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New to Microscopes and loving it!

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#1 oasmith

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Posted 23 March 2019 - 09:44 AM

Hi All,

 

Clouds and a full moon have stymied the galaxy bonanza for this time of year for me. So, i turned towards going tiny. I got an AmScope B120C, and have found myself fascinated just with cheek cells. I hope to get some other cell types at some point, but cheeks are easy. The scope has a camera attachment, and i took a few pictures hoping to see what you guys have to say about possible organelles. I have been able to identify the neucleolus, and vacuoles, and general cytoplasm, but was hoping to find mitochondria. I don't have the methylene blue yet, so i improvised with food coloring. It came out ok. One question i had was, how do i tell the difference between bacteria sitting on the cell membrane, vs mitochondria inside the cell? Aren't they going to be basically the same size?

 

Any suggestions for things i can experiment with at the organelles level? The scope goes up to 2500x, but i can never get in focus (just like how i can never get Jupiter in focus with my 2.3mm eyepiece (maybe if i lived in Atacama)).

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#2 TOMDEY

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Posted 23 March 2019 - 10:03 AM

Nice!

Yep, 2500x is ~empty magnification~ for even the best optical microscopes, even with oil-immersion, matching condenser, dark-field, etc. Will still be blurry because it's impossibly high mag (Heisenberg Uncertainty Principle). To go that high, need a good electron microscope!    Tom


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#3 B 26354

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Posted 23 March 2019 - 10:17 AM

Yep, 2500x is ~empty magnification~ for even the best optical microscopes....

Thank you, Tom! I've been looking into different sorts of microscopes in the $200-$500 range for a while now (haven't used one in about forty years), and wondering about the realistic usefulness of these (seemingly impossible) higher magnification levels. I value and trust your opinions on optical performance... so your comment is truly helpful.



#4 EJN

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Posted 23 March 2019 - 02:04 PM

On each objective, there is a number printed or stamped called the numerical aperture (NA).

For standard achromatic or plan objectives, the sequence is usually this:

4x - 0.10

10x - 0.25

40x - 0.65

100x - 1.25

 

The rule of thumb is that the maximum useable magnification is NA * 1000,

so for the 100x oil immersion objective the maximum useable magnification is 1250x

 

I've found that using eyepieces more than 10x is a waste. For example, to get 200x,

a 20x objective with a 10x eyepiece is far superior to a 10x objective with a 20x eyepiece.

Amscope sells 20x objectives, I have one.

 

As far as seeing mitochondria, they are at the limit of resolution of a light microscope,

and normally require either special staining techniques, or the use of differential

interference contrast, which requires a specially equipped microscope which is very expensive.


Edited by EJN, 23 March 2019 - 02:17 PM.

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#5 Microscopy

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Posted 24 March 2019 - 04:09 AM

Hi,

 

Mitochondria are very (I mean VERY) small and thus dificult to observe in a light microscope. If it's really necessary to see them, prepared slides of fixed and stained samples are used. Preparation techniques for mitochondria exist, but they're very demanding.

Also: squamous epitelium cells from the cheek/tongue are a poor model for animal cells. They're readilly available, that's their main advantage and that's why they're always used in biology classes and such.

But those cells are usually long dead. You can tell that by their very small, condensed nucleus, showing little or no detail, which condition cytologists describe as "pycnotic". Nuclei in living, healthy cells (or permanent slides made from them) show abundant detail: nucleoli, a more or less dense chromatin network and so on.

 

A magnification of 2,500x... Without going into optical theory, a simple rule of thumb is this one: maximum usefull magnification of a microscope objective is it's numerical aperture * 1000. So for a regular achromat 100x, N.A. 1.25, used at it's full aperture (meaning oil immersion AND immersion oil between slide and condenser) usefull magnification is 1,250*. That's known as "Abbe's rule".

Such an objective used at it's full aperture, will result in a resolution of around 0.28 µm. So 2 points separated by 0.28 µm will be seen as two separate points when a 12,5x eyepiece is used. Now, there's an entire library written on sense and nonsense of Abbe's rule, but it's a good starting point.

 

Cell organelles: they're pretty much considered sub-microscopic... IMHO a light microscope isn't the best tool for that kind of work.


Edited by Microscopy, 24 March 2019 - 04:10 AM.

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#6 oasmith

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Posted 28 March 2019 - 12:14 PM

Thanks for the info, very interesting stuff. I think i'll try doing some staining, and i haven't tried the oil immersion yet. What experiments do you like to do at the cellular level? I looked at some pond water the other day and saw tiny little critters wiggling about, and they looked smaller than my cheek cells, so i assume they are single-cell.



#7 Microscopy

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Posted 01 April 2019 - 07:26 PM

- ... One question i had was, how do i tell the difference between bacteria sitting on the cell membrane, vs mitochondria inside the cell?...

 

- ... I don't have the methylene blue yet,...

 

- Any suggestions for things i can experiment with at the organelles level?... 

My two cents:

 

 

... how do i tell the difference between bacteria sitting on the cell membrane, vs mitochondria inside the cell?... 

 

1. when using oil immersion at it's full aperture (which is far more difficult than it sounds...), dept of field is that limited that it's not all that hard to distinguish things on the cell surface, versus things underneath the cell membrane. Furthermore: at those magnifications and N.A.'s it's possible to recognize the bacterial morphology, alltough that's in itself is not full proof, as for example cocci can easily resemble mitochondria.

 

2. to distinguish between live mitochondria and eh... other stuff, one can use vital staining with a dye which stains mitochondria very selective: Janus Green B, applied as an aquaous solution 1/10,000 - 1/25,000 strength.

 

... I don't have the methylene blue yet...

 

Don't buy it, it's a waste of money.
Methylene blue has a hughe reputation in microscopy, not because it's such a fantastic dye in itself (it isn't), but because it's a precursor for a whole range of dyes and staining solutions, very often used in cytology/histology/histopathology.
In those solutions, oxidised, alkaline methylene blue is combined with eosin, forming a whole range of dyes (the azurs, methylene blue-eosinate etc...), making it possible to stain in a wide range of colors/shades ("metachromatic staining") between blue and red, using only one staining solution.
That group of dyes/stains/staining protocols is commonly known as the "Romanovsky stains". These are very often used, for example to stain blood smears.
Examples of such staining solutions are Wright's stain, very often used in Brittain and the US, while Europeans prefer May-Grünwald's solution, Giemsa's solution or a combination of both.

 

Keep in mind that decent staining is only possible on pre-treated samples, meaning fixation and follow up treatment. 

The only exception to that rule is vital staining, using less harmfull (not the same as harmless!) dyes such as Congo Red, Neutral Red and the above mentioned Janus Green B.

 

.

... Any suggestions for things i can experiment with at the organelles level?...

 

Jee, where to start... 

 

Online magazines such as Micscape have a lot of interesting stuff.

And than there are these. If I remember correctly those are called "books"/"manuals" or something like that smile.gif : 

 

books3.jpg


Edited by Microscopy, 01 April 2019 - 08:04 PM.


#8 oasmith

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Posted 03 April 2019 - 06:53 AM

Wow, thank you so much for the information! I definitely have a lot to learn for getting the staining technique right, but i'm excited to go through that learning curve. Vital staining seems especially challenging. And books? What are those?



#9 Microscopy

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Posted 04 April 2019 - 03:15 AM

Maceration, dissociation and such, combined with simple staining techniques (vital or post vital) were the methods histologists and cytologists used, not only in the pre mechanical microtome era, but even long after rotary and sledge microtomes were readily availlable.

 

Much of the literature on biology, microscopy, microtechnique etc. of times gone is availlable as free download through https://archive.org, including such classics as Sass' "Botanical Microtechnique", Chamberlain's "Methods in Plant Histology", Gray's "Handbook of Basic Microtechnique", Peacock's "Elementary Microtechnique", etc. to name only a few in English. archive.org is a goldmine.


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#10 Microscopy

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Posted 04 April 2019 - 10:50 AM

Here's a nice challlenge: this is a picture I made while assessing a Zeiss Standard GFL microscope equipped for phase contrast I overhauled. 

The picture is made in phase contrast, using a 100/1.25 Zeiss achromat phase achromat, a Zeiss N.A. 1.25 phase condenser and a dark green filter.

The picture has been post processed slightly: converted to BW, adjusted for brightness and contrast. 

 

As I wanted a "test slide" with a lot of small details, I scratched my tonge very hard (it hurt) with a slide, resulting in some deeper laying epithelial cells being harvested. 

 

The cell in the picture is probably pretty much alive and kicking. In the nucleus, a nucleolus is clearly visible (at the 11 o'clock position). The cytoplasm appears heterogenious, as it contains lots of cell organelles.

The white droplets, surrounded by a darker aura, surrounding the epithelial cell are suggestive for fat droplets (I drank a glass of milk prior to the sample taking).

 

So here's the challenge: what are all those intracellular grains/cell organelles? (Don't ask me...).

 

secc3.jpg


Edited by Microscopy, 04 April 2019 - 11:00 AM.


#11 Charles Funk

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Posted 14 May 2019 - 06:46 PM

"Thanks for the info, very interesting stuff. I think i'll try doing some staining, and i haven't tried the oil immersion yet. What experiments do you like to do at the cellular level? I looked at some pond water the other day and saw tiny little critters wiggling about, and they looked smaller than my cheek cells, so i assume they are single-cell."

 

Pond water can be very interesting. First time I saw a water flea, it was in a folded up position and as I was observing, it all of a sudden threw its appendages out, I nearly jumped out of my chair. It's like the horror movie Hollywood hasn't thought of yet.

 

I haven't had the ole microscope out in a while, I need to recify that. Been wanting to attempt to find a tardigrade (water bear.)



#12 Microscopy

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Posted 22 May 2019 - 03:30 AM

[...]

I haven't had the ole microscope out in a while, I need to recify that. Been wanting to attempt to find a tardigrade (water bear.)

 

A pretty certain way to find tardigrades: after a rain shower, squeeze out some moss samples from a roof or something in a jar and examine.



#13 Charles Funk

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Posted 31 May 2019 - 07:50 PM

A pretty certain way to find tardigrades: after a rain shower, squeeze out some moss samples from a roof or something in a jar and examine.

Hey, thanks for the tip! I'll make an attempt soon as I get time :)



#14 Tom Stock

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Posted 21 August 2019 - 03:10 PM

Okay so you sucked me in. I don't know how I ended up in this section of the forum.. oh yeah I remember. I purchased a 14mm Meade series 4000 UWA, often called the "best" eyepiece Meade ever made and it's been clouded over ever since it arrived... going on 2 weeks now and I still have not looked thru it.

 

Anyway, I also just purchased the exact same microscope you did and it is my first as well.  I do have a 10x-20x AmScope SE400-Z for surface mount electronics and have generally been happy with it considering the price, so I figured $180 is reasonable to dabble in microscopy.

 

I'm hoping I can identify a urinary tract infection in my pug's urine, because it would save me at least $200 a month in vet bills.  That's the story I told my wife anyway.

 

Scope will be here Friday and my wife will be out of town for a couple days. Perfect!

 

oasmith is the camera attachment from your telescope or something which came with the microscope?

 

Tom


Edited by Tom Stock, 21 August 2019 - 03:14 PM.

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