Depth of field

[david_b]

I have been using a Motic BA310 with Motic's standard plan lenses.
The 10x objective (for purpose of comparison) has an NA of 0.25.
I have recently aquired an Olympus BHS with Splan oblectives.
The NA of the Splan 10x is 0.30.
Comparing the two, I notice that the resolution of the Olympus is greater, but that the depth of field is *much* less.
Is this normal?

[viktor j nilsson]

Yep, that is an inescapable optical property.
You can read more about the relationship between NA and depth of field here:
https://www.microscopyu.com/microscopy- ... h-of-focus

[david_b]

Yep, that is an inescapable optical property.
You can read more about the relationship between NA and depth of field here:
https://www.microscopyu.com/microscopy- ... h-of-focus

Good information, thank you.

[apochronaut]

If I can just add to that a little. Most of us have noticed that most 4 objective microscopes have a rather standard set of optics : 4X .10 , 10X .25 , 40X .65 and 100X 1.25 being representative. While the magnifications might stay the same , the N.A's. can go all over the place. Those quite standard N.A's. listed above have been found to give the best combination of resolution, depth of field and working distance for average microscopy. Higher N.A's. become more finicky in use achieving higher resolution capability and lower N.A's less finicky in use achieving lower resolution capability.
This is why microscopes for acholastic use often have an N.A. of .25 for the 10X but a reduced N.A. of .55 for the 40X. In student or beginner situations the sampling or lower quality slides can be excessively thick , so using a .55 objective with a naturally longer working distance , provides some relief from slide crunching while providing resolution quite close to a .65. That precaution isn't normally necessary with the 10X, so backing it off to .20 for instance isn't normally a consideration.

[75RR]

.
Additionally the Figures in Table 1 under Image Depth (of the above link) show why it is important to use a low power objective when setting up a camera to be parfocal with the eyepieces.

This is what Charles Krebs has to say in his Procedure for Making Camera and Microscope Eyepieces Parfocal pdf http://krebsmicro.com/parfocal/index.html

"It is best to use a 4x or 10X objective for this procedure. Higher power objectives will give less accurate results.
It’s somewhat counterintuitive, but the higher the power of the objective, the greater the depth-of-focus (at the film/sensor plane) even though the depth-of-field (at the subject plane) decreases dramatically.
Since the purpose of this procedure is to position the camera body for most accurate focus, it is desirable to perform it with minimal depth-of-focus, which is obtained with low power objectives."

[david_b]

It’s somewhat counterintuitive, but the higher the power of the objective, the greater the depth-of-focus (at the film/sensor plane) even though the depth-of-field (at the subject plane) decreases dramatically.

Indeed. I have been coveting Splan apo's, but as most of my observations involve 3D specimens, not so much now.

[MichaelG.]

Here are three pages of wisdom by Peter Evennett in 1996:
http://www.microscopist.co.uk/wp-conten ... -focus.pdf

MichaelG.

[75RR]

I have been coveting Splan apo's, but as most of my observations involve 3D specimens, not so much now.

The shallow depth of field also imposes stringent setup requirements, which can be challenging.

Yet high magnification coupled with high numerical aperture (NA) is where the fine details are reveled. I find it well worth the extra effort.

[david_b]

I have been coveting Splan apo's, but as most of my observations involve 3D specimens, not so much now.

The shallow depth of field also imposes stringent setup requirements, which can be challenging.

Yet high magnification coupled with high numerical aperture (NA) is where the fine details are reveled. I find it well worth the extra effort.

I can see that would be the case for some subjects & still photography, but (for example) making a video of a tumbling Phacus using the higher magnification Splans only catches fleeting moments of detail and sharpness, such is the narrow DoF. I'm guessing Apo's would be even more challenging/less rewarding.
The Motic is giving me a better result for this application, albeit with lower resolution.