🔬 How to get a 3D effect with your microscope | Amateur Microscopy

Oblique Illumination is an easy way to get a 3D effect using your microscope. It can be achieved by blocking the light on one side of the microscope lamp. The specimen is then lighted from the side. While the quality is not as high as from DIC (differential interference contrast), the big advantage is its low cost. For best results, it is important to very carefully align the oblique illumination filter.
Pictures comparing DIC and oblique illumination: In this German microscopy forum a user compared these two techniques with a side-by-side comparison of pictures (scroll down). The pictures labelled "DIK" are made using a DIC microscope and thoese labelled with "Schiefe Bel." are made using oblique illumination.
www.mikroskopie-forum.de/inde...
For a more theoretical and in-depth discussion of the technique:
www.microscopyu.com/technique...
www.olympus-lifescience.com/e...
Some historical background of oblique illumination:
www.microscopy-uk.org.uk/dww/a...
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[@negamerisltu299]

From my first days of microscopy I have been learning from you, my professor even used ur videos for presentations

[@AK-ee4lk]

Sucks knowing you have to pay for college when your professor is using a guy from KZbin to teach you.....

[@J-Physik]

@@AK-ee4lk Haha, it even more sucks, when you know that he's an Austrian teacher on a public school, so he's paid by austrian taxes^^ But he's good!

[@DavidEvans1958]

This is a brilliant technique. I just tested it on my Bresser TRM-301 Microscope by swinging the filter part of the condenser into a half way position. The 3D effect jumps out and makes even the smallest of details so clear to view. And all for free. Can't wait to capture a few test images. Thanks for the tip.

[@julioequinones]

the end was definitely glorious.

[@Mitch.Wasserman]

That long sliding triangle filter is such a great idea. Thank you

[@mgarcia4609]

I've had great luck with a swing-out filter holder and a poorly made dark-field illumination filter (electrical tape and plastic packaging). Haven't a made working dark-field filter, yet. But if I play around with the angle of the swing-out filter tray, it works perfectly for oblique illumination with all objectives. Thanks for your videos, Oliver. My wife got me a decent scope for Christmas and your videos have enriched my time with it considerably. If my primary and middle schools had microscopes with the resolution this new one has, there's no question I would have pursued biology in school. The first time I looked at a cross-section of a plant stem after unboxing the new microscope was one of the most profound experiences of my life! Seems like those moments are becoming increasingly rare with age... We're so lucky to be alive in a time where this technology is so easily accessible and have instant access to people like you who are willing to share a lifetime of experience with us.

[@Spacecomber]

For those with a mirror, instead of built in illumination under the stage, you can experiment with slightly tiling the mirror to get different lighting effects.

[@Ardeact]

i discovered this technique on accident my first few daysof microscopy with my swing out filter condenser, and wondered if this was an actual technique, well here we are

[@elenaacim]

Joy on the other side of the planet (Arizona) because I was able to apply alot of the information you generously shared enthusiastically! Your videos have helped me be successful in an area that I wanted to explore for years. Amazing! You are such a talented teacher! You were a treat while I needed to isolate . Joyous thank you and blessings to you and your family!🌱

[@ExperimentarEnCasa]

Excelent videos! Thank you for the great infromation

[@zane003]

nice explanation. saved me so much time. the oblique illumination can be made round like yin and yang, but facing each other. rotating one of them will give a wide range of illumination, and the shape of light would be perfect.

[@grim789]

These videos are the best I have found anywhere so helpful!

[@scanaccount5380]

Thank you for this great video. I made one and it's like I have a brand new microscope. I can see so much more detail in things I have seen many times before.

[@ianbcnp]

Many thanks Oliver, that was very useful and interesting. It encouraged me to work out where to try oblique illumination on my Motic which doesn't have a filter holder. Lots to discover still but I am already making progress and enjoying the results. All the best, Ian

[@bethechange4934]

Those are some stunning images. Well done sir and thanks for sharing.

[@alllowhat]

Your such a reference! Thanks for all your help!!

[@saeidehfathi6738]

Very helpful. Thank you!

[@vernonosier6610]

great A/V! i am not a beginner but found this both very informative and clear. enjoy your enthusiasm. will watch other presentations by you. thank you.

[@robertYTB78g]

Hi Oliver, here is something quite awesome that I have just got working. It is pretty simple - you get one of those narrow beam and moderately powerful "pen"pocket torches - not super powerful or anything - and mount it on a stand. I used this above the slide to shine downwards on my water sample, about 3cm from my 40x objective and at an angle of about 20-30 degrees to the slide. As I have a mirror based microscope, I also put in a black disk in the filter housing below the condenser. Wow!! this gave me a combination of oblique lighting and darkfield and the campanella umbrellaria and the rotifers I had only hitherto looked at in bright field were amazing - like moving milk bottles. PS Your site is helping me a lot, THANKS

[@Rafinade]

Soooo useful and easy to understand information! Thank you very very much!!!

[@salmacedonio3151]

Thank you , excellent information. Been working with microscopes for 25 yrs and they still fascinate me !

[@koushikbardhan3321]

This video is definitely worth watching...👍🏻

[@energymarketchile]

Great tip. Thanks for sharing!

[@jasonvictor9542]

Great video, thank you very much, I followed your dark field video and it worked great, I going to try this one also for sure!

[@ovalwingnut]

You have "magnified" my interest and have shown me the light of discovery... 🤗

[@robertYTB78g]

So useful and well presented. Grade A++ :) ,and thanks for posting

[@aktripathi48]

One of the greatest advantage of digital world, subscribing you channel, thanks brother, very valuable information are being given by you🙏🙏🌹🌹

[@MichaelLeightonsKarlyPilkboys]

Really great and helpful channel. Thanks so much

[@atharva-upclosingnature2423]

Wow I will today only try out oblige illumination........thank you oliver

[@Microscopy1]

Nice and instructive video (as always)! I have had a lot of fine microscopic experiences with oblique illumination (OL) and circular oblique illuminations (COL) as well. I happens to have both phase contrast and DIC in my zeiss axioplan. In many cases I prefer OL and COL before phase contrast. DIC is always my number one though. Thanks!

[@niggi4829]

Very good and informative channel, I learned a lot and it was fun

[@das250250]

Very nice ty

[@TheJMan152]

The end sequence is really nice. Something I like to do, I don't know what the technique is called, is using a flashlight. I turn off the microscope light and place a darkfield filter so it is a black background and point a flashlight onto my sample. It's fun because you can move the flashlight all around and see lots of different shadows and perspectives right away, very interactive. I also have a couple lights that can be stationary in the same way but I prefer holding the flashlight.

[@allanzephyrl.taguba1871]

This is so cool and innovative! =) I just needed this technique because I'm working on plant parasitic nematodes. Thanks microbehunter

[@MarieChardome]

i looooooove oblique

[@pdagaming7736]

Hi . I tried it using brown cardboard and it worked perfectly. Thank you so much

[@julioequinones]

Happy Easter!!!

[@wahabvlogger1651]

I am watching all of your videos.And i love it and very helpfull.i have microscope and i saw a micro world in single drope of water and its amazing. love form pakistan

[@markschofield576]

Can you show the magnification you are using in the images on the screen please?

[@limchai8404]

tq so much sir

[@teresashinkansen9402]

Ahhh yes, the poor man's DIC. Awesome, i hope to do this when i get new objectives. Ive tried it before but the phase contrast objectives i have don't work really well with oblique illumination, the image looks bad. But talking about experimental illumination, i have had some success doing fluorescence microscopy by changing the light bulb for a LED of the color needed to excite the fluorophore and then put a dark field stop on the condenser, finally i put a dichroic filter inside the female dovetail of the microscope were the head attaches, i guess it can also be placed inside the eyepiece like in telescopes. It works good enough i just need to get more fluorophores to test out. The good thing is that LEDs are now available from IR to UV and the condenser seems to work still down to 365nm so it should work with most fluorophores even DAPI. WARNING: some fluorophores are quite toxic and carcinogenic.

[@MicrobehunterMicroscopy]

I am also working on experimental fluorescence using blue LEDs ("blue light excitation"). It should be possible to excite chloroplasts (without addition of fluorophores). The plants/algae have to be placed into the dark for 30 minutes and then quickly observed in darkfield with the blue LEDs (without shining ambient light on them). The chloroplasts should light up red. It is only weak, so this can be captuered using long time exposure with a camera.

[@teresashinkansen9402]

@@MicrobehunterMicroscopy Well I just tried to see if i could see the fluorescence of chloroplasts and it seems its possible but I don't have good specimens, I guess an Elodea leaf would be perfect unfortunately I don't have one. I tried looking at an old algae culture I had, the chloroplast is about the same size as the whole cell 6um but they are a clumped mass and they no longer have a nice green color so Im sure they had very little active chlorophyll, only was able to get good exposures using the 10x objective so not enough magnification or resolution, tomorrow I will try to use a more powerful blue LED and get a good specimen to try again, also i will leave the specimen in the dark as you say before taking a sample. I will update my results as I experiment. Here its an image I took, I increased the contrast a bit, as can be seen it seems to work but this isn't a good image at all plus i need a better filter there is still some blue light leakage, still I think there is a significant potential in this technique. Happy microbe hunting! drive.google.com/open?id=1mkwOjLo3dniv0Tx9_32IblGiGDOU6Peb

[@teresashinkansen9402]

@@MicrobehunterMicroscopy Greetings Oliver, here it is an update regarding my experiments of fluorescence microscopy of chlorophyll: So today I changed the LED of the microscope for a more powerful one, the old LED was one of those cheap "3W" generic luxeon style driven at around 2W, the new one is a white LED (cree XD16) that i converted to blue by removing the phosphor (It took almost all day! its so hard to remove every grain from around the chip), it has a slightly shorter wavelength at around 450nm and its significantly more powerful, im driving it at about 5W. Also I placed some small dots of india ink on my filter because it had some pinholes that let blue light pass, its still not perfect but now i have much better contrast, also now i was able to use the 40x objective, again the pictures isn't as good as i would like but still significantly better than the last experiment. I will keep experimenting and if i have a breakthrough I will update so you and other people with the interest can benefit. The sample is from an old algae culture again because I haven't been able to get an Elodea, regardless it worked, in the image you can see clearly the cells that have chloroplasts in them, if you download the image use an image editing program to turn on and off the red and blue channels, you can see clearly the cell wall as blue and the chloroplast red so it does work. I share only two pictures because are the ones i think look the best and also i hope to update the gallery with more pictures as i experiment. Also I see you have a forum so probably I better make an account and update and post my experiments there. Happy microbe hunting and good luck with your experiments, I hope this info was helpful. drive.google.com/drive/folders/1NrnNvxwGrBoUoBqNxXiO4jcIhJi4ugjW?usp=sharing

[@karl3549]

Great work! Do you happen to have any, of what you considered to be your most successful, .stl files available for download?

[@atharva-upclosingnature2423]

Hello Oliver, I really miss those pond organisms and critters but due to lock down in India I am not able to go and collect any samples....please do you have any idea that can we cultivate those organisms at home Safely (specifically green algae)....

[@lotharmayring6063]

if the microscopes would have the possibility to move or bend their lightsources, OI would be better and easier to do, like the older with the sun and the mirror. By the way sunlight is the best lightsource for microscopy

[@shivaprakash2630]

Please make a vedio like microscope hacks where all microscope technic and tips are included , waiting for the vedio . Love form #India

[@gustavorad]

Yo he logrado enfocar un telescopio reflector de modo que la muestra quede a 1 metro del espejo para que funcione como un microscopio. La característica principal de este artefacto es que puede enfocar nítidamente todas las capas de la muestra al mismo tiempo. Ahora le voy a aplicar la luz oblicua según tu consejo. gracias

[@Bhodisatvas]

My goal is to get a DIC microscope, the images the technique produces are just stunning.

[@ermaindia1475]

Great teachings.. learnings.. on Microscopy techniques..thank you so much Sir.. ..need to get your expert technical advice and guidance on specific advanced microscopy techniques needs & problems..how do I get in touch with you Sir ..warm regards.. Er. Bhalla JS India

[@lotharmayring6063]

OI with good objektives is better than PH or DIC

[@bendirval3612]

You didn't specify which of the various tools there you used for the videos. The patch stop, presumably? That sliding thing looks like the best tool I could imagine for this. I have had problem with both oblique illumination and DIY darkfield because you have to have them just the right size and centered perfectly.

[@MicrobehunterMicroscopy]

I used the sliding device of all oblique illumination videos.

[@bananabestfruit]

I stared looking for DIC but noticed the price.. Now I don't need that, as oblique technique is dirt cheap! Thanks!

[@markkeyworth]

Notice you use your glasses when looking through the microscope. Is it best to use them and how does it impact on the view and resolution?

[@moralesriveraomar233]

It's a good idea if you plan to show the image to anyone else. If you want to see yourself only you can focus without glasses but you'll overcompensate for your messed up vision

I don't have oblique illumination always but when i put in small ounce of water i have it.

[@agri-lifeorganicfarm2466]

I purchased a used Hund Wetzlar Seiler v3000 microscope. I only paid $35. US so I didn't expect much. It was missing the eye pieces so I picked up two "Wf10X-18MM" from Amazon. I don't understand why they say 18mm when I ordered "AmScope EP10x23 Pair of WF10x Microscope Eye pieces (23mm)". Since I didn't know if the microscope was functional; I didn't want to spend much for the eye pieces until I know if the microscope worked. It seems the left view is out of alignment. I can see the object with my right eye but the left object is out of alignment ie off center. I guess I need to send the microscope in for repair??

[@andrealucecita]

I wanted to 3D print 32mm oblique filters but I don't know what measurements to use for the space slots, do you have any measurement recommendations?

[@MicrobehunterMicroscopy]

Depends on many factors and also on objective used. Size is very sensitive. I recommend a system that allows you to adjust it continually (eg swing-out filter holder etc) or to make a series of filters and then try it experimentally.

[@Microscopy1]

PS! It is important to mention that the 3D in both oblique illumination and circular illumination, (and DIC as well) is artificial. It does not illustrate the actual 3D structures, only the density of the different structures of the specimen due the refractive index of the structure.

[@honeypotusername]

What could illustrate the actual 3D structures?

[@Microscopy1]

@@honeypotusername Important question! 3D structures illustrate dense organelles and subcellular structures...

[@honeypotusername]

@@Microscopy1 ...... :)

[@AMadScientist]

9:50 that paramecium didn’t know the difference between DIC and your plastic filter.

[@m7mdax92]

Do you recommend zeny microscope for a Beginner? this one :ZENY 40X-1000X Magnification ,and thx

[@MicrobehunterMicroscopy]

Only if you do not want to spend more money. If you want to do things like oblique illumination, or darkfield etc. then you need a slightly better microscope with a condenser and a filter holder. A rotating ring (like in the link) is not a condenser.

[@julioequinones]

What makes you sure these are onion organelles and not endosymbionts?

[@danzender7665]

Hello Oliver, I am a beginner about to purchase a microscope. I have it narrowed down to a select few, but my question is: I have noticed that some of the microscopes have a trinocular tube that has the ability to focus. On the one I am interested in purchasing it does not have this ability. I am mainly interested in video/photos, will the "stationary" trinocular port still work with a DSLR or would it require buying additional parts? Thanks for all the advice and content.

[@MicrobehunterMicroscopy]

Hello, I made a video on this: kzbin.info/www/bejne/o5a9qpt_aLGabLM

[@danzender7665]

@@MicrobehunterMicroscopy Wow, I'm not sure how I missed this video. Thanks!

[@danzender7665]

I guess that'd be because you just made it. Thanks so much!

[@johnjohn-cs9eu]

Whats in snot?

[@johnjohn-cs9eu]

Can C19 attach to hair

[@pdagaming7736]

If I put light on the mirror, it is coming very dull image

[@johnjohn-cs9eu]

Can u look at a mosquito's blood food and see if they're transmitting C19

[@GLITCH_-.-]

Hm. Doesn't look "very" good, but does enhance contrast... Can you get rid of the fringing effects if you change the orientation of the filter? (Like, not pushing it in from the left, but from the front) BTW I always wonder why your shots are looking comparatively "bad". Sometimes I feel like my old 35$ toy microscope is equally good. (I don't have money) Most of the time it is because your Microscope is dirty, but that's not all. If I look at Journey to the Microcosmos' videos, they are much better. And they have a microscope in the same price range as yours. Is it because theirs is manufactured in the last few years and yours is an old model?

[@Microscopy1]

Even if this question is addressed to Microbehunter, allow me to comment: In my opinion, the direction does not matter. Yes, oblique illumination is a contrast enhancer. A lot of other factors determine the resolution, and the degree of chromatic and spherical aberration which you probably refering to when mentioning "fringing". Especially the numerical aperture (NA) of the objective is important.Plan-apochromat objective is the best but flourites is very god also. If you want the best, try to get hold of DIC. Second hand equipment is obtainable if you safe money....

[@GLITCH_-.-]

@@samueljackson1329 You're right. I recently got a good microscope and found that there are huge differences in image quality by even the smallest changes, so it can sometimes take a long time to get it looking good, and I guess he just didn't bother much here. I also got images in the range of DIC to toy-microscope with it.

[@zivkloda882]

How do you explain that you see so much best in your microscope compared to a Chinese microscope?

[@MicrobehunterMicroscopy]

You can get good results also with Chinese microscopes. If you do the setup properly. Proper lighting, proper filter use, contrast enhancement etc.

[@robertkeener2841]

BRILLIANT! BRILLIANT!! I love the "DIC look" but am not a millionaire to afford one. I have an older Leitz Laborlux III (black enamel) and it has the swing-out type of condenser filter holder (thanks for the handy tip!), so I cannot wait to try this technique!

[@johnjohn-cs9eu]

...continuatiom of my commentary .... well do you have (acccess to) electronic microscopy OR NOT

[@samueljackson1329]

What on earth? Do you mean electron microscopy? In that case, Oliver most likely doesn't.

[@benibuntu2052]

❤️❤️❤️⚘️🌷⚘️🙏🙏😊😊😊👍👍👍

[@negamerisltu299]

First!!!

[@johnjohn-cs9eu]

Can you see corona-virae in blood sneeze liquid snot pee ?

[@moralesriveraomar233]

Definitely not. Maybe with atomic force microscopy (which can be DIYd) but the pictures I've seen are all electron microscope