# fluorescent color and wavelength - which wavelength can my eyes see? emission/excitation wavelength (Jul/12/2007 )

Hi folks,

I dont know exactly the relationship between fluorescent color of the fluorochrome tagged in secondary antibody and its wavelength. If you see the table, TRITC for example, it excites at 547nm (green) and emits at 572nm (yellow?). I dont know exactly what color I can see under the microscope. Please help me!

-cancerous-

QUOTE (cancerous @ Jul 13 2007, 11:59 AM)
Hi folks,

I dont know exactly the relationship between fluorescent color of the fluorochrome tagged in secondary antibody and its wavelength. If you see the table, TRITC for example, it excites at 547nm (green) and emits at 572nm (yellow?). I dont know exactly what color I can see under the microscope. Please help me!

Excitation is the wavelength for light that excites or stimulates your dye. While emission is the wavelength of light that your dye emits after being excited, which you will see (if it is in the visible range) and the camera or confocal will collect to give pictures.

Thus, for example TRITC, it is excited at 547nm (green), so that the light the microscope will emit (if you look at the light that shines from the objective lenses) to shine through your sample will be about this range (depends on the filter of the microscope). But the emission wavelenghth is 547, that is what you will see if you put your eyes at the eyepiece lenses. Note that the emission wavelength is always longer that the excitation wavelength, because the longer the wavelength is the less energy it has. Your excitation light transfer its energy to the fluorochrome to excite it, bring it to a higher energy level. After that, the fluorochrome returns to its normal level of energy, thus releases (emits) the transfered energy in light. However, energy is lost as heat, thus the emission light is always of less energy than the excitation light and has longer wavelength.

The excitation light is important for you to set the filters at the light source, which will choose the light to shine through your sample. The emission light is important for you to choose the filter that will collect the light after your sample, which you will see, or the machine will collect.
UV, laser ---- (filter) -----> SAMPLE ---- (filter)----> eyepiece lenses, camera...
...........excitation.......................................emission

-Almasy-

or simplified
think of exitation as tickleing and emmision as the laughter produced
also dont forget that a lot of camera's on confocals will be producing a monochrome image and you then simply allocate the colour of your choice to that image

dom

-Dominic-