Microvesicles (MVs) or exosomes measured 30-1000 nm and all publications noted them through electron microscope but unfortunately EM is unavailable in our institute. Is it possible to visualize the MVs or exosomes using confocal or phase contrast microscopes? I mean to use a special/fluorescent stain for MVS to be easily investigated.
Since the size of MVs and exosomes close (or under) the physicial resolution limit of visible light microsocpys, you have to mark the vesicules with fluorescent markers to make them visible - so you will not see directly the subcell. ves.-s, but their fluorescent halo. The fluorescent markers cen be specific (such as fluorescent protein conjugated specific antibodies), or aspecific (using lipid markers, such as Annexin V for phos. serin, or other PKH stains). With the first method you can detect specific population of subcell. ves.-s, while with the other you can detect all of them. It also important to make a control of specificity of your stain, such as using detergent (e.g. Tween, Saponin, etc.), to eradicate the vesicles - in this case you wont see fluorescent marks. A third approach can be using intravesicule markers, as dr. Ganglum wrote.
By the and, let me recomend a good protocol oriented article: Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parametershttp://www.ncbi.nlm.nih.gov/pubmed/21041717
With best regs: Csaba Timar
We can, but you are not sure whether they are exosomes to convince yourself. When we performed GFP or RFP tagged secretory/exosomal protein, you can collected the culture medium to isolate exosomes containing GFP fusion protein and scan through confocal, you can determine the size and fluorescence, You can also perform, FACS analysis using aldehyde sulphate latex beads to adsorb exosomes and label the bead-exosome complex with primary antibody labeled fluorophores. Further more, westerns will help determine exosomal markers. Coming to Phase contrast, it is hard to define or characterize.
RKG
Since the size of MVs and exosomes close (or under) the physicial resolution limit of visible light microsocpys, you have to mark the vesicules with fluorescent markers to make them visible - so you will not see directly the subcell. ves.-s, but their fluorescent halo. The fluorescent markers cen be specific (such as fluorescent protein conjugated specific antibodies), or aspecific (using lipid markers, such as Annexin V for phos. serin, or other PKH stains). With the first method you can detect specific population of subcell. ves.-s, while with the other you can detect all of them. It also important to make a control of specificity of your stain, such as using detergent (e.g. Tween, Saponin, etc.), to eradicate the vesicles - in this case you wont see fluorescent marks. A third approach can be using intravesicule markers, as dr. Ganglum wrote.
By the and, let me recomend a good protocol oriented article: Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parametershttp://www.ncbi.nlm.nih.gov/pubmed/21041717
With best regs: Csaba Timar
Direct observation of these vesicles are tricky if they are below the resolution limit of your confocal microscope(generally~250 nm) but you can indirectly estimate the sizes of these vesicles from diffusion based methods like FCS or FRAP if you have access to a confocal microscope capable of making such measurements. Other option would be to try super-resolution imaging like STED etc.
Only a small addition to the answers already given: When you are going to try super-resolution like STORM/dSTORM, STED, SIM etc label density is very important. If you do not have enough Flurophores attached to you target you will not get good resolved structures. When you have a super-resolution microscope at you disposal, speak with the people running it for starters. Makes life easier for you and them.
We are working also on MVs IF staining. We wont to try with PKH approach on isolated MVs. It is necessary "to adhere" MVs onto slide or it is enough to put the drop onto slide?
- Badges
- Science method
- Similar topics
- Biological Science
- General Biochemistry
- Biochemical Techniques
- Cytology