My compound can't be excited using the shortest available wavelength (448 nm). Its absorption spectrum is within 300-360 nm. So, i appreciate if you could advise how to resolve this problem either by using FLIM misroscope or switching to another technique (hopefully it will be available in our labs).
Thanks in advance
Another alternative is to use frequency domain FLIM, because there are some LEDs and modulators available with 365 or 385 nm wavelength: it's not so expensive. If you desire to switch technique there are also LEDs with 310, 340, 360 nm wavelength, but it depends on what you want to see.
Thanks so much!
My question now is, those LED and laser diodes don't affect the vitality of cells??!!! I know UV light kill cells. Or i can perform cell imaging before cell death. My goal is to investigate the distribution of a drug with HeLa cancer cells.
Of course it will affect your cells. The general rule is; lower wavelength in excitation, higher energy pumped in them and subsequently higher cell demage. The question is, what do you really want to see? Are you interested in the lifetime information or just the fluorescence intensity distribution in the cell? If you were not interested in the lifetime then I would rather use wide-field (WF) microscope instead of confocal, since the cell demage is lower in the case of WF. Otherwise you should use the excitation intensity on the lower limit and maybe use phasor approach for lifetime analysis. It is more sensitive and informative for the lifetime data with low signal to noise ratio.
Yes. because i use broad band width (wide field).
Thank you all. I think the next step is to ask for a quote for 365 nm LED hoping it's not too expensive.
The problem of the cell viability is a difficult one, since the UV light is highly toxic to the cells. What you have to take in account is that, even if the cell doesn't die, so you don't see a major toxicity, the phototoxicity may bias the distribution of the drug you want to study. If you can switch to longer wavelengths, the longer the better, if you can't, as the other colleagues said, be as fast as possible, and maybe do some viability controls on your cells.
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