Introducción to a Laborlux D epifluorescencia.

[Sabatini]

Kind regards to all.
As we all know in this beautiful world of microscopes, our mind is always searching for better optical quality and this keeps us in the search and expectation of finding and obtaining better optics.
Now in this search, I managed to buy two nice microscopes a laborlux S and a laborlux D... both in excellent condition and with a variety of objectives among which there are several npl fluoters and 4 phase contrast objectives. I am very happy with this purchase. Among the things that came with the equipment is an epifluorescence system, a technique completely unknown to me. I have tried to get some image through it, but I can't get any image at all, it's all completely dark, reading here in several publications of this grest community, I see that there is possibility of modifications with leds, I bought a 100 watt led but I can't see anything either I am using a npl fluotar 2.5 NA 0.08 to start and tried with samples of plants and leaves...what recommendations can you offer me to get this to work...and what specimen would be easy to handle and within reach to avoid the need to use fluorophore dyes that are not available here. I was reading that some plants facilitate the possibility of using this technique.

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[Hobbyst46]

1. Choose an appropriate specimen. Assuming that your illumination wavelength (excitation, EX) is violet blue (400-430nm). Then a green leaf will emit red fluorescence (FL), wavelength 610-660nm or so. I would start with green algae - fibrous algae, or moss. A thin amount in water on a slide.
2. The EX beam must be intense. A 100W LED sounds good - if it is the correct wavelength (do tell us what color is its light). For best results it should be optimized by Kohler's method.
3. If the EX lamp is LED, you might do without EX filter. Just a dichroic mirror (cutoff wavelength ~500nm) and EM filter (long pass filter, cutoff say 500-550nm). Correctly positioned in a "filter cube " above the objective turret.
4. The objective should have a high NA - preferably, at least 0.75. The 2.5X is unlikely to gather sufficient FL light.
5. Brightfield, using the 2.5X, 10X etc can be used to locate the region of interest. Then turn off BF, switch to the appropriate objective and turn on the EX lamp . Turn off all ambient (room) lights if red FL is not visible.
6. Note, that FL is weaker (less bright) by orders of magnitude than EX.
7. The human eye is pretty sensitive to low-level FL light - sometimes more so than a camera.
8. Do not expose your eyes to direct EX light, for safety reasons. Never ever.
9. In general, white light is unsuitable as EX light. FL occurs by very specific EX wavelengths. If the EX light source is a white light LED, an EX filter must be added to the cube. Say, a shortpass or bandpass filter. Per the example above, it should transmit violet/blue light.
10. If there is no green leaf/alga/moss at hand, the specimen can be a piece of white tissue paper, saturated with virgin olive oil. Will show weak red FL.

Other specimens may require a different EX light and different filters.

Specimens mounted in various liquids and resins might give even weaker FL, because some EX light is absorbed by the mounting medium.

For most hobby microscopy, any objective can be used, provided its NA is high.

[Microscopy_is_fun]

I bought a 100 watt led but I can't see anything either I am using a npl fluotar 2.5 NA 0.08 to start and tried with samples of plants and leaves...what recommendations can you offer me to get this to work.

You need to take into account one of the basic laws of optics: If you have a light source and project it onto a target (in your case the slide with the object) you cannot increase the light intensity (meaning W/mm²) at the object beyond the light intensity of the source. Otherwise you would have a perpetuum mobile of second order.

The size of the observed object in your case is around 2x2mm², the size of your light source around 100x100mm². This means that you loose pretty much all your light since you cannot focus the large LED-array onto a small exposed area. You should rather look for a single LED with high power and take care of proper collimation.

[Hobbyst46]

Incidentally : Do the posted photos show that the light from the 100W LED comes from below the light port below the condenser ? if so, the setup is not epi-fluorescence but trans-illumination. If so, the chance of seeing any FL in the shown setup is zero, whatever the objective in use, whatever the specimen, whatever the wavelengths.

[Sabatini]

Incidentally : Do the posted photos show that the light from the 100W LED comes from below the light port below the condenser ? if so, the setup is not epi-fluorescence but trans-illumination. If so, the chance of seeing any FL in the shown setup is zero, whatever the objective in use, whatever the specimen, whatever the wavelengths.

Thank you very much for your detailed recommendations. So I will be experimenting with different types of leds until I find the right one. What do you think of the full spectrum used for indoor harvesting...I also have possibilities of different wavelength leds but I find them only 1w...will they work?...The idea is to have fun experimenting and solve the obstacles...I read this article and that's why I used the NPL 2.5 na 0.08.

http://www.microscopy-uk.org.uk/mag/ind ... luor2.html

I now update an image as I am actually using it.
Algaes. .I am leaving right now for the beach to harvest it in all shapes and colors.

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Yes the led 100w is white.

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[Alexander]

The 2.5/0.08 is a central part of your problem. This objective is totally useless for fluorescence. Use your Fluotars. They are much better suited to the job.

What filter cubes do you have and which one did you use?

btw: Nice microscope! This thing will give you a lot of pleasure and reward.

[Sabatini]

The 2.5/0.08 is a central part of your problem. This objective is totally useless for fluorescence. Use your Fluotars. They are much better suited to the job.

What filter cubes do you have and which one did you use?

btw: Nice microscope! This thing will give you a lot of pleasure and reward.

Thank you very much for your kind words and recommendation, I am sending you a picture of the cube I am using, I already got some algae and I will also use another objetives and do some tests and try some configuration with different LEDs.

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[Alexander]

The I2/3 is for blue light excitation in a band of 450 - 490 nm. Very good for specific fluorescence stains but somewhat limited on chlorophyl auto-fluorescence. A blue LED in the 460 - 470 nm range would be best for this filter cube. Buy some acridine orange. It is the perfect stain for this filter setup.

[Sabatini]

The I2/3 is for blue light excitation in a band of 450 - 490 nm. Very good for specific fluorescence stains but somewhat limited on chlorophyl auto-fluorescence. A blue LED in the 460 - 470 nm range would be best for this filter cube. Buy some acridine orange. It is the perfect stain for this filter setup.

Great advice!... appreciate your knowledge in this particular topic.
Of course I will be trying to get the necessary elements that you suggest. the idea is to learn with this new opportunity that this equipment gives me and to overcome its obstacles.

[Hobbyst46]

The link to David Walker's article is very relevant. His posted photos are very nice. Note, though, that the intensity of the fluorescence is proportional to the intensity of the excitation. Walker's excitation source was a 100W mercury lamp. Such lamp provides very intense light at specific wavelengths, and relatively weak light elsewhere. Mercury light spectra are well documented on the web. I was thinking of more humble lamps, say a 3W LED or a 60-100W halogen lamp. Unless a LED, the EX source needs a selective filter to provide the correct narrow-gap excitation light.
Since fluorescence is very sensitive to the NA of the objective (because the objective serves a double duty, as condenser in the epi-illumination), I suggested a high NA objective. Also, the samples I had in mind (who knows why...) where small cells and creatures that favor 20-40x objectives.

Under the appropriate conditions, low-mag objectives yield fluorescence, of course. For example, a stereo microscope with top (slanted) excitation; or even without a microscope at all. Take a small sample of virgin olive oil in a vial, hold it against a small UV lamp or "atmosphere" "black light" tube, turn off ambient light, and red fluorescence is clearly visible.

A couple of years ago, a forum member here or on Macrophotography presented a DIY fluorescence accessory based on 3W LEDs.

In accordance with above comments, the optimal wavelengths for green plant auto-fluorescence are 400nm or 425nm.