Fluorescent light theory for science and photography

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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axiverse
Posts: 8
Joined: Tue Jun 28, 2022 10:07 pm

Fluorescent light theory for science and photography

#1 Post by axiverse » Tue Jul 19, 2022 3:52 am

I've been learning about fluorescence for microscopy and have open questions about the science behind it. I'm doing my research primarily in the context of the BH2-RFC setup. For the BH2-RFCA attachment, there are a number of cubes which contain a exciter filter (band pass), dichroic mirror (acts like a long pass mirror I believe), and a barrier filter (long pass). There are a number of cubes available with different configurations of these elements available.
Understanding
- Fluorescent materials fluoresce if the incident light is of higher energy (shorter wavelength) than the fluorescent color by a certain amount.
- Fluorescent materials must be able to absorb the incident light in order to use it to fluoresce.
- We use band pass filter in order to only excite certain fluorescent materials while leaving others unaffected.
- Ideally the barrier filter is as close to the exciter light wavelength while maintaining low overlap to be able to capture fluorescence close to the incident light wavelengths.

Questions
- Is fluorescence microscopy useful for natural observation or is it primarily used with fluorescent dyes for science/medical use?
- Do materials fluoresce at any wavelength or are there a limited number of wavelengths that materials commonly fluoresce at?
- How common is it for microorganisms to fluoresce at all?
- How common is it for microorganisms to fluoresce differently in incident light wavelengths that are close together (i.e. will they fluoresce differently at 405nm light vs 430nm light)?

Also given the number of narrow wavelength LEDs now available, it is theoretically possible to make a carousel to emit light at a number of specific wavelengths without the use of an exciter filter (spaced about 20nm or less apart). I'm wondering if such a mechanism would be useful in seeing different things or creating interesting photographs or videos.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Fluorescent light theory for science and photography

#2 Post by Hobbyst46 » Tue Jul 19, 2022 5:43 am

axiverse wrote:
Tue Jul 19, 2022 3:52 am
Understanding
- Fluorescent materials fluoresce if the incident light is of higher energy (shorter wavelength) than the fluorescent color by a certain amount.
correct, although I would phrase it differently. When fluorescence occurs (not all materials, that absorb light, fluoresce, because there are competing processes that waste the energy of the excited state!), the emmision is at a higher wavelength (lower energy) than the excitation.
- Fluorescent materials must be able to absorb the incident light in order to use it to fluoresce.
correct
- We use band pass filter in order to only excite certain fluorescent materials while leaving others unaffected.
correct
- Ideally the barrier filter is as close to the exciter light wavelength while maintaining low overlap to be able to capture fluorescence close to the incident light wavelengths.
The first part of the sentence is incorrect. An ideal situation originates from the excitation and emission wavelengths, the filters should just match these. Ideally, the gap between the excitation and emmision peaks should be large (say, tens of nm at least), because the typical excitation and emmision peaks are fairly broad (look at popular respective spectra on the web).

The underlined section of the sentence above is correct.

Questions
- Is fluorescence microscopy useful for natural observation or is it primarily used with fluorescent dyes for science/medical use?
[/quote]It is useful for anything that fluoresces upon excitation. However, because of ongoing research and development to produce very specific dyes for science/medical use (as you say), the selection of such dyes is huge.
- Do materials fluoresce at any wavelength or are there a limited number of wavelengths that materials commonly fluoresce at?
In theory materials could fluoresce at any wavelength in the UV, visible and near infra red range. Again, the point is, that peaks are not narrow, so there is often overlap between peaks of different materials.
...
- How common is it for microorganisms to fluoresce differently in incident light wavelengths that are close together (i.e. will they fluoresce differently at 405nm light vs 430nm light)?
As an example of peak width: Chlorophyls (plant pigments) fluoresce when being excited at the 400-430nm range.

Also given the number of narrow wavelength LEDs now available, it is theoretically possible to make a carousel to emit light at a number of specific wavelengths without the use of an exciter filter (spaced about 20nm or less apart). I'm wondering if such a mechanism would be useful in seeing different things or creating interesting photographs or videos.
[/quote]Yes, and in addition, there are commercial multi-wavelength LED lamps for fluorescence microscopy.

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