Comet Labs WN591 Manual de usuario descargar pdf (Pagina 8)

Help! There is a comet in my computer! 5

When a slide is illuminated with green light, DNA-bound ethidium bromide on the slide

emits orange light in all directions (Figures 2B, 2C). During observation of an object

through eyepieces of a microscope, we see only light that is collected by the objective. We

now have two problems: the emitted light is very weak and some of the strong excitation

green light is reflected from the slide. To prevent excitation light disturbing our

observation of fluorescence, we need to place an emission filter between the object and

the eyepieces. This filter only passes light with longer wavelengths than the excitation

light. The emission filter for ethidium bromide passes orange light and blocks green light

(Figures 1, 2B, 2C). The second problem is easier to solve: because the intensity of the

emitted orange light is very weak compared to the intensity of daylight, we have to observe

the slide in a dark room.

Both the excitation and the emission filter are usually mounted together in a cubical filter

holder, which is typically located in a filter wheel just above the objective revolver. Each

fluorophore requires different combinations of excitation and emission filters to match its

specific absorption and emission properties.

Let us return to our task to measure the amount of DNA on the slide. We already know that

the relationship between the amount of DNA and the amount of ethidium bromide bound

to DNA is linear. The good news is that also the relationship between the amount of

DNA-bound ethidium bromide and the intensity of emitted light (the number of

emitted photons) is linear. We can thus conclude that the relationship between the

amount of DNA and the intensity of emitted orange light is linear. So all we need to do

now is to somehow measure the amount of emitted light, and we will know how much

DNA there is on the slide. This is where we need to start talking about images.

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Comet Labs WN591 Manual de usuario Pagina 8