MicrobeHunter.com Microscopy Forum

Hello,
If you were given complete control over microscope (using dslr camera) and objective design except for the absolute aperture size of the objective, what would you change to increase the depth of field at the magnification given by a 40x objective ?
Thanks

So far as I am aware you have only two viable choices:

1. Stacking of several digital images
or
2. Analogue 'light sheet' imaging which involves a long exposure.

MichaelG.
.

P.S. [2] was mainly used at 'macro' magnifications, and is probably impractical at 40x.

P.P.S. ... Here is a description:
https://www.mccrone.com/mm/constructing-a-scanning-light-photomacrography-system/
and, for completeness, the 1968 Patent is here:
https://worldwide.espacenet.com/publicationDetails/originalDocument?CC=US&NR=3398634A&KC=A&FT=D&ND=3&date=19680827&DB=EPODOC&locale=en_EP

_________________
Too many 'projects'

see:
https://www.microscopyu.com/microscopy- ... h-of-focus
for an explanation of the mathematical link between magnification, NA and depth of field.

_________________
Zeiss Standard GFL+Canon EOS-M10

is there nothing that can be done by changing lenses diameters and distances while keeping the aperture diameter and magnification?

I tried light sheet, on 4 and 10x it isnt super usefull because the depth of field is enough for stacking software. On x40 (stopped down) I didnt get enough light through and also got some artifacts (the light was coming only from one side, one side was too bright and the other too dark)

What is your specimen?

_________________
Zeiss Standard GFL+Canon EOS-M10

ant head, it is reflective, because the light moves with the Z axis , the overexposed speckle on the ant facing the light also spreads...

IMHO, this object is not transparent, so it is better photographed under top illumination, light coming from above. You might achieve it with a LED ring around the objective. And why use x40 objective for such a large object? am I missing something?

_________________
Zeiss Standard GFL+Canon EOS-M10

top & side light works fine on ants. The point is to try to get the best magnification possible. Got better results with x10 side illuminated (but not light sheet) here:
viewtopic.php?f=6&t=6044

Except as already mentioned ... The short answer is that 'Depth of Field' and 'Resolution' are mutually exclusive in light optics.

MichaelG.

_________________
Too many 'projects'

I get a little bit more by stopping down the condenser diaphragm, have to turn the lamp up to compensate.

In high resolution DF, the objective must be stopped down, either with a funnel stop or an iris. For this reason DF can give surprising depth of field, as well as surprising resolution.

"Surprising resolution" I happily accept ... but surely that resolution can never exceed the resolution provided by the full-aperture configuration of the same objective design.

Please correct me if you can demonstrate the error in my belief.

MichaelG.

_________________
Too many 'projects'

In my own diascopic microscopy, I use DF, Phase Contrast Bright L, Bright M, Dark L, Dark M, B-Minus L , B-MInus M, COL, Oblique and BF. Additionally I have used offset filters, and various conformations of gradient filters. Each has it's value for specific specimens and DF , is no less valuable than any of the others. It is particularly useful where inclusions occur in very small specimens and helps to resolve very small details in some specimens where no other technique can. When aqueous samples are flowing, the depth of field that it provides is quite informative and useful in providing a better overview of the processes going on in the sample as well, as the structures.
Certain samples do not benefit from the use of DF and in such cases, choosing another illumination technique is advisable but DF microscopes, which were originally called " the super microscope" do render fine details in certain samples very well indeed. I don't find DF all that useful for lower resolution microscopy...N.A.s below about .90. It's just pretty to look at.

High resolution DF is a tricky technique to use and master. Equipment needs to be very clean, precise and the illumination high and well focused. High N.A. apochromat objectives make a difference because chromatic aberration is the enemy of DF.......that's why the better oil DF condensers are reflecting condensers.

Hi Michael,
as far as I know the outer light rays of the light cone contribute more to the resolution than the inner light rays. A DF condenser provides just these outer rays.

Bob

@MichaelG
@MicroBob

The very simple theoretical Raleigh criterion for resolution is R = 1.22xLambda/(NA_condenser+NA_objective), where Lambda is the wavelength.
Supposing my condenser is of NA=1.4, and my objective is of NA=1.0(immersion), with iris.

1. Brightfield, using UNOILED condenser but OILED objective=> R_bf=1.22xLambda/(~1.0+1.0)
2. Darkfield, using OILED condenser AND OILED objective, but iris stopped down to 0.6, => R_df=1.22xLambda(~1.4+0.6)

thus R_bf is approximately equal to R_df - if the same Lambda applies!!.
All that is theory only. Other factors probably play a role in reality.

_________________
Zeiss Standard GFL+Canon EOS-M10

A very fair point, Bob ... But ...
[please forgive me if I am being excessively literal] I am aware of no method by which the resolution of a good objective can be increased by introducing aperture stops of any shape, size or position. [caveat: Yes, a bad objective can probably be made to perform less badly].

This thread started with the question of what might be done to improve depth of field; and it has now developed into a discussion of the trade-off between d-o-f and resolution. ... I currently remain convinced that it is impossible [except in the context of focus stacking] to increase d-o-f without decreasing resolution.

I would, of course, be delighted if someone could demonstrate that I am wrong !

MichaelG.

_________________
Too many 'projects'

Hi Doron,
I can imagine that this theory only applies to filled light cones to full extent. With a hollow cone there is much light with high n.a. and little light with low n.A. This might increase the actual "weighted n.a.".
Apart from that it's always nice when practice prooves theory right - but life is not always nice!

Bob

Why would you need to stop the objective down to .6? A DF condenser with an upper N.A. of 1.4, would have a lower N.A. of around 1.2. To obtain a dark background, one would require reduction of the N.A. of not less than about .90, possibly higher.

Thread gravedigger here,

One of the things I read referring to misunderstanding the resolution of objectives is that the true NA is only achievable with lighting such as extreme oblique, darkfield or COL. The reason as I understand it is that the initial lens NA is measured using a anaxial light source which can then use higher order diffraction bands for full resolution. If using standard brightfield with Kohler for instance, you are using an axial cone of light with fewer interfering diffraction bands available, also stopping down the condenser a little for increased contrast. Combined, these can effectively halve the stated resolution.

I believe that diffraction interference determines visibility and standard brightfield is poor in this respect, so in reality, darkfield should be highest on reaching true NA as it's all diffracted light with col and oblique following.

Correct me if I'm wrong. I'll try to dig up the original paper.