-142 (Jan2013) http://www.sciencedirect.com/science/journal/09684328/44/1

(NB-should you not be able to access or get the article most conveniently, just send me a short note via e-mail to < w dot muss at salk dot at >

Best wishes and good luck!

Thank you very much Wolfgang. I have the article in hand and thank you for your suggestions, as with Richard's - creates a great starting place!

All the best.

Dear Christopher, you're welcome!

G'day Christopher [and Wolfgang, good to see you] -- Regarding the use of TEM+stereology, I have just tried putting into PubMed. And the papers I remembered were all there. They are from the early 1980s, when TEM was still in fashion.

Regarding your live-cell strategy, let me check my understanding of what you're thinking of doing. You propose taking a near-membrane pH sensitive probe, labelling the plasma membrane of your cells with this, and triggering phagocytosis. Then probe bound to the plasma membrane will report on the pH of the external solution and be one colour, but internalised probe which arrives in a phagosome will report on the pH of the phagosome and so will be a different colour. And then, given some idea of the absorbances of the two probe species, you can get a ratio measure of the amount of internalised membrane compared to that in the plasma membrane. OK ... The stereological approach of course would provide absolute measures of membranes.

Ah, and while I think about it, what near-membrane pH probe had you thought of using? This is request for information not a preliminary to an informed suggestion by the way! I will sometime in the future being addressing such probes in a review article I'm due to write, so am seizing the chance of getting information!

Hi Christopher,

I think you can try live cell imaging using FM dyes available with life technologies, which will enable you to track complete endocytosis(http://products.invitrogen.com/ivgn/product/F34653). The beauty of FM dyes are they are not fluorescent outside the endosome and starts fluorescing as it is taken up by the cells thereby allowing you to track the complete endocytosis and also exocytosis. Once you track this you can add your dyed bacteria together with FM dye and look for co-localization of dye with your bacteria.

I am not sure if this helps you, but I thought this information would be relevant for you.

Thank you for the reply Richard. The main dye I had in mind is the exact same dye used on pHrodo bacteria. pHrodo is a succinimidyl ester that is pH sensitive. Essentially you would incubate your cells in a media of pH 7.4 (with the pHrodo dye), which neutralises the signal exterior to the cell's internal pH. Once the fragment is internalised, whether this be pHrodo dyed bacteria or pHrodo-dextran, the pH shift to more acidic conditions induces fluorescence, and also the level of fluorescence is correlative to the pH. Experiments using pHrodo to dye cells have been conducted previously but then analysed through FACS cell sorting.

So you are able to track the movement and maturity of the internalised fragment whether be in early endosomes or phagosomes maturing to later stages upto lyso-endosome fusion. I have been using this in conjuction with lysotracker for dual labelled live cell imaging in my phagocytosis assay for up to 5-10 hours using an ex vivo technique I developed in my PhD.

Rajesh, thank you so much for the suggestion of using FM dyes. I have conducted some pilot studies with FM464 dye, but am still to optimise the right conditions of concentration and incubation time before its removal. I am quite interested in FM dyes because I know that varying the length of incubation can lead to particular organelle membranes being marked, if I remember correctly? I will look into using this with the phagocytosis assays I am conducting. Its quite interesting because the one thing I would like to mark is the hybrid endo-lysosome (vacuole in some cell types), for many reasons but also because to my knowledge this is the largest organelle (in terms of membrane units) formed-degraded-reformed rythmically pending on stimulation.