Detecting fluorescence via CW or pulsed LEDs

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Discussion Overview

The discussion revolves around the design and functionality of a rudimentary fluorometer intended for detecting fluorescence in proteins. Participants explore the use of continuous wave (CW) versus pulsed LED excitation and the associated measurement techniques for fluorescence intensity.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant describes a DIY project involving a fluorometer that uses a high-power LED to excite proteins and a phototransistor to measure fluorescence intensity after the LED is turned off.
  • Another participant questions the effectiveness of optical filters in blocking light and seeks information on the best possible attenuation within a budget of $200.
  • A participant inquires about the short time constant of fluorescence, asking for clarification on the fluorescing material involved.
  • One participant distinguishes between fluorescence and phosphorescence, noting that fluorescence typically occurs in the nanosecond range and providing a reference link for further information.

Areas of Agreement / Disagreement

Participants express varying levels of understanding regarding the mechanisms of fluorescence and the technical challenges of measurement, indicating that multiple competing views remain on the best approaches and materials for the project.

Contextual Notes

There are unresolved questions regarding the specific fluorescing materials and the effectiveness of different measurement techniques, as well as the limitations of optical filters in practical applications.

Anton Alice
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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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I know its difficult.

For the continuous approach:
How well can optical filters block light? What is the best possible attenuation (below 200dollars)?
 
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?
 
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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