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Hi-speed flash fluorescence

 
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Linden.g



Joined: 06 Apr 2009
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Location: PA USA

PostPosted: Sun Mar 22, 2020 9:01 am    Post subject: Hi-speed flash fluorescence Reply with quote

I was curious to see if I could deliver enough UV emission from a standard xenon flash tube to take short duration fluorescence images of motile algae. I converted a mercury lamp source on a BH2-RFCA fluorescence set-up to a xenon flash. I tested the UV output with a B 490 excitation cube to see auto-fluorescence in the highly motile green unicellular algae Tetraselmis. The first image was taken at an iso setting of 500 and exposure duration of 0.6 seconds. As you can see the algae motion was easily seen as blurred trails. The second image was taken at a full power setting on the Yongnuo YN560 iii which is about 3 milliseconds. You can see the flash was able to stop the motion but with the cost of having to use an iso setting of 6500. The bottom line is that this conversions allows short duration fluorescence imaging at the cost of requiring high iso settings with corresponding signal noise. If I've done my calculations correctly, this comparison shows that the xenon flash is about 6 times brighter than the mercury vapor bulb.






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rjlittlefield
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PostPosted: Sun Mar 22, 2020 9:23 am    Post subject: Reply with quote

Nice work!

In this particular case, I find that I prefer the continuous exposure because the stationary individuals give me the structure while the trails give me trajectories and some indication of what fraction are moving.

But surely for smaller populations or more consistently moving organisms, the flash would be great.

--Rik
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Pau
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Joined: 20 Jan 2010
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PostPosted: Sun Mar 22, 2020 9:38 am    Post subject: Reply with quote

Interesting test!
Quote:
I tested the UV output with a B 490 excitation cube

Do you know the filters in that cube? The B490 naming seems more a Blue cube.

Red chlorophyll emission can be excited with almost any wavelength from near UV to green, being the most intense absorption peaks in the blue spectrum. Perhaps, depending of the filters you have, you could profit it with a cube with wide shortpass spectrum and red emission to gather more energy for excitation.

Another factor is the small size of the actual arch of the HBO lamp the lamphouse optics are designed for, leading to profit only a very small zone of the flash tube. If you can exchange this optics with the ones for a halogen lamp you could improve quite a bit.

Likely I would test the idea in the near future, not only for freezing the movement but also to minimize photobleaching...although of course this only will be practical if ISO can be lowered.
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Last edited by Pau on Sun Mar 22, 2020 9:59 am; edited 1 time in total
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Linden.g



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PostPosted: Sun Mar 22, 2020 9:55 am    Post subject: Reply with quote

Hi Pau, I'm really new to fluorescence microscopy so these are my first tests. So I really appreciate the advice. The cube is called "B" excitation and has a BP490 filter on the lamp side. You can see the graphs in here http://www.alanwood.net/downloads/olympus-bh-2-brochure.pdf on page 16. The excitation is intense blue and the emission filter on the viewing path is yellow but doesn't have markings. I have 3 other cubes, G+BP545, IB+BP495
U+UG-1 . This one gave me the brightest red with a black background. What cube do you recommend for auto-fluorescence?

All the best

Linden
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Pau
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PostPosted: Sun Mar 22, 2020 10:30 am    Post subject: Reply with quote

Linden.g wrote:
The cube is called "B" excitation and has a BP490 filter on the lamp side.
...
I have 3 other cubes, G+BP545, IB+BP495
U+UG-1 . This one gave me the brightest red with a black background.

Thanks for linking the info on your cubes
All your cubes have bandpass (BP) excitation filters. For maximum chlorophyll excitation a short pass or wide bandpass old style Ex. filter like the Schott BG3 could be better (just could, I've not tested the idea). From the ones you have the blue B cube seems the more convenient as you already have discovered.

The U cube is for UV excitation, the G cube is for green excitation and the IB cube for blue and cyan (or blue-green) excitation.

Quote:
What cube do you recommend for auto-fluorescence?

This all depends of the nature of your subject (and of your goals).
The U (UV) cube will produce the more colorful images as it can allow to gather all or almost all colors of the visible spectrum IF the sample has molecules emitting in different wavelengths.
With the B cube you can gather from yellow to red while with the G you only can get red
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Linden.g



Joined: 06 Apr 2009
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PostPosted: Sun Mar 22, 2020 11:53 am    Post subject: Reply with quote

Thanks Pau, thats very helpful
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