Most useful magnifications for phase contrast

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farnsy
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Most useful magnifications for phase contrast

#1 Post by farnsy » Tue Apr 20, 2021 8:48 am

I'm working on getting a working phase contrast microscope and considering which objectives toget. I'm interested in the opinions of the forum members here on what the most useful magnifications are for phase contrast, specifically. There are lots of little flagella to see at 40X, I'm sure. Do those of you who use phase contrast find it useful at 10X as well? Would you prefer 20X in phase to 10X? If you had just one or two magnifications for phase contrast, which would you want? 100X, despite the need for oil?

Secondarily, if your microscope has both phase contrast and regular objectives, does it make sense to double up and have 40X in both regular and phase contrast? I'm not sure how significant the quality degradation due to the phase plate is.

If you had a microscope with phase capability and 6 objectives (and couldn't have other microscopes), what mix would you want? 4X, 10X, 20X, 40X, 60X in regular and 40X phase? Or would you want a different mix with more phase objectives? Maybe skip the 4x and grab a 10X or 20X in phase?

Of course, this is all opinion and personal experience/need, but I'm interested in yours.

Hobbyst46
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Re: Most useful magnifications for phase contrast

#2 Post by Hobbyst46 » Tue Apr 20, 2021 9:47 am

You might find the comparison, including images, in this post

https://www.microbehunter.com/microscop ... UAR#p99207

of relevance.

IMHO 10x phase contrast is less useful than 20x or 40x, but that really depends on the object you want to see.

apochronaut
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Re: Most useful magnifications for phase contrast

#3 Post by apochronaut » Tue Apr 20, 2021 12:06 pm

If I think of my usage over the past years, I would say the 20X has been the most used but they all get used a lot. I don't use phase objectives for BF. They just aren't good enough. Instead, I sought out objectives of higher N.A. and better colour correction. They can produce imaging that rivals phase contrast but if I were limited to average achromat objectives in the standard 10 to 100X N.A.s, they wouldn't get used much. It wouldn't be worth it to change the objectives, which is a much bigger p.i.t.a. than wiping off a little oil. Most objectives marked achromat or planachromat that have a higher N.A. , also have some fluorite glass in them, so they have superior colour correction as well as resolution. You will also get superior DF with them, although in the AO systems the 10X .25 phase and 20X .50 phase objectives give good DF using the 100X diaphragm as a stop.

The settup below is based on the possibilities with an AO Diastar 6 objective microscope. In fact I have one similarly set up, which cost a grand total of about 750.00 as configured but the two high oil immersion objectives are planapo. One might be able to do such a setup for less, using planachros or Neoplans with diligent searching.

It is pretty hard to not have a high power objective that sports an N.A. of over 1 of some sort and that means immersion. Doesn't matter if it is a 60 or a 100 or somewhere in between or higher. Limiting yourself to dry objectives of .85 or less, limits your microscopy. You just don't get the resolution required that allows for the in depth microscopy of detail. If you are content to just gaze at the pretty colours and shapes, probably you can get by but if you are seeking the observation of details then your cheapest route is to use immersion objectives, so one of the 6 holes on your ideal microscope has an oil immersion 100 to 125X objective with an iris diaphragm and an N.A. and colour correction as high as you can afford. Next down is a 40 to 60X oil immersion with an N.A. of .80 unless it has an iris diaphragm in which case it can be higher. Next down a 40X phase objective. Next down a 20X phase. Next a 10X phase. Lastly a 2.5 X All must be plan. It is advisable to get a set of the correct 15X eyepieces for the microscope. The condenser has to be a phase carousel type with an achromat aplanat phase condenser that works at at least 1.25 when oiled and has an aux. lens that fills the field of the 2.5X objective. That gives you 25X BF, 25X DF, 100X phase, 100X DF, 200X phase, 200X DF, 400X phase, 400- 600X high resolution BF, 1000-1250X high resolution BF utilizing the one condenser option. With the 15X eyepieces you can add additionally, 37.5X BF, 37.5X DF, 150X phase, 150X DF, 300X phase, 300X DF, 600X phase, maybe 600X - 900X BF as well, depending on what your lower magnification oil immersion objective is. Add in an oil DF condenser and you would further be able to utilize the oil immersion objectives for high resolution DF with a quick condenser swap.

The microscope stand must have a 100 watt illuminator in order to effect the high resolutionoil immersion DF options well.
If those are not required, then most standard lower wattage illumination systems will work but a field diaphragm is a must.

dtsh
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Re: Most useful magnifications for phase contrast

#4 Post by dtsh » Tue Apr 20, 2021 2:45 pm

It depends on what I'm looking at, but I use the 10x the most for fast scanning and the 20x or 40x for most of my observations, rarely the 100x unless I'm dealing with bacteria a lot.

PeteM
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Re: Most useful magnifications for phase contrast

#5 Post by PeteM » Tue Apr 20, 2021 3:57 pm

AO/Reichert made a phase condenser with a single insert that could be slid in or out for phase contrast. They apparently thought a 40x was most useful, since this was the default configuration. For many really interested in cell details, it's probably a good decision.

Working with kids, I find a 20x better for larger pond critters due to the greater depth of focus and still-adequate magnification. I hesitate to say this, because we could really use another scope phase-equipped at 20x - and it's already hard to find the 20x annulli and objectives. This arrangement (the slide-in annulus to get an instant phase view at either 20x or 40x) is also a useful learning experience to share.

The 100x phase magnification gets used rarely if at all. Some inverted scopes will add a 4x phase objective for scanning a larger field.

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Re: Most useful magnifications for phase contrast

#6 Post by BramHuntingNematodes » Tue Apr 20, 2021 4:20 pm

Light requirements for 100x phase are high. Some things can be seen with it that cannot with lower power objectives.

I mean, it really depends on what you are looking at. That AO had 40x as a default also speaks to the type of microscopist that they expected to sell to. I don't have a 10x phase on my phase scope but don't miss it much with the 20x. I still use the condenser to achieve col and darkfield with the 10x and this combi action seems to work very well for the larger critters like ticks, mites, nematodes and mosquito larvae. Lower powers than 10 seem rarer in phase.
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Re: Most useful magnifications for phase contrast

#7 Post by BramHuntingNematodes » Tue Apr 20, 2021 4:28 pm

Oh, and like Phil I am not too keen on using phase objectives for brightfield. Since my scopes only have 4 slots, I use two scopes. I have seen some fancy phase objectives with fluorite or even apo corrections on them but I am not sure how useful these would be. Phase seems to really shine on achromats already. Phase specimens are often colorless too, so filtering can be used to good effect as well. If these are meant for double duty as both phase and BF objectives I wouldn't be too keen on them.
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Re: Most useful magnifications for phase contrast

#8 Post by zzffnn » Tue Apr 20, 2021 4:46 pm

It is very personal and depends on your preference and subjects of interest.

For me, my most useful phase contrast objective is 100x. Because at up to 20x NA 0.4-0.5 or 40x NA 0.65, I vastly prefer DIY darkfield, dedicated immersion darkfield (or DIC) for my protists viewing, over phase contrast. At 100x, contrast becomes very important.

Only you and your subject of interest can tell, whether 20x, 40x or 100x is more useful. For me, that magic number is between 40x-100x, depending on the subject.

Whether or not you can tolerate using phase contrast objective in bright field or darkfield, depends on your eyes. My eyes are ok with them, but keep in mind that I generally dislike the halo effect of phase contrast.

I will personally skip 4x if I have to.

I don't have a 6 slot nosepiece, but my scope accepts interchangeable nosepieces, so I have many 5-slot nosepieces:

one nosepiece for oil immersion + 4x-10x air,

one for water immersion + any air objective (I remove immersion water with cotton swap and go back to 40x air) ,

one for macro

maybe one for phase contrast, even though I generally don't like the effect of phase contrast, but for research purpose phase contrast can provide contrast others (such as DIC) cannot.

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Re: Most useful magnifications for phase contrast

#9 Post by apochronaut » Tue Apr 20, 2021 5:17 pm

PeteM wrote:
Tue Apr 20, 2021 3:57 pm
AO/Reichert made a phase condenser with a single insert that could be slid in or out for phase contrast. They apparently thought a 40x was most useful, since this was the default configuration. For many really interested in cell details, it's probably a good decision.

Working with kids, I find a 20x better for larger pond critters due to the greater depth of focus and still-adequate magnification. I hesitate to say this, because we could really use another scope phase-equipped at 20x - and it's already hard to find the 20x annulli and objectives. This arrangement (the slide-in annulus to get an instant phase view at either 20x or 40x) is also a useful learning experience to share.

The 100x phase magnification gets used rarely if at all. Some inverted scopes will add a 4x phase objective for scanning a larger field.
The only application AO would have thought it most useful for would have been for blood analysis and haemocytometer use. Not general purpose because the single annulus phase condenser was widely purchased for blood analysis and most likely came into being purpose built for that. The design of the single annulus phase condenser was a response to a market need.... customer driven . The haemocytometer uses .40 coverslips. The original infinity objective series # 1075 to # 1079 achromats included the #1078 45X .66 achromat objective, a magnification that is particularly useful for blood counts and working with the large well slides of the day which also used a .40 cover. The 45X .66 # 1078, with a .50mm w.d. is not always useful where the cover can float. They must have received some complaints because not long after they released the original achromat series, they added the almost identical 45X .66 # 1116 objective , with a .70mm w.d. The two objectives look and perform identically, except for that .20mm w.d.. You have to read the # on them to tell them apart. The #1116 out number the 1078 at least 10 to 1 on the used market today, such were the number of series 10 blood count microscopes sold. AO no doubt moved the 1116 into general use as well.
When phase was introduced for the series, the achromat dark phase series of objectives most commonly seen was/is 1224 10X 1225 20X, 1238 45X and 1227 100X. The bright phase series was/is 1234 10X, 1235 20X, 1239 45X and 1237 100X. Why not 1226 45X and 1236 45X? They probably exist, as phase versions of the 1078 but as with the 1116, given equal performance and a slightly longer w.d. those two out of series numbered 45X objectives dominate what is available, particularly in concert with the single annulus phase condensers. Most of those single annulus condensers have the 45X annulus too, even those with the series 100 dovetail.

100X phase is a bit of an anomaly for me. I use it sometimes by necessity in Bright and B-Minus phase, seldom in Dark phase, even with the slightly better plan objectives of later series microscopes. However, I had the opportunity to purchase a Reichert planapo 100X 1.32 phase objective a while ago for a good price. Even as good as the Reichert 100X 1.25 dark phase planachro objective is, there are details unseen with it that the planapo phase brings out with crystalline definition. It wss quite a revelation .

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Re: Most useful magnifications for phase contrast

#10 Post by farnsy » Thu Apr 22, 2021 4:51 am

I really appreciate all the great replies. What I'm kind of gathering from this is that 20X and 40X are, perhaps, most useful for phase contrast overall, though there are other magnifications at which it may be very relevant as well.

I also appreciate your input on preferred magnifications overall. Actually I just found out the microscope I have been looking at can be fitted with a 7-objective nosepiece, so I'm currently thinking 4X, 10X, 20X, 40X, 60X, 20Xph, 40Xph. I'm not 100% settled on that, but it seems like a pretty nice little roundup.

Apochromat makes some excellent points about 100X. I can easily see how that's an important aspect of microscopy. It's just that I have had a 100X on my scope now for a couple of years and *never* used it--well, not properly, with oil. I have the oil, still sitting like new in my drawer. Part of it is that I know I'm exactly the type of person who rotates a super expensive 60X lens right into the oil and doesn't notice until much later, when the objective is destroyed. Putting a drop of oil on the slide doesn't sound like a big deal, but it has always been just enough of a hurdle that I haven't ended up doing it--I may not be looking at the right stuff either. Plus a high quality 100X is really expensive. Still, I'll give it a shot on my old scope and perhaps it will grow on me.

By the way, do ya'll have an opinion on 100X water objectives? No one ever seems to talk about them, so I'm thinking maybe they aren't that good.

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Re: Most useful magnifications for phase contrast

#11 Post by PeteM » Thu Apr 22, 2021 5:41 am

It sounds like the scope you're considering takes interchangeable nosepieces. If so, you could have one turret for dry objectives and one for oil immersion. For example, with something like a 4x or 10x as a finder (with so much clearance it wouldn't come near oil on a slide) and a 50x-60x and/or 100x oil immersion objective. Longer working distance 20x and even 40x dry objectives (including phase contrast versions) could also stay clear. That should cover the concern of running a dry objective through oil by accident -- and put a high numerical aperture objective to work with little fear of contamination.

As for water immersion objectives, they tend to have lower numerical apertures than their glycerin or oil counterparts and somewhat specialized applications. The current high end water-dipping ones tend to be used for electrophysiology apps. My one example is an Olympus 40x UIS plan fluorite objective. Lomo made affordable ones in short barrel versions. You occasionally see used Nikon finite water-dipping objectives for sale. Phil has described some Chinese examples. But remember that water intrusion wouldn't be all that good for a dry objective either. Someone who can remember which pedal is the brake and which is the accelerator (and, yes, there are sometimes mistakes) can probably remember to wipe off the oil after observing or taking photos in those times they want higher magnification.

farnsy
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Re: Most useful magnifications for phase contrast

#12 Post by farnsy » Thu Apr 22, 2021 6:00 am

PeteM wrote:
Thu Apr 22, 2021 5:41 am
It sounds like the scope you're considering takes interchangeable nosepieces. If so, you could have one turret for dry objectives and one for oil immersion. For example, with something like a 4x or 10x as a finder (with so much clearance it wouldn't come near oil on a slide) and a 50x-60x and/or 100x oil immersion objective.
You are right, the nosepieces can be interchanged fairly easily on the scope I have in mind. It's absolutely on my list to consider (saving up for and) buying a second nosepiece for further microscopy adventures after I feel like I'm exploiting what I have 100%. You can get DIC with this scope as well, if your wallet permits. If I feel like my equipment, and not me, becomes the limiting factor (I'm pretty far from there now), I absolutely plan to consider getting a second nosepiece for special purpose microscopy, including potentially oil immersion, phase, or DIC, depending on where my interests go.

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Re: Most useful magnifications for phase contrast

#13 Post by Rossf » Fri May 07, 2021 12:30 pm

I’m preferring the 16x,25x,40x setup these days but if I had to pick just one phase mag it would be 25x. Easily the most used-but I’m not terribly fussed about seeing bacteria in super detail-extra detail doesn’t help that much in identifying the bacteria apart from are they rods,cocci,spirette-motile or immobile-I probably left something out there -I like the Choo Choo train rods that rock back and forth to move forward -it’s like a bacterial Congo line-I love my water immersion 100x-yes it not super sharp but allows me to glide across the slide with way less disturbance of the mount.and an oblique condenser can sharpen it up a bit.
Regards ross

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Re: Most useful magnifications for phase contrast

#14 Post by lorez2 » Sat May 08, 2021 5:47 pm

Thanks to all who have contributed to this topic. I enjoyed reading everyones ideas and applications. Keep up the good work.

lorez
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