Detecting fluorescence via CW or pulsed LEDs

In summary, the conversation discusses the building of a rudimentary fluorometer for measuring the fluorescence of proteins. They mention using a highpower LED to excite the protein and a phototransistor to measure the fluorescence, with a live time of a few nano seconds. They also mention the use of circuitries for this type of measurement and the difficulty of this task. In addition, they ask about the effectiveness of optical filters and the time constant for fluorescence. They also mention the differences between fluorescence and phosphorescence.
  • #1
Anton Alice
68
1
Hello forums,

in the context of a little DIY-project I am planning to build a rudimentary fluorometer, which is actually doing nothing but checking whether an amount of protein is fluorescing or not, and roughly measuring its intensity.

A highpower LED is used to excite the protein to the fluorescent state, while a phototransistor (specially sensitive to the fluorescent spectrum) starts measuring a short time after the LED has been turned off.
The live time of fluorescence is in the region of a few nano seconds. For example a nanosecond pulsed measurement should (tightly) be able to solve the problem. Do you know circuitries which can handle this measurement cycle?
 
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  • #2
I know its difficult.

For the continuous approach:
How well can optical filters block light? What is the best possible attenuation (below 200dollars)?
 
  • #3
Anton Alice said:
The live time of fluorescence is in the region of a few nano seconds.
Why is the time constant so short? What is the fluorescing material?
 
  • #4
One can distinguish between fast and slow decaying luminscence. The fastest kind of luminescence is fluorescence. There is also phosphorescence, which is more of an "exite, and wait until it decays", whereas fluorescence is more of a direct conversion of one wavelength to another.
http://nic.ucsf.edu/FPvisualization/#ref30
http://nic.ucsf.edu/FPvisualization/#ref30

Fluorescence is always in the nanosecond region.
 
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1. What is the difference between CW and pulsed LEDs for detecting fluorescence?

The main difference between CW and pulsed LEDs is the way they emit light. CW LEDs emit a continuous stream of light, while pulsed LEDs emit short bursts of light at regular intervals. This difference affects how fluorescence is detected and measured.

2. Which type of LED is better for detecting fluorescence?

It depends on the specific application. CW LEDs are better for measuring steady-state fluorescence, while pulsed LEDs are better for time-resolved measurements. Pulsed LEDs also have the advantage of being able to excite multiple fluorophores at different wavelengths simultaneously.

3. How does the excitation wavelength of the LED affect fluorescence detection?

The excitation wavelength of the LED is crucial for detecting fluorescence. Different fluorophores have different excitation spectra, so the LED must emit at the specific wavelength that will excite the fluorophore of interest. If the excitation wavelength is too high or too low, the fluorophore will not be excited and fluorescence will not be detected.

4. Are there any limitations to using LEDs for fluorescence detection?

While LEDs have many advantages for fluorescence detection, they do have some limitations. LED emission spectra can have peaks and valleys, which can lead to uneven excitation of fluorophores. Additionally, LEDs may not have enough power to excite certain fluorophores, requiring the use of more powerful light sources like lasers.

5. How is fluorescence intensity measured using CW or pulsed LEDs?

The fluorescence intensity is measured using a photodetector, which converts the light emitted by the fluorophore into an electrical signal. With CW LEDs, the photodetector measures the steady-state fluorescence intensity. With pulsed LEDs, the photodetector measures the fluorescence intensity at each pulse and then calculates the average intensity over time.

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