This video covers how to measure objects viewed through a light microscope in micrometers, using the diameter of the field of view as a measuring reference.
Thank you for putting that in plain English. I've read numerous definitions of the FOV, and they were worded in such a way that I think it was only for physicists to understand. Your video was extremely helpful.
This was great - Very succinct and clear. I learned this in Zoology, but I'm finding that I have a more specialized niche in the field that requires more accuracy than this method. I'm really into mycology and mushroom taxonomy. There are tons of new species to discover and research, so it's an exciting field for biologists. I've gotten good at identifying fungi based on physical features, but the only way to be sure of a species is through microscopy. As such, I need to produce accurate measurements of a spore's length and width as well as a solid description of a spore's physical features. This leads me to ask a couple of questions: 1.) How would one precisely measure something like a fungal spore? I mean, the texts get painstakingly specific; you need a range (in micrometers of course) of the spore's length and width. I've seen small slides that have rulers (increments of 0.01mm) used for microscope calibration. They're pretty cheap, so I figured I'd probe your brain to see if those could be used to produce more accurate measurements than "guesstimating" how many spores would fit in the field of view. That is a MUCH cheaper alternative to buying an optical reticle. If I can spend 5-10 bucks to get a little slide ruler that produces accurate measurements, then I'd rather do that than shelling out 50-100 for a freaking eye-piece. That somewhat leads me to my second question, which will take a lot more explaining: 2.) The only functional microscope I have at home is a toy that was given to me as a child. It's a tiny, ten dollar POS that has actually been quite useful up until I started to get frustrated with inaccurate spore-analysis. Maxed out at 250x, this little bugger can see the shape of most spores and with a LOT of brainwork and a LOT of guessing, I can produce an estimate to the spore size. However, I quickly learned that this simply is not sufficient for research or effective identification. We're talking, the difference that distinguishes one species from another is one or two micrometers in length. Furthermore, one has to be able to see the surface of the spores - are they smooth? rough? pitted? My little dinky toy just can't do that. So, it's time to upgrade. I've been doing my research and I'm torn between two microscopes. I'll link them here and then explain. 1. www.amscope.com/40x-2500x-led-digital-binocular-compound-microscope-w-3d-stage-5mp-usb-camera-1.html?gclid=Cj0KEQjw5Z63BRCLqqLtpc6dk7gBEiQA0OuhsBIMvVDOYWKE2ytKFn1GS3bxgrdwblLrb7xiRdfqq_gaAuXd8P8HAQ#product_tabs_additional_tabbed 2. www.amazon.com/OMAX-40X-1600X-Biological-Microscope-Illumination/dp/B004EBRCEI?SubscriptionId=AKIAJT4BRIUI6ZD32TNQ&tag=disccamera-20&linkCode=sp1&camp=2025&creative=165953&creativeASIN=B004EBRCEI My motives for the first are obvious: it's cheap, it goes to a high magnification, and comes with a 5 megapixel camera. The software included with this scope would allow me to do finite calculations like I described above. It has all the features that anyone in the field would expect a lab microscope to have and more. The only clear negative is the optics. They're industry standard DIN objectives. Everything else is amazing about this microscope, but as well all know - it's the optics you're really paying for n a scope. Hence the low cost of this one. After realizing that identifying the shapes of the pores isn't enough - I need to be able to see the physical attributes of the spore wall as well- I came to realize I might need a scope with better optics. This led me to consider PLAN objectives and darkfield condensers. PLAN objectives simply produce higher-quality images - they give a more "flat" appearance to the FOV and exhibit "crisp" resolution. A darkfield condenser essentially changes the way light interacts with objects on the slide - the background remains dark, whereas something like a spore on the slide would be lit up. The resulting contrast allows one to see details more clearly. That being said, these also significantly increase the COST of a scope. So, this leads to my second question (finally): If I was concerned about seeing fine details on a spore, would a cheaper microscope with DIN objectives be sufficient? I understand that staining may be necessary, and I may need to purchase some standard reagents. However, even with staining, do you think those details would be distinct enough to make out something like "pitting" on a spore? Or, is it just better to invest in the PLAN objectives and even consider a darkfield condenser right off the bat? Would it be better to get the darkfield condenser or the PLAN objectives (in other words, is it more important to produce a sharper image or to have more contrast when attempting to see details)? I understand that a lot of these microscopes are modular, but if you calculate the costs out - it's cheaper to know what you need from a scope and get it all in a package. It would cost an extra 300 dollars just to buy 4 PLAN optics for number 1 (600 dollar total after the scope), where I can get a scope with 5 plan objectives and a darkfield condenser on number two for six hundred. So for the same price, I get a much better brand and even get an expensive condenser. [I miss out on the camera, and the ability to perform measurements on the computer - but i have a digital camera, and if that slide ruler would work that's fine by me.] I just don't want to shell out 600 bucks if I really don't need all that tech. I just need to be able to do the goals I described. Sorry for the veritable novel, I just want to make sure I have the right tool for my needs, but there are no really good pieces of scholarship on this topic. You seem to know your field and respond to comments, so I figured I'd drop this here. I'll give you a "thumbs up" if you can help me out :/ this is a difficult decision.
You can physically measure the diameter of the field of view by putting a standard metric ruler on the stage and looking at the millimeter marks through the microscope or you can get a stage micrometer to measure field of view diameters that are less than one millimeter. Also, if you know the field diameter of one objective you can use a mathematical formula to calculate the field diameter of other objectives because the magnification of objectives is inversely proportional to the field size.
For a rough estimates you imagine how many of the object that you want to measure would fit across the field of view. For more accurate measurements you can use an eyepiece reticle and stage micrometer to improve your estimates. Search eyepiece reticle and stage micrometer to learn how these are used to improve the accuracy of measuring objects seen through a compound light microscope.
You have to know the diameter of the field of view in micrometers for all of the different magnifications (objective lenses). If you are using the low power objective, you use the diameter of the low power field of view as you measuring stick. If you are using the high power objective, the diameter of the field of view will be smaller but you measure the size of the object you are looking at in the same way. You just need to use the diameter of the field of view as your measuring stick in each case. As you increase the magnification the diameter of the field of view decreases.
This technique could be used to measure the size of the drop of water but, the nanoparticles would be to small to be visible with a light microscope, so you could not use this technique. You need to learn about how to calculate PPM. This technique only allows you to measure objects that are visible through the microscope.
Yes. The diameter of the field of view changes with the magnification power. The higher the magnification power the smaller the diameter of the field of view. You have to know the diameter of the field of view for each objective (magnification power) of your microscope. You then need to use the correct diameter for each objective when you are measuring an object.
Yes. The field of view changes when you change the magnification power. The diameter of the field of view decreases as the magnification power increases. The actual size of the specimen does not change but the size of the image you see compared to the size of the field of view changes.
+Vinay kumar Minajagi Yes, you can measure anything that you can see in the field of view of a microscope. If you can see dust particles in the field of view, you can measure their size using the procedure in this video.
+Glen Burger Dust particle size varies from 0.1 microns to 1000 microns. Can such small measurements be made? Are they visible in the field of view of microscope?
+Vinay kumar Minajagi Microns and micrometers are both used to represent the same unit of measurement. If you can see the dust as distinct particles in the field of view of a compound light microscope you can measure them. The resolving power of a light microscope is limited by the size of the wavelength of light passing through and around the specimen. Research resolving power of a compound light microscope.
it's 2020 and this video is still out here helping others. :'3
Thank you for putting that in plain English. I've read numerous definitions of the FOV, and they were worded in such a way that I think it was only for physicists to understand. Your video was extremely helpful.
I have a bio exam on Thursday and I’m pretty sure this is in there. Thanks for explaining it so well, Glen!
I've been through so many videos searching for a simple explanation, this one definitely helps! Thank you!
A rather very simple but detailed lesson.
Thanks a lot!!!!!!!!!
Great video! My college professor explained it so confusing.
This is awesome, thanks for making a clear and captivating video!
This was great - Very succinct and clear. I learned this in Zoology, but I'm finding that I have a more specialized niche in the field that requires more accuracy than this method. I'm really into mycology and mushroom taxonomy. There are tons of new species to discover and research, so it's an exciting field for biologists. I've gotten good at identifying fungi based on physical features, but the only way to be sure of a species is through microscopy. As such, I need to produce accurate measurements of a spore's length and width as well as a solid description of a spore's physical features. This leads me to ask a couple of questions:
1.) How would one precisely measure something like a fungal spore? I mean, the texts get painstakingly specific; you need a range (in micrometers of course) of the spore's length and width. I've seen small slides that have rulers (increments of 0.01mm) used for microscope calibration. They're pretty cheap, so I figured I'd probe your brain to see if those could be used to produce more accurate measurements than "guesstimating" how many spores would fit in the field of view. That is a MUCH cheaper alternative to buying an optical reticle. If I can spend 5-10 bucks to get a little slide ruler that produces accurate measurements, then I'd rather do that than shelling out 50-100 for a freaking eye-piece.
That somewhat leads me to my second question, which will take a lot more explaining:
2.) The only functional microscope I have at home is a toy that was given to me as a child. It's a tiny, ten dollar POS that has actually been quite useful up until I started to get frustrated with inaccurate spore-analysis. Maxed out at 250x, this little bugger can see the shape of most spores and with a LOT of brainwork and a LOT of guessing, I can produce an estimate to the spore size. However, I quickly learned that this simply is not sufficient for research or effective identification. We're talking, the difference that distinguishes one species from another is one or two micrometers in length. Furthermore, one has to be able to see the surface of the spores - are they smooth? rough? pitted? My little dinky toy just can't do that. So, it's time to upgrade.
I've been doing my research and I'm torn between two microscopes. I'll link them here and then explain.
1. www.amscope.com/40x-2500x-led-digital-binocular-compound-microscope-w-3d-stage-5mp-usb-camera-1.html?gclid=Cj0KEQjw5Z63BRCLqqLtpc6dk7gBEiQA0OuhsBIMvVDOYWKE2ytKFn1GS3bxgrdwblLrb7xiRdfqq_gaAuXd8P8HAQ#product_tabs_additional_tabbed
2. www.amazon.com/OMAX-40X-1600X-Biological-Microscope-Illumination/dp/B004EBRCEI?SubscriptionId=AKIAJT4BRIUI6ZD32TNQ&tag=disccamera-20&linkCode=sp1&camp=2025&creative=165953&creativeASIN=B004EBRCEI
My motives for the first are obvious: it's cheap, it goes to a high magnification, and comes with a 5 megapixel camera. The software included with this scope would allow me to do finite calculations like I described above. It has all the features that anyone in the field would expect a lab microscope to have and more. The only clear negative is the optics. They're industry standard DIN objectives. Everything else is amazing about this microscope, but as well all know - it's the optics you're really paying for n a scope. Hence the low cost of this one.
After realizing that identifying the shapes of the pores isn't enough - I need to be able to see the physical attributes of the spore wall as well- I came to realize I might need a scope with better optics. This led me to consider PLAN objectives and darkfield condensers. PLAN objectives simply produce higher-quality images - they give a more "flat" appearance to the FOV and exhibit "crisp" resolution. A darkfield condenser essentially changes the way light interacts with objects on the slide - the background remains dark, whereas something like a spore on the slide would be lit up. The resulting contrast allows one to see details more clearly. That being said, these also significantly increase the COST of a scope. So, this leads to my second question (finally):
If I was concerned about seeing fine details on a spore, would a cheaper microscope with DIN objectives be sufficient? I understand that staining may be necessary, and I may need to purchase some standard reagents. However, even with staining, do you think those details would be distinct enough to make out something like "pitting" on a spore? Or, is it just better to invest in the PLAN objectives and even consider a darkfield condenser right off the bat? Would it be better to get the darkfield condenser or the PLAN objectives (in other words, is it more important to produce a sharper image or to have more contrast when attempting to see details)?
I understand that a lot of these microscopes are modular, but if you calculate the costs out - it's cheaper to know what you need from a scope and get it all in a package. It would cost an extra 300 dollars just to buy 4 PLAN optics for number 1 (600 dollar total after the scope), where I can get a scope with 5 plan objectives and a darkfield condenser on number two for six hundred. So for the same price, I get a much better brand and even get an expensive condenser. [I miss out on the camera, and the ability to perform measurements on the computer - but i have a digital camera, and if that slide ruler would work that's fine by me.] I just don't want to shell out 600 bucks if I really don't need all that tech. I just need to be able to do the goals I described. Sorry for the veritable novel, I just want to make sure I have the right tool for my needs, but there are no really good pieces of scholarship on this topic. You seem to know your field and respond to comments, so I figured I'd drop this here. I'll give you a "thumbs up" if you can help me out :/ this is a difficult decision.
This video was super helpful, good job and thank you.
Thank You! this explained everything
Maybe if u would have done it with a specfic microscope but over all a good video. Pretty basic and simple. Thanks!
Great video. Thank you so much for the help.
Saved my life! lol. Thank you
Thank you for explaining this!
man that teacher is badass!!
Thanks!
thank you so much sir
Helpful, thank you.
How can you explain why calibration factor decreases as the magnification increases?
Very informative
good deal. thanks
thanks
How do I find out the diameter of a view field??
You can physically measure the diameter of the field of view by putting a standard metric ruler on the stage and looking at the millimeter marks through the microscope or you can get a stage micrometer to measure field of view diameters that are less than one millimeter. Also, if you know the field diameter of one objective you can use a mathematical formula to calculate the field diameter of other objectives because the magnification of objectives is inversely proportional to the field size.
Thanks!
Thx
but for this to happen then all cells must be in line?? or randomly arranged in the microscopic field??
For a rough estimates you imagine how many of the object that you want to measure would fit across the field of view. For more accurate measurements you can use an eyepiece reticle and stage micrometer to improve your estimates. Search eyepiece reticle and stage micrometer to learn how these are used to improve the accuracy of measuring objects seen through a compound light microscope.
but how do you determine the size based off the magnification you are using???
You have to know the diameter of the field of view in micrometers for all of the different magnifications (objective lenses). If you are using the low power objective, you use the diameter of the low power field of view as you measuring stick. If you are using the high power objective, the diameter of the field of view will be smaller but you measure the size of the object you are looking at in the same way. You just need to use the diameter of the field of view as your measuring stick in each case. As you increase the magnification the diameter of the field of view decreases.
ohhh i see thank you very much
how can i use this method, to determine how many PPM of nanoparticles are in a single drop of water.
This technique could be used to measure the size of the drop of water but, the nanoparticles would be to small to be visible with a light microscope, so you could not use this technique. You need to learn about how to calculate PPM. This technique only allows you to measure objects that are visible through the microscope.
ok thanks
is magnification power important in this case?
Yes. The diameter of the field of view changes with the magnification power. The higher the magnification power the smaller the diameter of the field of view. You have to know the diameter of the field of view for each objective (magnification power) of your microscope. You then need to use the correct diameter for each objective when you are measuring an object.
though the magnification power has increased, the size of the specimen will not be changed as a result of smaller field of view?
BTW. GREATTT VIDEO
Yes. The field of view changes when you change the magnification power. The diameter of the field of view decreases as the magnification power increases. The actual size of the specimen does not change but the size of the image you see compared to the size of the field of view changes.
Can we measure dust particle sizes using microscope?
+Vinay kumar Minajagi Yes, you can measure anything that you can see in the field of view of a microscope. If you can see dust particles in the field of view, you can measure their size using the procedure in this video.
+Glen Burger Dust particle size varies from 0.1 microns to 1000 microns. Can such small measurements be made? Are they visible in the field of view of microscope?
+Vinay kumar Minajagi Microns and micrometers are both used to represent the same unit of measurement. If you can see the dust as distinct particles in the field of view of a compound light microscope you can measure them. The resolving power of a light microscope is limited by the size of the wavelength of light passing through and around the specimen. Research resolving power of a compound light microscope.