I am truly a beginner, looking at a Leitz Orthoplan and an Olympus BX51 microscopes and trying to use them for the detection of microplastics in environmental (sediment) samples. Please help. Thanks in advance!
Here is a silly question - For the Leitz Orthoplan, I put lamp housing at the upper position in the back. I see the light beam but it seems "stop" in the middle inside, and no light reaches to the specimen. Is there any switch to turn on?
Hello from a beginner
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gastrotrichman
- Posts: 54
- Joined: Mon Nov 12, 2018 4:04 pm
- Location: Oregon, USA
Re: Hello from a beginner
The upper light path is for observing objects with reflected light. The light reaches the object through the objective. If you don't have objectives made for this type of illumination (known variously as epi-illumination, incident light illumination, reflected light illumination, etc.), the illumination light cannot reach the object. I think Leitz reflected light objectives were all for an infinity tube length and are marked with the infinity symbol "∞". Also, some reflected light microscopes have a solid stage and no substage condenser. Leitz transmitted light objectives were for 170 or 160mm tube length, and are marked "170" or "160." Check the markings on your objectives. If objective markings don't resolve the question for you, and your Orthoplan has a substage condenser and an opening in the center of the stage to allow light from the lower light path to reach the object, try moving the lamp to the lower light path. If you get good images with the lower light source, but not with the upper light source, you probably have transmitted light objectives. If you have reflected light objectives, remove the upper light source and peer down the upper light path to detect possible obstructions or other conditions that might prevent the light path from reaching the objectives.
My suspicion is that you have transmitted light objectives.
My suspicion is that you have transmitted light objectives.
gastrotrichman
Nikon Microphot
Leitz Orthoplan
Wild M8
Bausch & Lomb MicroZoom
Nikon Microphot
Leitz Orthoplan
Wild M8
Bausch & Lomb MicroZoom
Re: Hello from a beginner
I'm guessing that your best results will be with transmitted light (brightfield) and polarization. Many transparent or semi-transparent plastics will show up with strain colors under polarized light.
I'd think you'll want to collect samples and filter them for size (calibrated mesh screens). The big stuff will be caught on the screen -- and you'd likely want a stereo microscope to inspect it.
For the small stuff (gets through a fine mesh screen) - then to a compound microscope. Could be reflected light illumination is what you want; but for things like plastic fibers coming off in a clothes dryer, the transmitted light and polarizer works best. Around 40x objective (400x total with a 10x eyepiece) you should be able to pretty clearly see the difference between organic material (cells) and plastics.
As for your first attempt with the "epi" illumination on your Leitz -- there are also irises in the light path you may need to open and align.
Hope you'll tell us more about your project -- this sounds like the kind of thing kids in our "Micronaut" program might be interested in.
I'd think you'll want to collect samples and filter them for size (calibrated mesh screens). The big stuff will be caught on the screen -- and you'd likely want a stereo microscope to inspect it.
For the small stuff (gets through a fine mesh screen) - then to a compound microscope. Could be reflected light illumination is what you want; but for things like plastic fibers coming off in a clothes dryer, the transmitted light and polarizer works best. Around 40x objective (400x total with a 10x eyepiece) you should be able to pretty clearly see the difference between organic material (cells) and plastics.
As for your first attempt with the "epi" illumination on your Leitz -- there are also irises in the light path you may need to open and align.
Hope you'll tell us more about your project -- this sounds like the kind of thing kids in our "Micronaut" program might be interested in.
Re: Hello from a beginner
Some additional ideas.
I guess that the major challenge of your project is to differentiate between polluting microplastic and other particles or background.
In other words, to identify the specific features of the microplastics.
One direction might be the outer morphology or "form" of the microplastic. That might be attacked with image analysis software, pattern recognition etc. Such methods have proliferated in recent decades, for example, for auomated tracing of the borders of a live cell in culture, in studies of live cells in motion. For this purpose, transmitted light and phase contrast or DIC seem appropriate.
Still another direction might be fluorescence. If that plastic is fluorescent under specific light illumination, AND the fluorescence differs from that of the background, then one has a very sensitive tool. Fluorecence is obtained under epi-illumination excitation, with either the Orthoplan or (better IMO) BX51; if the excitation is in the UV region, objectives that transmit UV are the best. In any case, for fluorescence ordinary objectives are used, and NOT the objectives used for reflected light.
If the microplastic can be stained with a specific fluorescent die, so much the better, possibly.
I guess that the major challenge of your project is to differentiate between polluting microplastic and other particles or background.
In other words, to identify the specific features of the microplastics.
One direction might be the outer morphology or "form" of the microplastic. That might be attacked with image analysis software, pattern recognition etc. Such methods have proliferated in recent decades, for example, for auomated tracing of the borders of a live cell in culture, in studies of live cells in motion. For this purpose, transmitted light and phase contrast or DIC seem appropriate.
Still another direction might be fluorescence. If that plastic is fluorescent under specific light illumination, AND the fluorescence differs from that of the background, then one has a very sensitive tool. Fluorecence is obtained under epi-illumination excitation, with either the Orthoplan or (better IMO) BX51; if the excitation is in the UV region, objectives that transmit UV are the best. In any case, for fluorescence ordinary objectives are used, and NOT the objectives used for reflected light.
If the microplastic can be stained with a specific fluorescent die, so much the better, possibly.
Re: Hello from a beginner
Hi,
welcome to the forum!
Your topic is really interesting an of great importance today.
In most places micro plastic is not highly concentrated so you might have to look for a way to concentrate it. Most plastics have a density of around 1 while sand has one of around 2. Plastics can withstand some treatments biological matter can't withstand. So there might be ways to concentrate the microplastics.
For digital image analysis a mentioned above you might have a look at the programms ImageJ and Fiji, scientific imag softrare for free.
Your Orthoplan is a true research microcope where "research" means that all componenst are interchangeable against other variants. So "Orthoplan" just gives a rough sketch of what you have in front of you.
With which name can we adress you?
Bob
welcome to the forum!
Your topic is really interesting an of great importance today.
In most places micro plastic is not highly concentrated so you might have to look for a way to concentrate it. Most plastics have a density of around 1 while sand has one of around 2. Plastics can withstand some treatments biological matter can't withstand. So there might be ways to concentrate the microplastics.
For digital image analysis a mentioned above you might have a look at the programms ImageJ and Fiji, scientific imag softrare for free.
Your Orthoplan is a true research microcope where "research" means that all componenst are interchangeable against other variants. So "Orthoplan" just gives a rough sketch of what you have in front of you.
With which name can we adress you?
Bob
Re: Hello from a beginner
"For digital image analysis a mentioned above you might have a look at the programms ImageJ and Fiji, scientific imag softrare for free."
You must have a 64 bit Windows for ImageJ to work.....
BillT
You must have a 64 bit Windows for ImageJ to work.....
BillT
Re: Hello from a beginner
I am so glad to see these experts' replies from you all. I will be back in the lab soon, first to check the infinity symbol, find any irises in the light path, and see if the fluorescence possible on either of my microscopes.
Our sediment samples need to be treated to remove organic matter by oxidation, followed by density-separation to isolate plastics particles. Because the final "specimen" is a white glass-fiber filter (not a transparent glass slide), I guess transmitted illumination may not work.
Thanks again, everyone! Also thanks in advance for any further comments and suggestions!
Our sediment samples need to be treated to remove organic matter by oxidation, followed by density-separation to isolate plastics particles. Because the final "specimen" is a white glass-fiber filter (not a transparent glass slide), I guess transmitted illumination may not work.
Thanks again, everyone! Also thanks in advance for any further comments and suggestions!
Re: Hello from a beginner
Hi BillT, I might be wrong, but I see downloadable 32-bit versions of ImageJ. Perhaps not the latest updated version, but still functional. 64-bit vesrions are faster thorugh.billbillt wrote:"For digital image analysis a mentioned above you might have a look at the programms ImageJ and Fiji, scientific imag softrare for free."
You must have a 64 bit Windows for ImageJ to work.....
BillT
Re: Hello from a beginner
1. I guess that your oxidation methods will be gentle, so as to leave the microplastics intact! not concentrated oxidizing acids. Peroxides maybe?anliuic wrote:Our sediment samples need to be treated to remove organic matter by oxidation, followed by density-separation to isolate plastics particles. Because the final "specimen" is a white glass-fiber filter (not a transparent glass slide), I guess transmitted illumination may not work.
2. Speaking of fluorescence, please note that some filters show auto fluorescence, probably in the violet light region.
I would love to see more details of your study in posts here!
Re: Hello from a beginner
Thanks for the heads up.. I guess I didn't look at it all enough..Hobbyst46 wrote:Hi BillT, I might be wrong, but I see downloadable 32-bit versions of ImageJ. Perhaps not the latest updated version, but still functional. 64-bit vesrions are faster thorugh.billbillt wrote:"For digital image analysis a mentioned above you might have a look at the programms ImageJ and Fiji, scientific imag softrare for free."
You must have a 64 bit Windows for ImageJ to work.....
BillT
The Best,
BillT
Re: Hello from a beginner
May be a bit late, but just found this thread. We have developed some methods to detect microplastics with any basic microscope. Here is the link https://pubs.acs.org/doi/10.1021/acs.jchemed.0c00518. Supplementary files are freely downloadable. Private message me if you want access to the main text.