Hi Shafqat,

I think TIRF would be suitable (often used for single molecule studies). I tried to check if someone ever used it on isolated mitochondria and it seems they did. Check out this paper:

http://www.nature.com/srep/2011/111214/srep00195/full/srep00195.html

Best of luck!

Michał

STED can be used too:

http://www.sciencedirect.com/science/article/pii/S1567724911002856

Radif

If you are worried about EM artifact try looking up high pressure frozen EM images. Isolated maybe just plunge frozen. 

I published two articles on monitoring single mitochondria (isolated) metabolic activites by inverted fluorescence microscopy. As previously mentioned in this thread, TIRF and STED are also can be used for imaging. It depends upon which kind of information do you really want from isolated mitochondria. For example, confocal/fluorescence microscopy is the simplest option for counting the no. of mitochondira, following their NADH autofluorescence and, membrane potential variations, ROS etc.... using extrinsic dyes. You may not get the information about the "real size" of mitochondria and "shape changes" which are triggered by apoptosis for example. For studying mito protiens and shape changes and other in detailed information, I would suggest you to go for advanced microscopy.

Here are the links of my publications. Hope they will provide you the information you required.

     https://www.researchgate.net/publication/253334865_Optical_microwell_array_for_large_scale_studies_of_single_mitochondria_metabolic_responses

https://www.researchgate.net/publication/236249599_Monitoring_Metabolic_Responses_of_Single_Mitochondria_within_PDMS_Wells_Study_of_their_Endogenous_NADH_Evolution

Cheers

Article Optical Microwell Arrays for Large-Scale Studies of Single M...

Article Monitoring Metabolic Responses of Single Mitochondria within...

Dear Shafqat Rasool,

It is not only nuclear physics where the law of relativity is in action. In Biology, mitochondria are particularly sensitive to the experimentalist actions.  Mitochondria are the Fundamental organelle and they are very dynamic. In addition, mitochondria from every organ and tissue, even in the same cell may vary significantly. For example, mitochondria from brain do not contain any substrates. Therefore without providing external substrates you cannot visualize mitochondrial NAD(P)H fluorescence. Addition of a fluorescent dye will also cause a response from mitochondria. Therefore, you have to compromise your goal and the method. I think that we already have a rather reliable understanding of what mitochondria may look like, much less we know about how their function. Visual information is not the best approach to understanding mitochondrial functions.

Alexander: I get what you re trying to say…I am more interested in structure at the moment, plus as far as I know, the functions that I am interested in are in intact in isolated mitochondria…and i am more into flourescent proteins than dyes anyway