Why Fluorescence is detected @right angle to the excitation?

In summary: Near the sample, the incident beam and the emitted beam pass along the same optical path but in different directions. The source and the detector cannot be in the same place so the two beams must be combined or separated at some point.This is an issue with fluorescence microscopy because the emitted light from the sample is often too bright for the detector. The emitted light can be reduced by using a half silvered mirror, but that also reduces the detected signal. A wavelength sensitive dichroic mirror is then used to effectively brighten the response.
  • #1
Shi
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Hi everybody,
I would like to know what would be the best angle (or best geometry) to put the detector relative to the light source in order to observe the emission radiation from the fluorescent materials?
As I know the most common geometry used for fluorescence is right angle observation. Detector is placed at 90 deg to incident light to minimize the risk of transmitted or reflected incident light reaching the detector (from wiki). Why?!
If we place detector at 45 deg or at smaller angle to the source, what would be happened?
 
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  • #2
Various materials tend to reflect under shallow angles, or directly back to the source. I don't know details about it in terms of fluorescence measurements, but that effect is well-known in astronomy.
 
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  • #3
Shi said:
Detector is placed at 90 deg to incident light to minimize the risk of transmitted or reflected incident light reaching the detector (from wiki). Why?!
Near the sample, the incident beam and the emitted beam pass along the same optical path but in different directions. The source and the detector cannot be in the same place so the two beams must be combined or separated at some point.

For the right angled illumination case, a half silvered mirror mounted at 45° would combine the beams. But the half silvered mirror would reduce the detected beam to 25%, since half the illumination is lost on the first pass, before half the emission is lost on the second pass. Since the two beams have different wavelengths, a wavelength sensitive dichroic mirror is used to effectively brighten the response for the same illumination source.

I believe that right angled illumination with a 45° mirror is used because it leads to simple compact engineering and avoids the Brewster angle. https://en.wikipedia.org/wiki/Brewster's_angle

mfb said:
Various materials tend to reflect under shallow angles, or directly back to the source. I don't know details about it in terms of fluorescence measurements, but that effect is well-known in astronomy.
The astronomical phase curve is best explained by the relative brightness of our moon in different phases. A full moon has more surface illuminated by the Sun that is also visible from Earth. The new moon returns little light because we cannot see much of the illuminated surface. The shape of the curve is determined to some extent by surface texture. That cannot be related to the florescence microscope situation.
 
  • #4
Baluncore said:
A full moon has more surface illuminated by the Sun that is also visible from Earth.
That is not the only effect, and it is not the effect I was discussing. The full moon is much brighter than you would expect based on illuminated area. The linked article explains those brightness excesses and opposition surges.
But I see that wavelength filters work fine to stop light from the initial light source, so this is not an issue here.
 

1. Why is fluorescence detected at a right angle to the excitation?

Fluorescence is detected at a right angle to the excitation because it allows for better separation of the fluorescence signal from the excitation light. This is because the excitation light, which is often much stronger than the fluorescence signal, can be easily filtered out at a right angle detection.

2. Can fluorescence be detected at any angle to the excitation?

Technically, fluorescence can be detected at any angle to the excitation, but detecting it at a right angle is the most common and efficient method. Other angles may be used for specific applications, but they may require more complex instrumentation and may not provide as clean of a signal as right angle detection.

3. How does right angle detection improve the sensitivity of fluorescence detection?

Right angle detection improves the sensitivity of fluorescence detection by minimizing the amount of excitation light that reaches the detector. This reduces background noise and allows for a more accurate measurement of the fluorescence signal.

4. Are there any disadvantages to using right angle detection for fluorescence?

One potential disadvantage of using right angle detection is that it may not be suitable for all samples. Some samples may have a low fluorescence signal or may emit fluorescence in a different direction than the excitation, making right angle detection less effective. In these cases, alternative detection methods may need to be used.

5. How is right angle detection used in different fluorescence techniques?

Right angle detection is commonly used in various fluorescence techniques, such as fluorescence spectroscopy and microscopy. In spectroscopy, right angle detection is used to measure the fluorescence emission spectrum, while in microscopy it allows for the visualization and localization of fluorescent molecules in a sample.

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