There are several booklets that you can look at. But the light bulb and the combinations of, excitations filer with "blocking" filters are most important. Also, you must have a reference slide to check the calibrations with.

Good luck!

FITC is very sensitive to pH changes and also to temperature. You could check this link:

http://tools.lifetechnologies.com/content/sfs/manuals/mp36915.pdf, page 2, Fig. 2 shows The pH-dependent absorption and fluorescence emission spectra of FITC.

Apart from the obvious (using proper filters, exposure time, clean objectives, etc.), you could use multicolor fluorescent microspheres (e.g. TetraSpeck) or dye solution of known fluorophore to check if with proper excitation you will be able to observe through eyepieces or with the camera the corresponding emission.

Thank you very much for your replies, they have given me some insight into this fascinating technique.

So from my understanding a fluorophore gets excited by a particular wavelength and emits a different, lower wavelength fluorescent light, which is then captured by a CCD or viewed in the eye piece. In the case of fluorescein diacetate (FD) cell esterase activity degrades it to fluorescein, which is the compound with the fluorescence. In this way whether the cell is alive or death can be assessed. But the excitation difference between these two seems to be not very high. FD is excited by 490 nm while propidium iodide by 535 nm. And, in my images through the eyepiece sometimes I can see both emissions simultaneously using the same filter, both green from the FD (emission at 526) and orange from the PI (617 nm); what I was assuming is that one filter excite only certain wavelengths and receives light from certain wavelengths. In other words, with one filter I would excite only one of the fluorophores and see its emitted response.

The attached image, for example, is a FD-PI staining taken through the eye piece with the cellphone. The filter used has excitation BP filter between 450 - 490 nm. Shouldn't only the green part be visible with one filter and the orange/reddish part with the other?

Now, for a image to be created the microscope available has a black and white CCD, which I guess is then corrected by using the false color features and try to match the image we create with what we see through the eye piece. The software (Leica Application Suite) allows me to put a false color to one or more filters, take a picture and then create a nice overlay with the images for publication, which shows what has a response to what. However since in a single filter I can see two different colors, the software does not recognize this because it paints everything in a single false color. Further on, the image you take is highly dependent on the settings for brightness, gamma and gain. How do researchers manage this parameters when taking a picture, is there a scientific way to select the best parameters or just moving the sliders until one is satisfied with the result?

Hello Luis,

Would you please give me a bit more info: the propidium iodide cat. number and the company; what microscope are you using (wide-field or confocal, company); do you have any idea what are the characteristics of the filters (excitation and emission) you are using to detect FITC and PI.

Sure, thank you Jana for your help. The PI is from Fluka, 81845. We are using a stock of 1 mg in 50 mL of artificial sea water (our sample is also resuspended in ASW), to which we use 30 uL (0.6 ug) in 2 uL of sample. Then, together with the FD incubate 3 minutes room temp in darkness, then ice until visualized on microscope.

The microscope is a confocal Leica DMR and the filter (filter cube I3) excitation band pass filter is 450-490 nm.

Hello Luis,

The I3 filter set you are using have following characteristics (if it is a standard filter set, not modified one):

Emission: BP 450-490; Excitation: LP 515 (which means it will transmit all the light after 515 nm).

I also plotted the FITC and PI spectra and corresponding emission and excitation filters spectral ranges (see below).

It is clear that with 450-490 Ex. filter, you excite simultaneously both FITC and PI and with the corresponding Em. filter (LP 515) you could register both emissions as well. In case you are using multiple flourophores in one sample it is important to consider not only their max excitation and emission wavelengths, but also the whole spectra in order to avoid bleedthrough.

In order to separate FITC from PI, I would suggest using two different filtersets:

1) for FITC - Filter cube L5, excitation filter: BP 480/40, dichromatic mirror: 505, suppression filter 527/30

2) for PI - Filter Cube TX2, excitation filter: BP 560/40, dichromatic mirror: 595, suppression filter 645/75

To estimate if you have bleedthrough, you could prepare two samples, one stained only with FITC and the other – only with PI.

Image the FITC sample with Filter cube L5, and then take another image with Filter Cube TX2 with the same imaging conditions you used for L5 filter cube. In the image, taken with TX2, specific fluorescent signal should not be detected.

Repeat the procedure with the sample, stained only with PI: image fist with Filter Cube TX2 and then take an image with Filter cube L5. In the image, taken with L5, specific fluorescent signal should not be detected.

You could also measure only artificial sea water (without a sample) to verify with both filter cubes if you have background fluorescence from it.

Good luck