new member and zeiss questions.

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solartje
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Post by solartje »

solartje wrote:ok now i understand the lengths..

my goal is checking my blood in live blood smears for educational purposes. me and my wife have positive serology for several bacteria. i've been studying them for 2 years now and now i want to see them with my own eyes (even if it means small odds without dna staining or cultivation). hence the darkfield and the 400X and 1000x need.

i'm gonna start with darkfield live view and safe staining techniques in brightfields.

But honestly... now i found this forum and i look at all the pictures ,maybe photomicrography could become a new hobby. its fascinating. i'm constantly looking for things to put under my microscope and amazed at the new worlds i'm seeing :-) diatoms especially are amazing and i love the compositions of them. Does this mean i'm getting hooked? :roll:

ps with the NA of 1.00 i could get max of 1000x without losing resolution right? or is that only in theory. thats why i was thinking of the combo 60/0.90 and X16 instead of the 100/oil/iris and the X8. both will give almost same resolution and almost 1000X total mag. but it would save me the trouble of having to oil the objectives.


PS2, my next problem will be the light. it has the standard 6v 15w halogen lamp and i guess it wont be strong enough for 100x darkfield?
i'm reading alot about that, and not sure whats the best option. diy led replacement , find a used 100W 12V old zeiss parts but that will be costly, or find a 30W 6V but guess thats not enough either. (http://zeiss-campus.magnet.fsu.edu/arti ... logen.html ) guess i need a led with 4k lumens wich is ALOT. ) pau did you use led lightning and if not is the 15w lamp really a big problem for darkfield or only like 10% less or something in contrast?)


Edit: my slides arrived and did a test on 40x. set the köler correct so all planes are equal. in BF the 40X works nice. So i decided to make a darkfield stop and test the 40x/65 in df with the 0,9 cond it came with. It worked , because without specimen it was black and with i saw something, but the light intensity is WAY to low. even if i turn my 15w to max, i barely can see something in 40x, so i can only imagine with the 100x obj this will be even worse. sight. the big Q, how many lumens is needed :-) anyone with a 100x obj in ultradf that can tell me how many lumens there setup has?

Pau
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Post by Pau »

Very likely your home made DF adaptation isn't working well. Zeiss condensers are not good for use DF stops because its construction: a DF stop must be placed very close (or ideally at) the diaphragm position that in these condensers it's placed inside the condenser close to the upper flat glass. Also at 40/0.65 DF with a dry not dedicated condenser is very tricky even when placing the stop at the right position. With a phase condenser it's easier to place a DF stop at the right position.

With the ultracondenser you will get excellent (although not very intense) illumination when immersed (not light without oil!) and well centered and at the right height. Until very recently I was using the 15W rear tungsten lamp or the 10W built in halogen in my other stand with no problems

Here can see a DF image taken with this lamp:
http://www.photomacrography.net/forum/v ... php?t=9256
Pau

Pau
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Post by Pau »

solartje wrote:...
my goal is checking my blood in live blood smears for educational purposes. me and my wife have positive serology for several bacteria. i've been studying them for 2 years now and now i want to see them with my own eyes
I hope that you'll not be able to see any bacteria in your own blood!. This will be indicative of a very severe infection, if you have antibodies this means that you have ben infected in the past or that you could have a cronic infection, or at most you could have very few bacteria circulating. If you were affected by a septicemia I'm sure we werent be discussing about microscopes at this moment.
I can be wrong, I'm not a medicine doctor, but would be very surprised....
Pau

solartje
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Post by solartje »

Hi pau, that DF picture looks amazing. If i can achieve that with the 15w then i'm one happy man.

Still you are comparing a 40x vs a 100X . The Light-Gathering Power of Selected Objectives should be 4x smaller on the 100X at equal NA. I think only way to tell for sure is just test it. A cheap 2000lumens led flashlight on bateries that can be focused might be an option. its small enough to fit into the socket. it wont be perfect, but it might work. buying one made for the zeiss is to expensive.

About the bacteria, i did find 3. i didnt put oil around the side of the coverglass, so the slide got dry after some hours and the RBC are bursting and putting so much debris that it has not much sense to continue looking for more, but i found 3 when the plasma was clear. it wasn't brawnian motion. everything was sitting still (smear was well spread) and i had to move a complete field view away about every 30sec with the stage knob to follow him.

I have been ill for some time and i had to give up my dayjob. I have stage 3 borrelia infection ( and some others) for 2 years and my wife for 6 years. i tried to film it, but my 30$ camera and the low light makes it horrible. its visible in the movie, but not worth posting. setting up a good webcam diy is a project for later.

I wasn't expecting to see so much motile bacteria to be honest. i though id only see biofilms and cysts (ive spend 3 months on IV and 3 months of oral AB to get red of most of the motile borrelia) and expecting to only see movement with timelaps, but in BF without a contrast method its impossible to say what im seeing, but the little bacterie was moving so fast that it was pritty easy to see in between all the rest . it moved in and out of focus several times , swim next to white blood cells like if he didnt care and just swimming around in the plasma. curious to do some staining later

I really want to thank you for you help. i've seen you react and help almost in every post i was following. you are a greath + for this forum.



Ps; i'ven been looking for planapo 100x with iris, and they are so rare, the prices are redicoulos. ive seen asking prices of 500$ up to 1600$ for a used one. Where-as planapo 40x/1.00 oil are way more common and alot cheaper (and more usuable for diatoms i i want to study them later on). As i'll have a NA of 1.00, i guess max total magnification will be x1000, gues i'm cheaper of with a 40x oil and also buy a 16x or 25x eyepiece , then to use my 8x eyepiece and buy a 100x. same detail/total magn but alot cheaper. i will lose some field view, but thats not my main concern. i need to be able to see the sop/cists/biofilms as close as possible. i can always scan with the 10x or with the 40x and 8x eyepiece. Am i right in this assumption?

Pau
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Post by Pau »

solartje wrote: Ps; i'ven been looking for planapo 100x with iris, and they are so rare, the prices are redicoulos. ive seen asking prices of 500$ up to 1600$ for a used one. Where-as planapo 40x/1.00 oil are way more common and alot cheaper (and more usuable for diatoms i i want to study them later on). As i'll have a NA of 1.00, i guess max total magnification will be x1000, gues i'm cheaper of with a 40x oil and also buy a 16x or 25x eyepiece , then to use my 8x eyepiece and buy a 100x. same detail/total magn but alot cheaper. i will lose some field view, but thats not my main concern. i need to be able to see the sop/cists/biofilms as close as possible. i can always scan with the 10x or with the 40x and 8x eyepiece. Am i right in this assumption?
Take a look at:
http://www.olympusmicro.com/primer/anat ... ation.html towards the end of the page
The range of useful total magnification for an objective/eyepiece combination is defined by the numerical aperture of the system. There is a minimum magnification necessary for the detail present in an image to be resolved, and this value is usually rather arbitrarily set as 500 times the numerical aperture (500 × NA). At the other end of the spectrum, the maximum useful magnification of an image is usually set at 1000 times the numerical aperture (1000 × NA).
They are not absolute numbers but a very good approach. With a 100X working at NA 1.00 you can't get any advantage from 15X eyepieces, except in the case of some visual problems.
Because resolution is a function of system (mainly objective) NA, you could in princiiple get the same with a 100X objective and 10X eyepieces than with a 50X objective and 20X eyepiece if both have 1.00NA (personally I prefer the 50X with 10X eyepieces though)
Pau

solartje
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Post by solartje »

i filmed this today with my old objectives and scratched eyepieces and crappy camera. my 10w lightbulb in brightfield is so soft at 100x it just shows every single scratch in every single optic.

I was told i won't find bacteria.

then this vid will be interesting. Looks like a groep of borrelia burgdorferi forms with blebs at the end invading/escaping my wifes platelet (or small rbc?). i'm not a docter but this doesnt look like artifacts in brownian motion or some fat particuls to me. That form is a known form for bb. to bad my stain kit hasn't arrived yet. i'd love to stain this with giemsa.

https://vid.me/bJVd

Pau
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Post by Pau »

As I said I'm not expert, this seems both interesting and disturbing
i filmed this today with my old objectives and scratched eyepieces and crappy camera. my 10w lightbulb in brightfield is so soft at 100x it just shows every single scratch in every single optic.
This isn't related tho the lamp power but with the low effective aperture you allways get with a high magnification objective. The eyepiece damage, in special at it's upper lens is more visible with low effective aperture systems, both visually and with the camera working afocal (it also applies to dust on sensor and even to the particles or defects suspendend inside your own eyes (eye floaters)
Pau

solartje
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Post by solartje »

yes, the upper lens of my eyepieces are the worst. even with good cleaning, the scratches are just all over the place. the condenser is not perfect either, but by moving it just slightly lower or higher then perfect they are hardly visible.

I haven't found a good deal yet on new eyepieces. found 16x, but like you said not ideal. I did find a PL fluotar 100x with iris for sale for 200€, but was hoping to get even better deals . guess i'll wait for bit longer to see if better deals come up, and if not, i think the pl fluotar will be a nice objective paired with 10x widefields.

and yes, the vid was interesting but disturbing. i was happy and sad at the same time. she has been on 12 months of pulsating different Antibiotics, but borrelia burgdorferi and the other coinfections are a pain in the *** to get rid off. she was getting better and is now worse again.

Pau
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Post by Pau »

A Pl fluotar 100 1.32 for $200 seems a good deal to me if it's the diaphram bundled version and it's corrected for 160 tube, usually ebay sellers ask more for this objective...but often they remain unsold for long time. I have one (ph version) and it's nice. For the Zeiss microscope it will work at its best if paired with Leiz Periplan eyepieces, but you want thelonger ones for 160 microscopes. Another good option is the Zeiss Neofluar, not full flatfield corrected but very good.
Pau

solartje
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Post by solartje »

yup, diaphragm and 160. i'm tempted but if i need leitz eyepieces then my other objectives wont be optimized. I think i'm better at staying with all zeiss pieces. That and my budget can't go that far. I got 100€ for an objective and 50€ for the eyepieces.

I did buy an objective though but the deal was to good to leave it. 40x plan apo with iris for 80€ no delamination. gonna use that one now and then sell it with enough profit hopefully so i can afford a zeiss 100x plan apo with iris when one comes up. (or try to save up some more so i can use both the 40x and 100x plan apo. that would be a killer setup for what i want.

solartje
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Post by solartje »

haven't recieved my darkfield yet, so while waiting, here another vid in brightfield. sorry for the quality, but i'm more interested in what i'm seeing atm. i'll hope to be able to post better things soon

https://vid.me/WMVx

looks like a spyrocheate having fun.

and string of pearls formation (he is lying in the focal plane in the last 10 seconds)

https://vid.me/ae84


i'd love to do a time-lapse in the future and see if they change forms. i'm not sure but i think they change from blebs , to rods, to string of pearls the older they are and the longer they are in the bloodplasma.

Ps my ultra darkfield condenser arrived, and... WOW ! single best buy by far ! It is as good as new, not a single scratch to find.It took some tuning, but once well centered, the detail is amazing ! i can't use my leitz 100x, but with the zeiss plan 40x 0,65 and the ultra the quality is already amazing. i can even see spiro's swim at 320x !! it's amazing (plan 40x and the 8x kpl). They also "pop up" alot more in darkfield, wich makes it easyer to find them. i wasn't expecting much to be honest, but this is amazing.


To bad i can't take good pictures yet. i'm looking for some kpl's on ebay.

solartje
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Location: Belgium -Europe

Post by solartje »

decided to buy a used webcam so i can do some time-laps on a budget. (still haven't found a good deal on new eyepieces)
found a used logitech C920 and i read good things about it, uses a small 1/3" sensor, and has 3Mp true image resolution. has some special thing that is supposed to be good for low light (like my darkfield).i saw a video on youtube of a microscopist that uses it and it looked pritty good. no idea of in darkfield the quality be as good. the sound is not working on the webcam, and comes with no box, paper, or whatever, so i'm probably getting it for a good price. i think its my best option for time-laps in 1080p


Also i have a crazy idea. not sure if its possible?
:
I would use the same technique (using a used stage with a very fine point) that is used to pick up diatoms so you can put them on a separate slide , but use this to pick up specific red blood cells and maybe even spirochetes? i know they are allot smaller then diatoms, but would it be possible to "fish" them?


i also have a question:

when i turn the iris of my 40x planapo to the max (NA of 1.00 and even as low as 0.85) i find i get TOO much light from each red blood cells, and it kinda gives too much brightness to see even the smallest fat particles in the plasma. (like looking at stars in a city full of light). for video's with my digital camera it helps to be able to increase the NA ,but when looking with the eye i almost always turn the NA to around 0.80. is my darkfield not setup correctly ( i think i got it well centered and at the right hight) or is this normal? just a shame to spend alot of money on a objective that has a high NA, if I'm not using it.


I'm having another "problem", but i guess nothing i can do about it, except not using the full wide length. with the oil underneath the glass, when i move the stage around, as my slides are wider then the gap in the stage, the stage gets oiled if i move my preparation too much around and my glass kinda gets vacuum sucked and wont move after some time. (ow and i broke my first glass :roll: guess i'm not a virgin anymore. forgot to oil the objective and i focused too far ) :oops:

discomorphella
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Post by discomorphella »

I just noticed this thread, sorry for not responding earlier. If you are trying to find specific pathogens, such as a spirochete or other bacteria for example, then you are much better off obtaining some specific primary antibody (or antibodies) against those species, and a good fluorescently labeled secondary antibody and using indirect immunofluorescence rather than trying to see things under DF. First of all the IFA staining is very specific, under DF you won't know if you're seeing the actual bug you're interested in or something unrelated that looks similar, and secondly, its a far more sensitive technique.

David

Edit:
You can also use a similar technique, similar to affinity chromatography, to selectively adhere pathogens to small particles or a slide or coverslip using immunoglobulins attached to a slide for example. This way you can collect a large fraction of the pathogens in a small volume of blood and selectively stain them, examine them etc.

solartje
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Post by solartje »

hi disco, thanks for your reaction.

I've been thinking about immunofluorescence, but I'm afraid this might be over my head. i have no medical background, just a very big curiosity and appetite to understand my illness better and i have a science grade, just not in biology or anything related. I've talked to my Md about my findings and he also suggest IF.

Is this IF staining, something you would advice or not recommend for a "in the basement experiment at home" ?


I'm having success with giemsa staining (found a parasite (worm) but no bacteria yet but i think its due to the PH of my buffer solution not being corrected for bacteria like Bb? or maybe i just to need to do alot more samples to increase the odds of finding one) but thats kinder-garden compared to IF right? i'm not afraid of a challenge, but I need to know my limits.

the big downside of staining and i guess its the same with immunifluorescence is that the samples must be fixed , and i won't be able to see movement, shape shifting, etc. also 'i guess I'll need other equipment and my $$ piggy bank is at its limit with this dark-field microscope already.

discomorphella
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Post by discomorphella »

IF is actually a lot safer than most microscopic protocols, in that the potentially toxic fluorochromes are covalently bonded to protein molecules that are used in very very dilute concentrations. The actual procedure is not much more complex than Giemsa staining. The main disadvantage of IF is the expense of specific immunological reagents. However, consider that the price/slide is actually only on the order of a USD or two. For example, to stain cilia using IFA one would need some anti-tubulin antibody, say mouse anti-tubulin...approx 200 - 300 USD. The usual quantity of a 1mg or so it sufficient for at least 200 to 500 slides depending on the final dilution. The next step will require a goat or rabbit anti-mouse fluorescently labeled antibody, which will be <=200 USD and again, good for 100's of slides. Some goat or rabbit normal serum for blocking is another 40 USD, and some Triton or similar nonionic detergent is perhaps 10-20 USD. This is enough material for easily 300+ slides, so for a few dollars/slide you're set. You can store the antibodies for a decade if you dilute them to ~50% glycerol and store them at -15C or so (i.e. most freezers, ~0 F). Fixing a smear is easily done with some ice cold methanol, at least for your bacteria / blood. I can send you a typical protocol if you want. The main fixed expenses will be a decent fluorescence microscope and some micropippettes, misc lab supplies like glycerol to mount the coverslips etc.

I honestly don't think regular BF or DF (or phase or DIC for that matter) is of much use for finding live blood-borne pathogens, at least without some way to specifically culture or concentrate them first. If you have anywere near enough bacteria / mycoplasma / rickettsia etc in your blood to see readily you are pretty much septicemic, and potentially not long for this world. Additionally those beasties are so small as to be close to the resolution limit for optical microscopy, and motion is a difficult thing to quantify. DF will show you a myriad of things, which you can't ID easily. IFA on the other hand, is highly specific, easily amenable to controlled experiments (e.g., omit the specific antibody, if the secondary fluorescent antibody still stains it you know its not what you thought). You can also stain specifically for nucleic acids using conventional fluorescence microscopy, helping to confirm that what you stained is actually a biological object and not some dust that shows up in DF. A Giemsa or Wright's stained smear is also nice, but suffers from the same lack of concentration for most hematological samples. Although there's some variation in staining effects with pH, that's mostly a concern for sections, for bacteria in smears anything around pH 6 will work just fine. pH 6.4 Phosphate buffer is a fine diluent, for this kind of staining, so is distilled water. I don't think its that crucial for simple blood smears so long as you are around 6.5 or so. But its not nearly as specific as IFA, and anything that resembles a bacterium will stain like one. IFA combines the detectivity of DF with specific antibodies. That's why its used in clinical and research labs.

David

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