Week 14- Confocal Microscopy!

Hey..this week’s my turn, and I have interesting PICS to share from my research work.

As I have mentioned before, my project is basically delivering DNAs into cancer cells using synthetic carriers such as polymers for future gene therapies. Hence, I’m doing the last part of my project which is ‘localisation of polymer-DNA complexes’ in the cells.

For the localization studies, my main aim is to determine whether my polymer goes into the nucleus. Hence I have to label my polymer, nucleus and DNA. However, since my DNA labeling kit has not arrived, and I want to train myself using the confocal microscope, I started my experiment first using my labeled polymer and nuclear labeling.

The cell line I chose is HepG2, it is a human hepatocellular carcinoma cells, mainly because it does not detached easily from the tissue culture plate. I label my polymer with FITC, and to stain the nuclei, I used Hoechst dye that is specially used to stain ‘live’ cells.

Why use CLSM?

In order to view my cells, Confocal laser scanning microscopy(CLSM) is used. The CLSM used is LSM 5 LIVE, the latest edition for imaging of living cells from ZEISS technology. CLSM uses laser to excite the fluorochromes, unlike fluorescence microscope(FM) which uses fluorescence light.

1)CLSM allows 3D view of the sample, hence if there is an overlapping of different fluorescent signals, only CLSM can help differenciate them.

2) CLSM allows viewing of distinct and sharp images by removing out-of-focus light, unlike FM that usually gives blurry images and some signals cannot be viewed distinctly.

If you wanna know the principle of CLSM and FM, just give me a comment, as it is more like ‘physics’, afraid some of you might not understand.

Ok back to my experiment, I prepared 4 different samples at different time interval: 1, 2, 3 and 4.5 hr, these time intervals are chosen after reading some science papers and also FITC ‘photobleaches’ easily, hence I have to prepare samples at different time intervals. The polymer-DNA complexes are given(transfected) to the cells at 0 hr, while the nuclei are stained with Hoechst 40 mins before viewing under CLSM, and the cells are washed 5 times with colourless DMEM medium before viewing to prevent auto-fluorescence. Hence, that is why i used HepG2, because of the washing steps.:)

One important precaution: sample preparations must be done in the dark, once exposed to light, the fluoresce intensity can greatly decrease, especially for FITC.

GREEN(FITC): polymer ( note: DNA is still bound to the polymer, but cannot be seen as it is not labeled)

BLUE(HOECHST): nucleus

The following pictures are a bit blurry because I magnify them 8-12 times from the original one.

1 hr after Transfection: on the cell membrane

Q: Why do the polymers 'stick' onto the membrane?

A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!

2.5 hr after Transfection: in the cytoplasm



Q: How does my polymer enters the cytoplasm?

A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.

4.5 hr after transfection: in the nucleus!!



Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?

A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.

Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :(
But still, research is FUN! :))))

Nisha
TGo2
0503254E