In order for a fluorochrome to be excited, it is necessary that the electrons receive enough energy to be able to level up.
Classic fluorescence light source is HBO Mercury bulb.
New sources have been developed starting from the LEDs normally used in microscopes.
However, it is necessary to clarify the main differences between the two light sources currently used in fluorescence: HBO and LED bulbs.
First of all, let's look at the emission spectra of an HBO and an LED bulb
LED has a maximum energy at 450 nm (blue region), but starting from 480 nm it has an excitation energy of about 50%.
The excitation energy around 350 nm is 0.
An HBO bulb has a lower excitation energy around 450 nm (about 80%) but has good energy peaks at 350 nm (90%) and 520 nm (about 60%).
Another important aspect is the average life time of the two sources: LED has a lifetime of around 50.000 hours, while HBO has 200 hours of lifetime.
After 200 hours HBO bulb must be replaced.
LED bulb is pre-centered in the light path
HBO must be re-centered every time a new bulb is installed in order to achieve the maximum performances.
More: LED can be turned on and off several times without affecting the lifetime.
When using HBO, user must wait 5-10 minutes before the bulb properly warms up and, once the bulb has been switched off, must wait at least 15 minutes before switch on again. This is because we must allow the bulb to cool down.

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