The DHYology: Stem Cell Concepts: immunofluorescence (IF)

I've been quite lazy with these posts, and now there's a lot of pictures and ideas just tucked away somewhere and in my computer. I have 21days before i go back to Cornell, lets see how many i get up before i go :D

The concept i will introduce today is Immunofluorescence (IF).

Basically, IF is a labeling or tagging of specific sub cellular structures with fluorescence.
It is called "immuno-" fluorescence because it tags antibodies and antigens which is part of the immune system. I am not too knowledgeable about this, though, so i don't think i should try to explain how IF works.
I do know, however, why IF is used.

The reason why IF was developed, and all the other methods to 'tag' molecules, is very much related to the properties of light microscopy. Well, more like limitations of light microscopy. It is a general knowledge that cells or sub cellular under microscope do not have much color. The structures do not have much color to begin with and since the cells are often so thin they do not retain much color. To overcome this, many scientists have devised some methods to stain the cells so that it would be easier to make out the structures. 'Tagging' can be understood as more advanced form of staining. Researchers can now 'tag' specific types of structures with different type of tags to help that study their specimens. One of the shortcomings of staining was that it often requires the cells to be dead and treated, but tagging can be done to living cells, which allows the tracking of specific molecules or structures within the cells. This is rather significant because one could expose cells to different conditions and follow the tagged structures within the cells.

btw my explanations are rather amateur, and i apologize for that but stay with me, i still have some more to say :D

Another advantage to tagging is the specificity of the tagging, which is generally much more than what staining to offer. If a stain could stain an entire cell blue, tagging can tag just the DNA in the cell. (There might be a stain that could stain just the DNA, but i got my point across right?) And probably more significant than the other advantage is that the tagging can be read by computer very efficiently, while stained objects can not be read so efficiently. Tagging is done often with fluorescence, but this fluorescence cannot be seen by normally, it has to be first excited by laser than go through a filter before it could be seen. This information could be read by a sensor or a receiver which could be stored as a data of only the tagged molecules. Whilst if this was attempted with a normal stain on light microscopy would be hugely inefficient. Also staining is often only done once, but tagging can be done multiple times. If the staining is done multiple times the color of the stained area might change, but in tagging it won't happen because each fluorescence has to go through a different filter before it could be seen.

I'll just show you what i mean
these are pictures of Hela cells that have been tagged with DAPI and FITC. The second picture is the cell seen through the FITC filter and the third picture is seen through the DAPI filter.
The first picture is the composite of both pictures done on computer.

As you can see. DAPI stains the nucleic area while FITC stains the entire cell apart from the nucleic area. If you look closely, the area where neucleous would be in the second picture is a little less green.