I wanted to do a write up for those who are interested in using a microscope to check their blood for Lyme and co-infections. There’s a lot of information to absorb when looking at buying a microscope, and I wanted to create a post that people can refer to if they want to get into microscopy. I really think microscopes are an invaluable tool when it comes to diagnosis.
Mods, if you’re not into microscopes, you don’t have to read this lengthy post.
First, A Quick Disclaimer: I am not a microscope expert. Six months ago, I knew literally nothing about
microscopes. With some research online and some guidance from others who have more experience, I’ve been able to learn enough to check my blood for infection. Therefore, if something on this page isn’t accurate, let me know so I can edit my post accordingly.
Also, I will be talking about
compound microscopes, which are the type we use to view tiny organisms. Other microscopes, like stereo microscopes, have no use for Lyme diagnosis, so are being completely ignored. Please don’t buy a stereo microscope and expect to see your blood cells. Onward!
Misconceptions about Microscopy• Borrelia is NOT difficult to see under a microscope. They are fairly unique in their appearance, and after a bit of practice, you can pick them out quite easily under magnification. More on that later.
• You do NOT need to spend an insane amount of money on a microscope to see Lyme and co-infections. A basic microscope in the $200 - $500 range will work just fine.
• You do NOT need any prior training or experience in biology or medicine.
• It is NOT difficult to use a microscope once you learn a few simple techniques.
• Just because you don’t see spirochetes in your blood, doesn’t mean they aren’t there. They could be hiding in blood cells, or any other part of the body for that matter. They could also be in their round body forms (cysts), at which point they become harder to differentiate from other round “things” in the blood.
Reasons To Start Using A Microscope• It’s relatively inexpensive. It’s cheaper than doing a full IGENEX panel (I’m NOT advocating skipping IGENEX, but I’m just putting it into perspective).
• It’s definitive. If you see spirochetes in your blood, well then, they’re there. You’re not relying on testing based on antibodies, which remain in your system for years even after the infection is cleared.
• It’s easy. As mentioned, no special training required.
• You can test as much as you like, and don’t have to differentiate between strains. For example, if you’re doing a Babesia FISH test from a lab, a few samples are tested, and if nothing’s there, the test is negative. If you have one of the dozens of Babesia species that the lab doesn’t test for, the test is negative. On the other hand, under a microscope, you can check as many samples as you like. It might take 20+ samples before you find a Babesia parasite in your blood. No lab will work that hard for you! Plus, it doesn’t matter what strain of Babesia you see, it’ll look like Babesia, and give you confirmation.
Different Types of MicroscopesThere are 3 basic types of microscopy we’ll take a quick look at:
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Brightfield Microscopy: The most basic and cheapest option available. The sample is lit directly from below. Spirochetes can sometimes be difficult to detect, though with a bit of practice, completely do-able. Brightfield is great in conjunction with stains (like a wright-giemsa stain) for detecting co-infections like Babesia, Bartonella, Mycoplasma, and Anaplasma. You can easily make out larger parasites like nematode larvae. No special requirements for everyday viewing.
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Darkfield Microscopy: This technique blocks out light from directly below the sample, and instead, illuminates it from the edges of the sample. The resulting image is a black background with organisms lit up like a Christmas tree. Spirochetes are very easy to detect in darkfield, though it’s less commonly used to look for co-infections. Darkfield is more expensive than brightfield as it requires a few special components: a stronger light source, a darkfield condenser (oil condenser if planning on 100x magnification), and an objective lens with an adjustable iris (only if planning on 100x magnification).
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Phase Contrast: I actually don’t know much about
phase contrast. I can tell you that phase contrast objective lenses are much more expensive than regular ones, but the image quality is very good. It provides much better contrast than brightfield, so it’s easier to see organisms. While it would be cool to have, the cost often makes it prohibitive for beginners.
Parts of a MicroscopeOk, let’s do a quick primer on the different parts of a microscope. Here’s an image that labels all the major components:
1.bp.blogspot.com/-97yIqXkoOHY/Vhal14UU9-I/AAAAAAAAD_I/riRHcnCDIZc/s640/label-microscope-answers.jpg•
Light Source/Illuminator: This is what illuminates your sample from under the stage. Older microscopes use halogen bulbs, while newer microscopes use LED bulbs. Both work fine. If you’re using a halogen bulb, you might want to buy a blue filter to put under the condenser to cool the red hue of the final image. If you’re planning on doing darkfield microscopy, you will need a brighter bulb than if you were just doing brightfield work.
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Condenser: The condenser sits between the light source and the stage. It gathers light from the light source, and concentrates it into a circle of light that illuminates the specimen on the stage.
The most basic condenser is the Abbe condenser. It has 2 adjustments. The first adjustment moves the condenser closer or further away from the stage. The second adjustment
opens or closes an iris diaphragm within the condenser, which adjusts the diameter of the beam of light allowed onto the specimen. Using these two adjustments, you can manage the brightness and contrast of the final image.
The NA of a condenser notes its maximum numerical aperture. This is important because, for optimal image quality, the NA of your most powerful objective should not be more than the NA of your condenser. So for example, if you have an oil-immersion 100x objective with a NA of 1.25, you’ll need a condenser with an NA of 1.25.
Any condenser with an NA of 0.95 or greater should be used with oil. This involves putting a small drop of immersion oil on the condenser lens, and then raising the condenser until the oil on the lens comes in contact with the underside of the slide sitting on the stage. This ensures that all light rays are concentrated onto your sample.
If you want to do darkfield microscopy, you’ll need to ensure that you have a darkfield condenser. Darkfield condensers block out light from directly behind the sample, and instead illuminate it from the side. This gives the effect of light-colored organisms on a black background. Again, make sure the NA of your condenser is at least that of the NA of your strongest objective. Most budget darkfield microscopes only come with a dry darkfield condenser, which is really only useful up to 40x. Again, if you want to do 100x darkfield, make sure you have an oil darkfield condenser.
There are other, more expensive types of condensers besides Abbe (aplanatic and achromatic) which make optical corrections for a better image. However, I don’t have any experience with these.
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Stage: This is what your sample slide sits on. Using adjustment knobs, it moves in two directions so that when you’re using the microscope, you can move the sample around quite precisely.
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Objective Lenses: These are the most important (and expensive) parts of your microscope, as they do most of the magnifying. Often, a microscope will have 3 or 4 objective lenses to allow for different magnifications. They are locked onto the nose piece, and then revolved into place depending on which one you want to use. The three most common magnifications for our purposes would be a 10x, 40x, and 100x, with 100x being the highest magnification available.
At 100x, you’ll want to ensure that it’s an oil immersion lens with an NA above 1.0 (typically 1.25). Like with the condenser, the oil immersion will vastly improve the quality of the image at the high magnification. More on the specific technique for this later.
The cheapest and most basic objective lenses are achromatic lenses. These lenses have great focus in the middle of the image, but lose some of their focus near the outer edges. Plan-achromat objectives are a little more expensive, but retain their focus much better to the outer edges of the image. There are other, even more expensive types of objectives that create even better quality images (fluorite objectives), but I don’t have any experience with these.
If you want to do darkfield at 100x, you’ll need a special objective that has an adjustable iris in it. This adjustable iris can reduce the NA of your objective while retaining the strong magnification. A 100x oil immersion darkfield objective with an adjustable iris will be much more expensive than typical 100x oil objectives, and generally aren’t included on low-budget darkfield microscopes. However, all objectives are easily replaceable, so you could always buy it separately if you were so inclined. No special iris objectives are needed for darkfield at lesser magnifications like 40x and 10x.
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Eyepiece: This is what you look through to see the sample. The most basic eyepiece is a monocular. This means that you look through a single tube with one of your eyes. I highly recommend spending a little more money and getting a binocular however, as looking through two tubes will make viewing much more pleasurable. You can also get a trinocular if you want a third tube for someone else to look through, or more commonly, to hook up a camera to, in order to capture video of your discoveries.
The most common magnification for the eyepiece is 10x. A 10x eyepiece combined with a 100x objective lens, will give you a total magnification of 1000x. You can also get 20x eyepieces. However, upping the eyepiece magnification is akin to digital zoom on a camera: you’re not gaining any additional resolution, you’re just making the image larger. I would suggest starting with a 10x eyepiece, and upgrading to a 15x or 20x later if you feel the need.
You can also choose the eye relief of you eyepiece, measured in mm. The larger the eye relief, the larger the area of the actual eyepiece lens. A larger eye relief makes it a little easier to look down the tube.
Certain microscopes also have adjustments for inter-pupillary distance, and independent focus on each of the binocular lenses (for those whose vision differs from one eye to the other).
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Focus Knobs: There are generally two focus knobs: a course adjustment and a fine adjustment. These knobs raise or lower the objective lens, thus affecting the focus of the final image.
What to Look for In a Microscope and Where to BuyTraditionally, the “Big 4” microscope manufacturers have been Olympus, Nikon, Zeiss, and Leica. Unfortunately, buying a brand new microscope from one of these manufacturers is going to cost you an arm and a leg. You have several other options however:
• You can buy a “Big 4” used. There are a ton of microscopes on eBay, but on eBay you risk getting a microscope that has not been taken good care of. If you go the eBay route, try to buy from a reputable reseller or refurbisher with a history of microscope sales. Alternatively, there’s a number of microscope forums online where you can inquire about
resellers/refurbishers. People who are involved with these online communities really know their stuff, and will sell you a quality product at a fair price.
• Often, even a used “Big 4” will be cost-prohibitive. There are a few companies which no longer make microscopes, but were, back in the day, very reputable. Two of the most prominent were American Optical (Spencer) and Bausch and Lomb. Again, look on eBay or check out microscope forums for resellers. These microscopes usually cost in the $200 - $500 range. Don’t worry that the microscope you’re buying is 40 years old; if it’s well taken care of, or refurbished, the image quality will be just as good as a brand new microscope. In the 60’s to early 80’s, these microscopes were found in doctor’s offices and labs across the world; they were very popular.
• If you really want to buy a new microscope, you can check out Chinese manufacturers like Omax and Amscope. These are in the $200+ range for basic setups, and sell on Amazon and eBay. Microscopy experts would tell you to steer clear of these, because the image quality and build quality will be inferior even to a 40 year old American Optical, and certainly to a “Big 4” microscope. However, I’ve seen some pretty decent quality videos on YouTube of blood smears viewed with these cheaper brands.
Honestly, I prefer the tried and true manufacturers, but I don’t think Omax or Amscope are completely garbage either. Just a word of warning: sometimes Omax or Amscope will sell microscopes as “Darkfield”, but what they don’t tell you is that the darkfield is only good up to 40x. If you want to do 100x darkfield, you need to buy an additional oil darkfield condenser and an additional oil darkfield 100x objective with an adjustable iris. Even then, the built-in light source may not be strong enough.
Other Things You’ll Need• immersion oil
• rubbing alcohol
• slides
• cover slips
• a diabetes lancet
• giemsa stain solution if you plan of looking for co-infections
How to Look at a Basic Blood Smear Under BrightfieldIt’s probably easier to watch someone explain this stuff on YouTube than have me write it out, but I’ll do my best and you can always go watch a video later:
• Prepare your slide by cleaning it with rubbing alcohol. If you're planning on using a stain later, instead of rubbing alcohol, use soap and water to clean the slide. Alcohol can fix any pre-existing bacteria on the slide, giving you a false-postive when you stain it later (thanks TOOTY!).
• Next, use the diabetes lancet to get a small drop of blood from your finger.
• Carefully put the drop of blood from your finger directly onto the slide. Don’t push your finger into the slide or smush your blood droplet. Also, don’t put the blood droplet in the middle of the slide. Instead, put it 3/4 the way towards one edge.
• Take a cover slip, and push one edge of the slip up against the slide. Angle the slip to 45 degrees, and move the edge that’s touching the slide towards the blood droplet. When the edge of the slip comes in contact with the blood droplet, the blood will run along the entire edge of the slip and create a line of blood. Keep the slip in contact with the slide.
• Next, quickly drag the edge of the slip across the slide, dragging the line of blood with it. This will create a blood smear. It will take some practice, but soon you’ll be able to make your smears thinner or thicker, depending on your viewing preferences.
• What you do next depends on what you’re looking for. For co-infections, you’ll want to let the smear dry, then stain it (explanation on this later), and then view it. If you’re looking for spirochetes, right after you smear the blood and it's still wet, you’ll want to apply a cover slip over the smear. This will allow you to use oil immersion without making a mess of your blood sample.
• Next, make sure your condenser is in it’s lowest position, using the knob to adjust. Using a dropper or a thin dowel, apply a small drop of immersion oil to the lens of the condenser. Place the slide you just created (blood side up), into the holder on the stage. Then raise the condenser until the oil on the condenser lens makes contact with the underside of the slide. Note: using oil on the condenser is not necessary if you’re going to use a magnification less than 100x.
• Then, place another drop of immersion oil on top of the cover slip. Rotate the 100x objective into place, and use the course adjustment knob to slowly lower the objective until the lens just makes contact with the oil on top of the slip. DO NOT use oil on objective lenses that aren’t meant for oil (i.e. less than 100x). If you're using less than 100x, you don't need to apply oil, and can go to the next step.
• Now, switch on the power source to light up your sample. The light source usually has different levels of illumination, so you can fool around with this. If your microscope uses a halogen bulb, don’t keep it on full power for longer than you have to, as you’ll burn out the bulb quicker. Instead aim for 3/4 power. Don’t forget, with a halogen light source you might do well with a blue filter for underneath the condenser; it’ll cool the reddish image. Now, you won’t see anything right away. Using the course and fine adjustment knobs, you’ll need to find the proper focus, slowly lowering the objective lens until your blood cells come into view.
• When the image comes into focus, you can further adjust the iris on the condenser to fine tune the image. For darkfield at 100x, you’ll have to adjust the iris on the objective as well. For further information for darkfield, check out some YouTube videos.
• Now that you have your image in focus, you can use the stage adjustment knobs to move the stage back and forth to see different parts of your blood smear. Note that as you look around your smear, you’ll constantly have to re-adjust the fine focus. This is especially true at 100x magnification.
What You’re Looking For In The MicroscopeWell there’s TONS of information and pictures online, not to mention TONS of home microscopy videos on YouTube, but I’ll try to post some basics here.
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Red Blood Cells: These are the circular things that take up most of your view. Depending on if they got damaged when the cover slip went on, they may look spiky and deformed.
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White Blood Cells: Slightly larger than red blood cells, somewhat clear, with dots in them and visible nucleus. There are less white blood cells than red blood cells. This picture shows a single white blood cell surrounded by red blood cells (note: this sample has been stained):
images-mediawiki-sites.thefullwiki.org/08/2/8/1/7483332245899785.jpg•
Spirochetes: These will look like very thin, tiny worms, 1-2 red blood cells in length approximately. In brightfield they will be very faint. They will be moving in Browian motion, meaning erratic, random movement. Sometimes they will look like a string of pearls, or a thin line with a bulb on each end, or other times like a short dumbbell. Look for pictures and videos online for confirmation; I don’t want to post YouTube videos without permission.
Once you get some practice and see a few, they’re easy to pick out, since they don’t really look like anything else in your blood. Interestingly, I often don’t see spirochetes in my blood smear right away, but if I leave the smear over night with a cover slip on, the next day I see them in my blood. I think they’re hiding in the blood cells, and come out to play when the blood cells start to die.
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Babesia: You will only see Babesia if you stain your sample. They reside inside red blood cells, and the stain will make them darker. They appear as halo’s, tetrads, and various other shapes. Check out a bunch of images here:
/www.cdc.gov/dpdx/babesiosis/gallery.html•
Bartonella: Like Babesia, you will only see Bartonella if you stain your sample. They are much smaller than Babesia and Borrelia, and tend to stick to the outside of red blood cells. They look like tiny dots basically, and can be difficult to differentiate from other things in the blood. Here’s a page with a bunch of Bartonella pictures, and some Babesia as well:
www.babesiabook.com/articles/bartonellabloodsmears.html•
Other Co-infections: That’s for you to look up online. You should be able to see stuff like anaplasma and mycoplasma with some practice.
For larger parasites like nematode larvae, I tend to look at my blood under 10x magnification and scan the entire smear. If I see a worm-like object (much, much bigger than spirochetes, like several dozen red blood cells in length), I then zoom in with the 40x or 100x. No stains are necessary for these larger parasites.
Well, I think that’s it for now. I’d love to do a post on staining but I’m still building my experience in that area so I’m going to hold off.
Happy hunting everyone!
Post Edited (Mustard Seed) : 12/9/2016 7:14:51 PM (GMT-7)