thank you for teaching me so much on this hobby I have spent hundreds of dollars on this hobby and have almost no regrets I have seen so many incredible things and learned so much more and all because I happened to stumble onto your channel Thank you again.
Hi, I am a microbiologist and I think I can give some insights to the lack of resolution on the second slide, I am almost certain that you placed the slide upside down, when I was a student I made this mistake sometimes and that explains the presence of dust suspended in the oil , further the first sample preparation was focused correctly.
Спасибо! Очень информативно. Недавно приобрел микроскоп и ваши видео дают очень много информации для исследований. Я плохо понимаю английский язык, но визуальное наблюдение дает очень много. Огромное спасибо!!!
@14:13 You can fiddle with your condenser so you avoid the eye piece focus, that's what is not moving. Also changing the light affects so much when focusing at hig her magnitudes.
I think it is fair to say, that our dear Microbehunter, giver of all things Pond-Enthusiastic (very contagious!!!) is not a big fan of oil emersion. He has said in many videos his reasons:1) pond water living things are can move too fast for 1000x, 2) the complete set of olympus objectives that oliver has ends with 60 which is perfect for pond water, 3) he does not like the risk of oil getting all over the place, 4) he is very sensitive to sales-related microscope hype and the 100x oil is clearly a selling point used to take advantage of unsuspecting newbies who might be better off with a set ending in 60 (600x)
@@Microbehunter Actually, the 100X objective was initially developed for oil immersion and high resolution imaging. But today, this has been achieved by the 60X objective in most instances, with some 60X (oil immersion) objectives even achieving a N.A. of 1.4 (this used to be the upper limit of 100X objectives in the past), making the 100X objective redundant in some fields of microscopy. Actually, if we look at the N.A./magnification factor as an evaluation metric for objective performance, we can see that increasing the objective N.A. is not exactly proportional to the magnification - for instance, considering the best oil immersion 40X objectives which have a N.A. of 1.0, we would expect the 100X oil immersion objective to have an N.A. of 2.5 (100 * 1.0/40 = 2.5), but in reality, the best 100X objectives only achieve a N.A. of 1.6-1.7 (with most 100X objectives having a N.A. of 1.25-1.45). The 60X oil immersion objective which has a N.A. of 1.4 is closer to its desired N.A. of 1.5 (60 * 1.0/40 = 1.5), allowing the 60X oil immersion objective to realize an image resolution & clarity which is parallel to that of the 40X oil immersion objective, & thus does not disappoint the user, when stepping up to the 60X oil immersion objective from the 40X oil immersion objective. But the 100X objective still has a role, albeit only in very specialized applications, where magnification is crucial (e.g. in metrological applications, bacteriology, SR imaging, etc). In this regard, the 100X objective may be preferred to the 60X objective, though the latter may have a relatively high NA and an ability to achieve resolutions matching the 100X objective.
I was thinking of the dust-sticking-to-oil-residue problem just before you mentioned it and I envisioned a tiny cup-shaped cover for the objective to keep dust out (and perhaps also prevent an inadvertent crash).
you need to adjust the iris diaphragm when using the oil immersion objective to improve the resolution, otherwise you will get that poorly detailed image of your specimen at 1000x
Indeed, a way to adjust the iris is to use a phase contrast alignment eyepiece or just take out an eyepiece and and then close the iris until you start seeing the iris, stop when it has covered about 20% of the objective area. If you close it too much you not only lose brightness also resolution!
Actually it will start to soot if you hold it to far away from the burner do to the build up of unburned carbon. Closer the better. Also a blue flame is characterized as complete combustion which means less unburned carbon from the hydro carbon gas causing less chance of soot. Its probably best to use your stove top as it is the best solution no matter the kind of stove top. If it's natural gas chances are your getting a bunsen blue flame which is perfect. And if its electric place it directly above or in contact with the electric burner and use the same feel method.
Hi Microbehunter, can I use a household lighter to heat fix blood cells before staining? I am new to microscopy and am trying to look at blood cells with a nucleus stain and oil of immersion.
Thank you so much for ‘‘tis very informative video! I was making a lot of mistakes as a beginner. It is actually comforting to know that 1000X is pretty much the.limit. Now I won’t be drooling for expensive higher power objectives. I had no idea that oil Emerson is not to be used for prepared slides. Is there an optimal highest power one can use for prepared slides?
You should make a new video on this, this video seems to be outdated and it's important because most mid/beginner tier microscopes come with 100x oil and people don't know how to use them
Thank you for your excellent video. Is it possible you could advise me please. When I try to use the 100x objective it touches the slide before I can get into focus, what am I doing wrong? Using 0.13-0.17 cover slips. My instrument is a Prior PL032.
Make sure that it is in focus before you use the 100x. Also make sure that the objectives are from the same series, otherwiese they might not be parfocal.
I have a question, for a microscope I am about to buy I see specs as Objectives: Achromatic DIN 4X, 10X, 40X(S), 100X(S, Oil) for objective. does 100X(S, Oil) what does the 'S' means here?. can 100x be used for general use with out oil immersion as well?
S stands most likely for "Spring Loaded". This means that the bottom part of the objective retracts when it crashes into the slide to prevent damage. do NOT use oil on regular objectives, only on those that are labelled with "oil". If you use 100x without oil, then the quality will be bad and you risk crashing it into the slide becasue it will be blurry any you try to find the focus.
Microbia In my micro lab classes we never used a cover glass for any of the gram stains, acid fast stains, oil immersion slides, or anything we made. Just a variation of this method.
Hi mate, thank you very much for your great videos and information. It is appreciated. I have a question if you could please answer. I need a proper microscope for Gemology, to see through stones like quartz, emerald, etc... and around what prices please? cheers mate.
You need a stereo microscope. There are so many models available that I can not give you a specific recommendation. If you are totally unsure, then I would buy a relatively low-cost one and see how far you can get with that and use it to find out its advantages and limitations. Buy a better one (if needed) later. This is better than investing a lot of money at the beginning and then discovering that it was wasted, becasue a cheaper one would also have done it.
HP red inkjet refill ink works ok as some types of stain. It is water soluble. You rammed the objective directly down into the oil in the last part of the video. Should it be slid in sideways to prevent air bubbles? DC 704 silicone oil works well as immersion oil and is cheap and easily available. It cleans up with CRC brake cleaner cleaner spray (red can) from any auto supply store.
Oliver, I have a question. Why do some videos on RUclips show oil placed beneath the slide as well as on top of it? I've seen at least two videos that say you should place oil on the condenser lens and move it up to meet the underside of the slide as well as place it on top of the slide. Could this be a reason for the lack of resolution?
There are some systems where oil is also placed on the condenser lens. If the condenser has a numerical aperture that is lower than that of the objective, then it will limit the resolution. With oil on the condenser lens, you get a higher condenser NA. But I would only do this if the condenser is specifically designed for this. In my case this is not so much of an issue. The subjectively perceived lack of resolution is becasue the magnification goes up faster than the resolution. So objectively you will be able to see more, but subjectively it will appear more blurry because we are used to crisp images.
You won't like my answer: The microscope that you will be using most often is the best one. In other words, the microscope that fulfills your needs the best. The most modern, expensive, advanced microscope won't be the best for you, if you won't use it because it does not fulfill your observation needs. If you talk about general quality of construction, optics etc, disregarding everything else, then you have to get one from the big 4 microscope manufacturers, who target the research/university/medical market (Olympus, Nikon, Zeiss, Leica). You will be paying significantly more. An introductory microscope from Olympus the CX43 costs over EUR 1000. Most basic features, that you can get also for EUR 250. And you won't see more. No limits concerning price. I suggest that you get a microscope for about EUR 250-350. You can always spend more later.
Yes. Heat fixing makes the cells stick to the slides . Without heat fixing, the cells will get washed off during staining. If you don't stain the cells, then you can skip this step :)
Yes, methylene blue the standard stain. The problem is, that methylene blue is not always easily available as it is also a bit toxic. I once wanted to buy some but was not given it, becasue they only sold it to registered laboratories. For amateur microscopy use I found that some inks also work.
You need to use cover glass with oil immersion, and this is why you got terrible results without it. The .017 cover glass changes the refraction of light rays into the oil. This is also why the .017 is printed right on the objectives. Any deviation from this thickness results in a degradation of resolution. Putting the objective directly into the oil, minus cover glass, puts you .017 closer to the specimen than you should be, again, resulting in degraded resolution.
Thank you for the comment, but this is not the case. The refractive index of the cover glass and the oil is (nearly) the same, therefore there will be no refraction of the light rays into the oil, as you say. The cover glass is necessary when not using oil, as the difference of refractive index between glass and air is much larger. For the A100x olympus oil objective that I use, the working distance is 0.20 mm, which is the distance between the front lens of the objective to the specimen. This is independent on whether a cover glass is present beneath the oil or not (ideally the cover glass should be optically neutral as it touches the oil). Placing a cover glass on top of the specimen would reduce the clearance between the objective and the cover glass down to 0.03mm. It would be difficult, if not impossible to focus on the deeper parts of the specimen. A cover glass would make sense, if the specimen were in water, as in this case the oil and water have to be separated. The focus would be changed, if a cover glass were missing in an air objective (thereby shifting the focus as well as changing the resolution), but for oil it is irrelevant.
I love your website but I have to disagree with your evaluation on not using a cover glass on 100x oil immersion objectives. There are very expensive 100x objectives with adjustable variable range built in to compensate for the very slight differences (+/- .001mm or more) in cover glass thickness. All of the major microscope manufacturers (Nikon, Olympus, Leica) address this, stating that a deviation as little as 0.001 in your cover glass from your objective recommended cover glass thickness will begin to degrade your image. There is even #1.5H high performance cover glass for keeping the 0.17 specification to +/- .001. In your video you state you always get "poor results" and "this is why I don't do oil immersion much". Not hating on you, I like what you do. I just find it odd that you don't use a cover glass with 100x oil immersion. Could we do an experiment and try one of the #1.5H or at least a #1.5 cover glass, viewing the same slide, with the cover glass and without the cover glass, and see honestly if there is a noticeable difference or not? The would make a great video and maybe put to rest if the cover glass is absolutely necessary or not for good oil immersion microscopy.
@@KevFlaAVCHD Hi, the cover glass is not necessary, unless the sample is fresh, and to give you an example, it is very common in clinical practice to place oil over slide directly for differential blood tests.
thank you for teaching me so much on this hobby I have spent hundreds of dollars on this hobby and have almost no regrets I have seen so many incredible things and learned so much more and all because I happened to stumble onto your channel Thank you again.
Hi, I am a microbiologist and I think I can give some insights to the lack of resolution on the second slide, I am almost certain that you placed the slide upside down, when I was a student I made this mistake sometimes and that explains the presence of dust suspended in the oil , further the first sample preparation was focused correctly.
exactly
Your channel is amazing. I am just starting as a hobbyist microscopist an I find your videos very informative and really delightful to watch!
Спасибо! Очень информативно. Недавно приобрел микроскоп и ваши видео дают очень много информации для исследований. Я плохо понимаю английский язык, но визуальное наблюдение дает очень много. Огромное спасибо!!!
@14:13 You can fiddle with your condenser so you avoid the eye piece focus, that's what is not moving. Also changing the light affects so much when focusing at hig
her magnitudes.
Thanks ♥ You save my slides. I didn't know I can't use x100 for permanent slides. I get my microscope tomorrow, so lucky me I find this video
HE goes over everything, thanks!
I think it is fair to say, that our dear Microbehunter, giver of all things Pond-Enthusiastic (very contagious!!!) is not a big fan of oil emersion. He has said in many videos his reasons:1) pond water living things are can move too fast for 1000x, 2) the complete set of olympus objectives that oliver has ends with 60 which is perfect for pond water, 3) he does not like the risk of oil getting all over the place, 4) he is very sensitive to sales-related microscope hype and the 100x oil is clearly a selling point used to take advantage of unsuspecting newbies who might be better off with a set ending in 60 (600x)
You summarized it perfectly!
@@Microbehunter Actually, the 100X objective was initially developed for oil immersion and high resolution imaging. But today, this has been achieved by the 60X objective in most instances, with some 60X (oil immersion) objectives even achieving a N.A. of 1.4 (this used to be the upper limit of 100X objectives in the past), making the 100X objective redundant in some fields of microscopy. Actually, if we look at the N.A./magnification factor as an evaluation metric for objective performance, we can see that increasing the objective N.A. is not exactly proportional to the magnification - for instance, considering the best oil immersion 40X objectives which have a N.A. of 1.0, we would expect the 100X oil immersion objective to have an N.A. of 2.5 (100 * 1.0/40 = 2.5), but in reality, the best 100X objectives only achieve a N.A. of 1.6-1.7 (with most 100X objectives having a N.A. of 1.25-1.45). The 60X oil immersion objective which has a N.A. of 1.4 is closer to its desired N.A. of 1.5 (60 * 1.0/40 = 1.5), allowing the 60X oil immersion objective to realize an image resolution & clarity which is parallel to that of the 40X oil immersion objective, & thus does not disappoint the user, when stepping up to the 60X oil immersion objective from the 40X oil immersion objective. But the 100X objective still has a role, albeit only in very specialized applications, where magnification is crucial (e.g. in metrological applications, bacteriology, SR imaging, etc). In this regard, the 100X objective may be preferred to the 60X objective, though the latter may have a relatively high NA and an ability to achieve resolutions matching the 100X objective.
I was thinking of the dust-sticking-to-oil-residue problem just before you mentioned it and I envisioned a tiny cup-shaped cover for the objective to keep dust out (and perhaps also prevent an inadvertent crash).
you need to adjust the iris diaphragm when using the oil immersion objective to improve the resolution, otherwise you will get that poorly detailed image of your specimen at 1000x
Indeed, a way to adjust the iris is to use a phase contrast alignment eyepiece or just take out an eyepiece and and then close the iris until you start seeing the iris, stop when it has covered about 20% of the objective area. If you close it too much you not only lose brightness also resolution!
Actually it will start to soot if you hold it to far away from the burner do to the build up of unburned carbon. Closer the better. Also a blue flame is characterized as complete combustion which means less unburned carbon from the hydro carbon gas causing less chance of soot. Its probably best to use your stove top as it is the best solution no matter the kind of stove top. If it's natural gas chances are your getting a bunsen blue flame which is perfect. And if its electric place it directly above or in contact with the electric burner and use the same feel method.
Hi Microbehunter, can I use a household lighter to heat fix blood cells before staining? I am new to microscopy and am trying to look at blood cells with a nucleus stain and oil of immersion.
Yep, that's what we used in clinic. Regular Bic lighter.
Thank you so much for ‘‘tis very informative video! I was making a lot of mistakes as a beginner. It is actually comforting to know that 1000X is pretty much the.limit. Now I won’t be drooling for expensive higher power objectives. I had no idea that oil Emerson is not to be used for prepared slides.
Is there an optimal highest power one can use for prepared slides?
Thanks you, great lesson!
You should make a new video on this, this video seems to be outdated and it's important because most mid/beginner tier microscopes come with 100x oil and people don't know how to use them
Thank you for your excellent video. Is it possible you could advise me please. When I try to use the 100x objective it touches the slide before I can get into focus, what am I doing wrong? Using 0.13-0.17 cover slips. My instrument is a Prior PL032.
Make sure that it is in focus before you use the 100x. Also make sure that the objectives are from the same series, otherwiese they might not be parfocal.
@@Microbehunter Thank you very much for your help, very much appreciated.
Cooooool
I have a question, for a microscope I am about to buy I see specs as Objectives: Achromatic DIN 4X, 10X, 40X(S), 100X(S, Oil) for objective. does 100X(S, Oil) what does the 'S' means here?. can 100x be used for general use with out oil immersion as well?
S stands most likely for "Spring Loaded". This means that the bottom part of the objective retracts when it crashes into the slide to prevent damage. do NOT use oil on regular objectives, only on those that are labelled with "oil". If you use 100x without oil, then the quality will be bad and you risk crashing it into the slide becasue it will be blurry any you try to find the focus.
i like so much your channel.
Thanks for your video! And why didn't you use cover glass? Can 100x objective be used with a cover glass?
Microbia In my micro lab classes we never used a cover glass for any of the gram stains, acid fast stains, oil immersion slides, or anything we made. Just a variation of this method.
Hi mate, thank you very much for your great videos and information. It is appreciated. I have a question if you could please answer. I need a proper microscope for Gemology, to see through stones like quartz, emerald, etc... and around what prices please? cheers mate.
You need a stereo microscope. There are so many models available that I can not give you a specific recommendation. If you are totally unsure, then I would buy a relatively low-cost one and see how far you can get with that and use it to find out its advantages and limitations. Buy a better one (if needed) later. This is better than investing a lot of money at the beginning and then discovering that it was wasted, becasue a cheaper one would also have done it.
Nice video. Can I reuse the slide or it must be every time new?
If you are able to clean the slide properly (and remove all the oil), then you can reuse the slide.
@@Microbehunter Ok, I will clean it with ethanol.
HP red inkjet refill ink works ok as some types of stain. It is water soluble. You rammed the objective directly down into the oil in the last part of the video. Should it be slid in sideways to prevent air bubbles? DC 704 silicone oil works well as immersion oil and is cheap and easily available. It cleans up with CRC brake cleaner cleaner spray (red can) from any auto supply store.
Your microscope objective n eyepiece need cleaning. You'll need to fix the stain by ph buffer so that stain is retained properly.
Oliver, I have a question. Why do some videos on RUclips show oil placed beneath the slide as well as on top of it? I've seen at least two videos that say you should place oil on the condenser lens and move it up to meet the underside of the slide as well as place it on top of the slide. Could this be a reason for the lack of resolution?
There are some systems where oil is also placed on the condenser lens. If the condenser has a numerical aperture that is lower than that of the objective, then it will limit the resolution. With oil on the condenser lens, you get a higher condenser NA. But I would only do this if the condenser is specifically designed for this. In my case this is not so much of an issue. The subjectively perceived lack of resolution is becasue the magnification goes up faster than the resolution. So objectively you will be able to see more, but subjectively it will appear more blurry because we are used to crisp images.
Thanks for the reply. I like your channel very much.
HI, Very interesting video, AT 10:44 The Cell in the Center Measures Roughly how
many Nano Meters. THANKX in Advance, JOHN
What microscope do you suggest is the best???
You won't like my answer: The microscope that you will be using most often is the best one. In other words, the microscope that fulfills your needs the best. The most modern, expensive, advanced microscope won't be the best for you, if you won't use it because it does not fulfill your observation needs. If you talk about general quality of construction, optics etc, disregarding everything else, then you have to get one from the big 4 microscope manufacturers, who target the research/university/medical market (Olympus, Nikon, Zeiss, Leica). You will be paying significantly more. An introductory microscope from Olympus the CX43 costs over EUR 1000. Most basic features, that you can get also for EUR 250. And you won't see more. No limits concerning price. I suggest that you get a microscope for about EUR 250-350. You can always spend more later.
How about the Swift 350T
Is the Swift 350B good?
Microbehunter how about the Swift 380B
Microbehunter plzzz answer is the swift 380B good?
is that tap or Distilled water?
Imaging chromosomes sounds like an interesting possibility for a future video.
💜
Can you clean the objective with glasses tissue?
yes, blot if there is oil
@@Veronica.John10-10 Thank you very much!!
Was kann ich tun wenn ich aus Unkenntnis mein 40X objektiv in Immersionsöl eingetaucht habe ?
Is that ink methylene blue?
Do you need to gaslight the cells if it is a temporary slide?
Yes. Heat fixing makes the cells stick to the slides . Without heat fixing, the cells will get washed off during staining. If you don't stain the cells, then you can skip this step :)
I know that slide would melt at
bunsen burner. :3
Normally we use methylene blue alkaline stain.
Yes, methylene blue the standard stain. The problem is, that methylene blue is not always easily available as it is also a bit toxic. I once wanted to buy some but was not given it, becasue they only sold it to registered laboratories. For amateur microscopy use I found that some inks also work.
Can I use alcohol burner ?
yes
Pl type magnification on sides.
But if you cook them you wont see them in their true form.
Is there any other way to see them without baking and killing them?
I believe you could fix with absolute methanol instead of heat
You need to use cover glass with oil immersion, and this is why you got terrible results without it. The .017 cover glass changes the refraction of light rays into the oil. This is also why the .017 is printed right on the objectives. Any deviation from this thickness results in a degradation of resolution. Putting the objective directly into the oil, minus cover glass, puts you .017 closer to the specimen than you should be, again, resulting in degraded resolution.
Thank you for the comment, but this is not the case. The refractive index of the cover glass and the oil is (nearly) the same, therefore there will be no refraction of the light rays into the oil, as you say. The cover glass is necessary when not using oil, as the difference of refractive index between glass and air is much larger. For the A100x olympus oil objective that I use, the working distance is 0.20 mm, which is the distance between the front lens of the objective to the specimen. This is independent on whether a cover glass is present beneath the oil or not (ideally the cover glass should be optically neutral as it touches the oil). Placing a cover glass on top of the specimen would reduce the clearance between the objective and the cover glass down to 0.03mm. It would be difficult, if not impossible to focus on the deeper parts of the specimen. A cover glass would make sense, if the specimen were in water, as in this case the oil and water have to be separated. The focus would be changed, if a cover glass were missing in an air objective (thereby shifting the focus as well as changing the resolution), but for oil it is irrelevant.
I love your website but I have to disagree with your evaluation on not using a cover glass on 100x oil immersion objectives. There are very expensive 100x objectives with adjustable variable range built in to compensate for the very slight differences (+/- .001mm or more) in cover glass thickness. All of the major microscope manufacturers (Nikon, Olympus, Leica) address this, stating that a deviation as little as 0.001 in your cover glass from your objective recommended cover glass thickness will begin to degrade your image. There is even #1.5H high performance cover glass for keeping the 0.17 specification to +/- .001.
In your video you state you always get "poor results" and "this is why I don't do oil immersion much". Not hating on you, I like what you do. I just find it odd that you don't use a cover glass with 100x oil immersion. Could we do an experiment and try one of the #1.5H or at least a #1.5 cover glass, viewing the same slide, with the cover glass and without the cover glass, and see honestly if there is a noticeable difference or not? The would make a great video and maybe put to rest if the cover glass is absolutely necessary or not for good oil immersion microscopy.
@@KevFlaAVCHD I would assume what's *under* the cover glass would affect things as well.
I got a very clear result with my Omax microscope using standard microscopy stain and a cover glass.
@@KevFlaAVCHD Hi, the cover glass is not necessary, unless the sample is fresh, and to give you an example, it is very common in clinical practice to place oil over slide directly for differential blood tests.
💚🤘🏿
The oil immersion images are a blur.
clean your lenses ffs
Just clean the dammn microscope man !!
Hi sir
I am Dharamjeet -I am adding sir
@@dharamjeetkumar158 WTH
your immersion techniques is terrible.. lol. how did you get such terrible focus on such large cells??
thats a terible example of oil emmersion