Viewing Red Blood Cells

[BrassCat]

Hello the Forum, I have prepared my first slide, live blood. Want to look at red blood cells. I have a few questions. Microscope is SRL. For this microscope, idle for many years. its first light.

1) I have viewed thru all the objectives; 4,10,40, and 100x. Eyepieces are W10X. RBC are visible at using 40 and 100x objectives. The 100X objective reads MICRO PLAN, Swift, 791287(1979??), HI 100 1.25. It has no makings stating use with oil. It certainly does focus on the specimen. With some distance between the lens and slide, not almost touching. Is this objective in fact an oil type objective? If it is, how much better performance could I expect.

2) I have not replaced the grease in the focus mechanism. I notice something like a backlash in the focus or dead zone in reversing focus rotation , is this normal? Or normal for a microscope from ~1979.

3) Concerning slide preparation: I put a very small blood drop on the slide, then put top cap on. I did not dilute with saline. Pressed down cover plate. Viewing showed blood cells was to densely packed, too thick. I then pressed the cover glass and also slide it back and forth, smear the blood flatter. Now I could see what appears a single layer of rbc, flatten down, no rbc on their sides. If there was any rbc aggregation, would this have separated the stacked rbcs? Someone today suggest making one edge of the cover plate touch the slide, making a slopped or wedged cavity. What is the way to get proper cover plate spacing??

Thanks for any assistance, Stan.

[MichaelG.]

Stan,

Here's a good description of how to make a blood-smear slide:
https://www.uvm.edu/~jschall/pdfs/techn ... smears.pdf

The supporting photos can be seen here:
https://www.uvm.edu/~jschall/techniques.html


I think you will find this technique gives you the view that you want.

Best Wishes
MichaelG.
.
P.S. I am rather surprised that your Swift 100x MicroPlan is not marked Oil
... The Vickers version is so-marked, and I think it's safe to say that NA 1.25 requires Oil.

[lorez]

It certainly does focus on the specimen. With some distance between the lens and slide, not almost touching. Is this objective in fact an oil type objective? If it is, how much better performance could I expect.

When this condition exists your objective is full of oil. It is making an image, but not a good one at all. Not all 100X objectives are marked as "oil", but in the entry level instruments it seems to be the norm. Maybe erroneously so, but it seems to work.

If your scope has seen no maintenance since 1979 it definitely needs attention. When everything is working properly the focusing mechanism is a very smooth system. There are wear points that make it necessary to do a more complete job.

lorez

[zzffnn]

The 100X objective reads MICRO PLAN, Swift, 791287(1979??), HI 100 1.25.

Welcome to the forum, Stan.
HI = homogeneous immersion. It basically means an immersion oil bridge is needed between:
1) objective top lens and cover slip; and
2) bottom of slide and condenser top lens.

[MichaelG.]

HI = homogeneous immersion.

.
Thanks for that 'Pearl of Wisdom'
The day is always better when I learn something.

MichaelG.

[apochronaut]

2) bottom of slide and condenser top lens.

... but make sure it is a complete condenser. Some condensers are separable and the top lens can be removed for use with the low power objectives. Condensers that require oil, in order to achieve peak performance are all marked with an N.A. higher than 1.

[BrassCat]

Thanks for the responses,

While this microscope is about 1979 vintage, I do not know it's history. It is a recent acquisition for me. I am assuming (yes, I know) that the grease stiffness is a bit excessive. Especially the condenser control and the left eyepiece now nonrotating adjustment. Glade to know what the HI on the 100x objective, I guessed it just meant the high power objective!

Thanks for the point about the removable top to the condenser, yes it does come off. But, what a pain, I need to move the whole condenser module to do that, and that is a tight fit. That makes going to the 40x to scan around and the use 100x to see better a little more difficult, unless you can use the 100x condenser top while using the 40x objective.

Did not have good results with the blood smear technique. But practice might improve my technique. I did get improved results pressing down on the top glass off center, more to one side. Using this technique, I could see single rbcs, then where there is more room the rbc aggregation.

At a med lab I was told yesterday, RBC aggregation occurs very quick with blood, it need to be drawn into container with edta, that is at ~body temp, called a hot blood draw. Never heard of that before, but should not surprise me, microscopes are a recent interest to me.

Thanks again.

[Crater Eddie]

Here is a good vid showing how to make a proper blood smear slide:

https://www.youtube.com/watch?v=UjSXOjVAGqE

CE

[BrassCat]

Hello Crater Eddie, thanks for the response. I tried that and was successful. It smeared from multiple layer to monolayer of the rbcs. In this blood smear I did not find much of any rbc aggregation, where as smearing the blood out by pressing down the top cover showed lots of aggregation. One of the techniques either produced the aggregation or the other broke them apart.

Seems like 1000 (100x + 10x eyepiece) is typically used for viewing blood, what do you do concerning that 100x objectives are supposed to be oil immersed?? I really do not know, just asking. Somehow if one was to be viewing blood much, a perhaps a 60x objective 15x eyepieces would be better. any thoughts?

Stan

[Hobbyst46]

Hello,
Oil immersion is a requisite of the numerical aperture, rather than the nominal magnification of the objective. A 60x or 63x obective with 1.25 or 1.4 NA will still need immersion. Otherwise the actual NA is less than 1.25 or 1.4, respectively, so the resolution is poorer than when using oil immrsion. I have a 25x 0.8NA oil, designed for immersion, and similarly a 40x 1.0NA oil for immersion.
I have succesfuly used a Zeiss 63x 1.4NA oil objective for red blood cells. Alas no photos to show.

[apochronaut]

Hello Crater Eddie, thanks for the response. I tried that and was successful. It smeared from multiple layer to monolayer of the rbcs. In this blood smear I did not find much of any rbc aggregation, where as smearing the blood out by pressing down the top cover showed lots of aggregation. One of the techniques either produced the aggregation or the other broke them apart.

Seems like 1000 (100x + 10x eyepiece) is typically used for viewing blood, what do you do concerning that 100x objectives are supposed to be oil immersed?? I really do not know, just asking. Somehow if one was to be viewing blood much, a perhaps a 60x objective 15x eyepieces would be better. any thoughts?

Stan

Check back to your 3rd reply from zzffnn. HI = Homogenous Immersion. This is a condition where all of the elements between the condenser and the objective, aside from the sample have the same or as close as possible, the same refractive index.

The light would therefore pass through 5 distinct layers , while transiting to the front lens of the objective from the condenser. .
Leaving the condenser the light would pass through 1) immersion oil 2) the slide 3) the sample 4) the coverglass 5) immersion oil. 1,2,4 and 5 should be homogenous and the closer 3 can be brought into homogeneity, the better the image will be.

[MichaelG.]

... what do you do concerning that 100x objectives are supposed to be oil immersed?? I really do not know, just asking.

Stan

.
Stan,

According to the notes that I linked in a previous post ... Immersion Oil can be applied directly to a dry specimen of Blood, with no coverslip.

I haven't tried it [yet] but it's an interesting possibility.

MichaelG.

[Tom Jones]

.. Immersion Oil can be applied directly to a dry specimen of Blood, with no coverslip.

Clinical labs do it all the time for CBC's and body fluid smears. No one coverslips grams stains or the like, either. A thin layer first to substitute for the coverslip, then a larger drop when using the oil immersion lens. No need for the coverslip unless you want to make a permanent mount for teaching or reference. When the finished, the slides are soaked in Xylene, or a citrus-based substitute to remove the oil, then stored. If you need to reexamine the slide, just add another drop or film of oil and off you go. They will last for many, many years that way.

And it's not just high NA lenses that can be immersions lenses. I have a BX-40 set up with all immersion lenses, even a UPlanApo 20x/0.80 and a UPlanApo 10x/0.40

[MichaelG.]

Thanks for that reassurance, Tom

MichaelG.

[apochronaut]

.. Immersion Oil can be applied directly to a dry specimen of Blood, with no coverslip.

And it's not just high NA lenses that can be immersions lenses. I have a BX-40 set up with all immersion lenses, even a UPlanApo 20x/0.80 and a UPlanApo 10x/0.40

Many mfg. give a nod to the de facto circumstance of users getting involved in fairly high magnification oil immersion microscopy yet often having the need to back out of that objective to take a broader field view again. Having lower magnification oil objectives in the nosepiece, greatly facilitates that process.