I Lose Subjects When I switch Objectives

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DonSchaeffer
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I Lose Subjects When I switch Objectives

#1 Post by DonSchaeffer » Thu Sep 23, 2021 12:35 am

When I use the microscope I usually just switch between to 10X and the 60X objectives. The only trouble is when I make the switch I always lose the subject that I spotted with the low power. It's particularly hard when I spot an interesting ciliate in the low power. No matter how I try to center the subject in the low power, I completely lose it in the high power and typically never find it. When I switch objectives, I have to refocus and move the stage. By the time I make these adjustments there is no hope of finding the subject again. Is there away of improving the precision of centering in the low power and speeding the process of finding these moving subjects.

Greg Howald
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Re: I Lose Subjects When I switch Objectives

#2 Post by Greg Howald » Thu Sep 23, 2021 1:03 am

I don't think so... But study it out. The problem you are experiencing is not uncommon, but the subject will have appeared to have moved in exactly the same place every time. For me (in the field of view) the subject is always down and to the right. I think you over come this by determining where you expect the subject to be and that is most likely unique to that microscope only. Another scope will most likely present a different, yet constant, result.
Greg

PeteM
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Re: I Lose Subjects When I switch Objectives

#3 Post by PeteM » Thu Sep 23, 2021 1:45 am

One benefit of the objectives on better scopes is that they are both parfocal and parcentered. Parfocal means when you switch to any of the objectives on a turret, precise focus is no more than a slight adjusment of the fine focus away. Parcentered means that an object perfectly centered in one objective will remain centered in all the others.

10x to 60x is a pretty good sized jump, especially for a fast-moving protist, so it can be a challenge with even parfocal and parcentered objectives.

You can probably make your objectives parfocal with thin shims meant for that task.

If you focus right to the center of your 60x objective, you might be able (as Greg has done) to know where to put your subject in the 10x field to have it more or less in the center of the 60x.

PeteM
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Re: I Lose Subjects When I switch Objectives

#4 Post by PeteM » Thu Sep 23, 2021 1:49 am

Might add that the nosepiece also has to register precisely -- and even the better objectives of all the same series will have slight deviations in focus and centering.

microcosmos
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Re: I Lose Subjects When I switch Objectives

#5 Post by microcosmos » Thu Sep 23, 2021 3:36 am

You could try installing a crosshair graticule in the eyepiece. It helps position the object dead-center in the field of view. If the optics are reasonably well-centered, you can switch directly from the lowest to highest objective and the object will likely still be in the field of view (unless it is moving, like your ciliate which may have moved).

If you also need a micrometer graticule for measurement, there are graticule patterns that combine both crosshairs and scales in the same graticule so you can have both.

DonSchaeffer
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Re: I Lose Subjects When I switch Objectives

#6 Post by DonSchaeffer » Thu Sep 23, 2021 5:44 am

Wow thanks for these ideas. lt's good to see I'm not alone,

DonSchaeffer
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Re: I Lose Subjects When I switch Objectives

#7 Post by DonSchaeffer » Thu Sep 23, 2021 5:44 am

Wow thanks for these ideas. lt's good to see I'm not alone,

MicroBob
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Re: I Lose Subjects When I switch Objectives

#8 Post by MicroBob » Thu Sep 23, 2021 6:22 am

Hi Don,
you can try to swap nosepiece positions and find the arrangement with the least error. Do you use your microscope with a USB camera? These often show only a small center portion of the image, posing high demands on the parcentricity.

Bob

apochronaut
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Re: I Lose Subjects When I switch Objectives

#9 Post by apochronaut » Thu Sep 23, 2021 1:21 pm

I am a stickler for parcentered objectives. Some microscope companies have guaranteed parcentering . Reichert published a tolerance , twenty microns or something like that? I think others too. It probably cost them $400.00, or the same price as an entry level microscope to parcenter a scope.
Entry level microscopes are notorious for this problem.
Older microscope designs with short objectives have less objective splay, so they are less prone to decentering. Older pre-60's microscopes also used smaller nosepieces and with precision machining parcentering could be specified with excellent hands on q.c. in the factory. As objectives became longer, weight on the nosepiece and splay increased so precision had to be upped, with ball bearing nosepieces but still q.c. in the factory was ever present.
In China and a low price point the q.c. only guarantees that the wheels turn, there is only average machining standards, worn out machinery, and long and heavy objectives, so modern inexpensive microscopes are a haven for misalignment demons of all kinds. Alignment is in the hands of the user.

Bob's idea is always a good idea with any microscope. Find out the best case for alignment. If you aren't picky about the rotation direction you have 2 X the sum of the objectives, as possible positions for the best case alignment. With cheaper microscopes I aim for the subject being at least in the field of view with all objectives , once centered for the low power objective. Almost always, moving the objectives around the nosepiece achieves that goal.

Occasionally an objective is really off. Almost 300 microns in one case with a 60X. Usually, in such cases there is a parfocality issue too, so shimming is a good practice. In this case hand filing a thicker shim down to the right thickness but tapering the shim solved the problem. Locating the tapered shim precisely, so that the taper counteracted the objective's extreme splay got it within about 60 or 80 microns.

A riskier but better option is to dress the objective shoulder a little in order to seat the objective securely and more centered with the others. This requires a little extra experience and foresight in terms of the slight change in objective rotation this may cause. Did it on some shorty objectives years ago and it worked.

Both of the above require that the objective stay in a chosen port, as do all the others once mutually parcentered.

In the o.p's case, one assumes that it is the 60X that is at fault due to it's extra length and the magnification factor. That needs to be determined exactly before anything is done be moving the objectives around the nosepiece and looking for consistencies in decentering. Is the objective decentered in the same direction always, for instance? Sometimes the nosepiece can be the culprit, or a combination of both. If a nosepiece port is found to be a bit off, it is best to locate the low power objective there, in order to keep the higher power objectives in as good relativity as possible.

eward1897
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Re: I Lose Subjects When I switch Objectives

#10 Post by eward1897 » Thu Sep 23, 2021 2:17 pm

Don:

Love your enthusiasm and your amazing images/videos you have obtained with your Amscope microscope. Your experimentation has paid off many times, and I'm sure you will improve your current situation. There may a couple of simple things that help some with your current issue:

- using a slide with stationary targets, find out which direction and how many turns of the handles are needed to get recentered when you switch from low to high power. Do the same for the focus difference. After a while you know where to look first after switching objectives, and moving the mechanical stage and focus in the right directions will be a reflex action.

- making your objectives focus the same depth (parfocal) on the same spot (parcentric) is harder, but some simple things might get you partway there, which is helpful. The special shims are hard to find. I have had luck with putting a wrap (or several) of thin, about 24 guage, wire around the threads of objectives that need to be extended (unscrewed a turn or so) to reach focus with the other objectives. I more recently use very small rubber bands (black ones used to hold hair turned to a figure 8 to wrap around the threads twice, or tiny dental rubber bands that go around once) to hold objectives in the right place even when not tightly screwed fully into the sockets. Put the rubber band on an objective then screw it in and out a tiny bit at a time looking through the eyepiece until it is in focus at the same level of focus as the adjacent objectives.

- getting parcentric is fussier and has more to do with the nosepiece (the rotating piece with threaded holes that holds the objectives) than the objectives themselves. Try to figure out which directions off center objectives are pointing toward (hard especially with lower powers) and try switching objective in different holes in the nosepiece until the highest power one is centered best you can get. Then fill in the lower power objectives (them being centered is less crucial). After getting your microscope reasonably parfocal (especially with rubber bands) and parcentric (perfect is the enemy of good enough), always use the nosepiece ring to move between objectives, not the objectives themselves, to avoid moving them slightly back out of place.

Good luck. Even with a perfectly adjusted microscope some ciliates are too fast to track, but you may improve things with the tips offered by the community.

Ed in Minnesota

PeteM
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Re: I Lose Subjects When I switch Objectives

#11 Post by PeteM » Thu Sep 23, 2021 5:54 pm

Just to add - you can also try to enforce a speed limit on the critters.

One approach is to have a bit of thin organic debris under the cover slip to provide speed bumps - places where they might congregate and not pass.

Another is to thicken the water solution with something like methyl cellulose.

Lomonaut
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Re: I Lose Subjects When I switch Objectives

#12 Post by Lomonaut » Thu Sep 23, 2021 7:42 pm

The gap between a 10x and a 40x is already pretty big, let alone a 60x. Don, this screams for a 20x, especially for pond life!

Excellent tips, my beloved Optiphot is certainly not perfectly parcentered! The DIC objectives will have to stay in their slots though, because their proper prisms are fixed (glued) in the nosepiece.

Poor parcentricity also shows by the field diaphragm image jumping around when switching objectives (Kohler). It's probably best to center the condensor for the highest power objective...

Dubious
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Re: I Lose Subjects When I switch Objectives

#13 Post by Dubious » Fri Sep 24, 2021 12:42 am

I second getting a 20x. I sometimes lose the subject going from 10x to 40x, but going from 10x to 20x then to 40x is relatively painless.

EYE C U
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Re: I Lose Subjects When I switch Objectives

#14 Post by EYE C U » Fri Sep 24, 2021 1:45 am

DON'T HAVE THAT ISSUE ON ANY OF MY ZEISS SCOPES...FOCUS CAN BE FIXED WITH SHIMS

DonSchaeffer
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Re: I Lose Subjects When I switch Objectives

#15 Post by DonSchaeffer » Fri Sep 24, 2021 2:27 am

Thanks guys. I really don't feel safe making substantive modifications. The problem isn't serious enough for that. I will complain but suffer.

Thanks Ed for your especially kind comment.

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