Proper Microscope setup for specific application. Help!

[micropath]

Hello all,

I’ve been lurking for a bit and now I’m getting ready to possibly make a microscope purchase. I would love everyones input on selecting the right scope and accessories for what I want to look at and also image.

I apologize in advance if the post is long. I just want to give you as much info as possible to start off with to hopefully make it easier to help me out. I appreciate the time and insight that anyone can give. Thank you all in advance.

As a preface let me just say that my interest in this is as a hobby as well as for personal interest, investigation, exploration and satisfaction.

As you are all well aware (much more-so than me I’m sure) this is a field that is overwhelming to the newcomer. I’ve read quite a bit but I’m left with the impression that I need to either spend thousands of dollars or that $400-$500 is sufficient. I need your help! Please help me sort out the right path. I’m on information overload aka analysis-paralysis.

I know I’m jumping in the deep end with my desired subject and I realize that I will need to build up my skills and techniques with larger specimens to start with, but I would like to make my initial purchase with my end goal as the requirements.

1. My main interest is in being able to see bloodborne pathogens in general and specifically Spirochete bacteria.

http://www.ucmp.berkeley.edu/bacteria/spirochetes.html

More specifically the Lyme spirochete Borrelia burgdorferi and other tick borne pathogens.

http://en.wikipedia.org/wiki/Borrelia_burgdorferi

2. My understanding is that darkfield would be my best option for seeing these specimens.
One thing I need to know is if a dry darkfield condenser would work well for these tiny specimens or do I really need to go with an oil condenser?

Below are two microscopes that I’m considering:
(It seems that Amscope and Omax are very similar. Any opinions about that?)

This one has a dry condenser. Would all the objectives work with this? Can I get enough dark field magnification with it for what I want to see?

http://www.amazon.com/AmScope-40X-2000X ... microscope

Or would an oil condenser type like this one be better suited? On this one there is a dedicated darkfield oil objective; is it necessary? It seems to already come with a 100x oil objective. You can see them both in the description.

http://www.amazon.com/OMAX-40X-1600X-Tr ... microscope

Here is the same scope with the oil condenser but without the dedicated darkfield objective. Why would they do this if you had to have it?

http://www.amazon.com/OMAX-40X-1600X-Tr ... microscope

Okay lastly (for now) if there was a choice between lamps what would be the best choice for a darkfield bulb in your opinion, a 3W LED or a 20W halogen bulb.

I’m sure there are many things that I didn’t think to ask but these were my main concerns:
Can either of those microscopes even see bloodborne bacteria like the ones I’ve mentioned, and if they can, do I really need the oil condenser to truly be able to get in and see them.

If neither of them will work please tell me so and if you have an alternate consideration please point it out. These scopes are more in my budget and thats why I’ve honed in on them but if they won’t work for what I want then it’s a waste of money.

Thank you all for reading and double thank you for any input and opinions.

edited: Mainly interested in seeing bacteria.

micropath

[Olympusman]

I think there are other microscopists on this website that can give you a more detailed answer, but bottom line:
1. You cannot see viruses with a light microscope, though some very sophisticated (and horrendously expensive) technology has approached rendering of viruses with light microscopy.
2. Darkfield imaging is not possible at extremly high magnifications (read 100x oil objectives). Not enough light gets through and the diffraction shoots down any hope of detail.
3. To render bacteria, you will need either a phase contrast setup or (ideally) DIC.
I think any pathologists in this group can provide you with more detailed information.

[micropath]

Thank you for the reply Olympusman I appreciate it. I would settle for being able to to see bacteria and take a pass on viruses if that's the case. Mostly I'm interested in seeing the spirochete bacteria. Thank you again.

[Tom Jones]

micropath,

Might I ask why you are so interested specifically in Lyme Borrelia?

You should be aware that the concentration of these organisms is so low, even in acute cases, that finding them in a blood specimen with a microscope is extremely unlikely, regardless of your method.

That's why nobody does it.

In general, the concentration of any pathogenic bacteria in blood, including Staph, Strep, and E. coli, even in fulminant cases, is so low as to render direct microscopic exam useless. Very rarely you might see an occasional bacteria or yeast cell during a routine CBC, but culture, or in the case of Lyme disease, serologic tests, are the only reliable methods.

Tom

[Pau]

To see bacteria the most useful and inexpensive technique is IMO the traditional brightfield in right stained slides.Another good approach is fluorescence with the adequate fluorescent dye. (but you can also see them alive with DIC or Phase Contrast)
Be aware that you can't identify bacteria with its morfology alone, you need specific techniques as bacteria have few morfological types and a big biochemical diversity. Inmunology or DNA analysys are the more adequate.
To identify them at the microscope you would need inmunofluorescence (specific monoclonal antibodies fluorescent labelled...) well beyond the amateur microscopist reach.

About darkfield, you will need a oil condenser and the objective must have NA under 1.1 . this is why the oil objective you linked has a diaphragm to lower the NA. Resolution will not be great but contrast will.

Any of the linked microscopes will be adequate to take pictures and none of them for very high quality (achromat objectives, Abbe condensers...). All them are chinese clones of the old entry level Olympus CH model (not a bad thing). I have a lot of similar scopes at the school.

[Eddie]

I believe the preferred method to view any spirochetes is with darkfield. The spirochete are between 20-30um long but a width of only 0.2-0.3um. Due to the small width being on the edge of brightfield microscopy, darkfield is need to view them properly.

It is best to get the dedicated DF objective or an objective that has an adjustable iris for best DF images. Also oil immersion gives you the best resolution. For best results and viewing, both the condenser needs to be oiled to the slide underneath as well as the slide needs to be oiled to the objective.

[Eddie]

Want to add that as Pau said, even though you may see spirochetes, you won't be able to identify which species it is without the more expensive equipment and procedures like immunofluorescence, unless the specimens are from a known culture. But for your initial venture into chasing spirochetes, DF is the least expensive way to go.

[Eddie]

Here is an interesting video of a Borrelia burgdorferi anchored to a platelete so they could calculate it's speed with use of a phase scope.
http://www.plosone.org/article/info:doi ... ne.0001633

[micropath]

I just wanted to say a quick thank you to all for the informative and helpful information and advice. I definitely have some more research to do. So off I go...

P.S. Any opinions about the light source for darkfield, brightfield, etc?
3W LED vs 20W Halogen

[micropath]

I guess I have one more question for those who would know if the eyepiece specs matter in relation to the objective.

Here are the two scopes that I am likely to choose between:

http://www.amazon.com/OMAX-40X-1600X-Tr ... microscope

and:

http://www.amazon.com/OMAX-40X-2000X-Da ... +darkfield

The only difference seems to be that one comes with:
Eyepieces: widefield WF10X/18 and WF16X

and the other comes with:
Eyepieces: widefield WF10X/18 and WF20X

Will one of these be better suited to the oil darkfield objective and condenser?

Thank you again for the help everyone.

micropath

[discomorphella]

Just to amplify (no PCR pun intended) what Tom has already stated. No clinical or academic lab uses direct visualization of unstained bacteria, be they bacilli, cocci, spirocchetes or any morphology really. Stained Brightfield or Epifluorescence with, as mentioned, the proper reagents and techniques (e.g, Gram's stain, or the fluorescent modern equivalent like ViaGram from Invitrogen, or fluorescently labeled Polymyxin, there's a large list) are the mainstay. You can see indeed see live bacteria with higher power brightfield, DIC or Phase Contrast, but other than saying, "Yup, those are bacteria", you're not going to get much more information. And as Tom said, even in really purulent material, e.g., something from a staph-induced abcess for example, you are not going to see that many clusters per high-power field in a Gram's stained smear. As for Darkfield, nearly everything in a real world smear is going to show up, and as Tom said, most of what shows up won't be the bacteria you're looking for.
Don't let this discourage you however. Gram's stain is easily done by the amateur, in fact on ebay you can find places that sell kits. If you are truly interested in epifluorescence, you can rig up a serviceable instrument using UV or blue LEDs and not all the dyes are impossibly toxic for a careful amateur. That being said, they are not inexpensive either. But enough reagents for Gram's staining 100's of simple smears will only set one back <= $50 or so. And even a simple single stain like Loeffler's Methylene Blue is perfectly good for amateur bacteriological staining.
http://www.photomacrography.net/forum/v ... highlight=