Microscopy: what is the depth of field of a microscope

[emma149]

what is the depth of field of a microscope and how is it different from the depth of focus of the microscope.

 

[Andy Resnick]

Technically, 'Depth of field' refers to object space while 'depth of focus' refers to image space. In practice, both terms are often used interchangeably to refer to object space- how much of the object is in focus.

http://www.microscopyu.com/articles/formulas/formulasfielddepth.html

 

[PAllen]

Technically, 'Depth of field' refers to object space while 'depth of focus' refers to image space. In practice, both terms are often used interchangeably to refer to object space- how much of the object is in focus.

http://www.microscopyu.com/articles/formulas/formulasfielddepth.html

And the answer is, for microscopes I can afford, at interesting magnification, "not enough".

 

[Andy Resnick]

And the answer is, for microscopes I can afford, at interesting magnification, "not enough".

Joking aside (because I feel your pain, what do you mean? I ask only because I may be able to offer some work-arounds.

 

[PAllen]

Joking aside (because I feel your pain, what do you mean? I ask only because I may be able to offer some work-arounds.

Well, at 400x, I don't get the top and bottom of a paramecium in focus at the same time. I assumed this was just something you lived with for conventional optical microscopes in the range of < $700. I can improve matters some with the diaphragm, but only decrease in brightness and unnatural appearance if I push it too far.

I would greatly appreciate affordable workarounds. (I have a basic achromat parfocal microscope with mechanical stage, substage lighting with abbe condenser; up to 600x without oil; 1000x with oil immersion (which I have tried and works nicely - but no depth of field)). With oil immersion I have tried higher mag eyepiece to 1500x, which seems to be empty magnification in color, but with filter to limit wavelength, gets pretty sharp (e.g. monochromatic green looks pretty sharp).

Of course, spoiled by gorgeous 3000x 3-d like scanning e.m. images.

 

[Andy Resnick]

What are the numerical apertures of the objective lenses?

 

[PAllen]

What are the numerical apertures of the objective lenses?

Well, I'm not sure. Here are the numbers on the 40x objective:

40/0.65
160/0.17

I assume 40 is obviously 40x (it is printed bigger than all the other numbers). This objective 100x immersion one have the front component on a spring mounting to protect from damage hitting a slide.

 

[PAllen]

The one I have seems pretty similar to this:

http://search.newport.com/?q=*&x2=sku&q2=M-40X

I have never measured (nor does my doc.) state the working distance or clear aperture. However, visually, this one looks very similar to mine.

 

[Andy Resnick]

Well, I'm not sure. Here are the numbers on the 40x objective:

40/0.65
160/0.17

I assume 40 is obviously 40x (it is printed bigger than all the other numbers). This objective 100x immersion one have the front component on a spring mounting to protect from damage hitting a slide.

Right- the numerical aperture is 0.65, it has a back focal length of 160mm (no tube lens is needed to make an image), and it's also designed for viewing through a #1 coverslip (0.17 mm thick). Not using a coverslip (or using an incorrect coverslip) will significantly degrade the imaging properties of the objective- specifically, spherical aberration will become intolerable. The 100X is most likely similar (except for a different NA- 1.3, perhaps?), and there is an additional wrinkle in that the index of refraction of the immersion oil (1.518, a standard) is the same as the coverslip, the embedding medium of the sample, and the glass slide. Using an oil immersion objective with aqueous samples also results in spherical aberration- there are water immersion objectives for this reason.

A 0.65 NA lens has a depth of focus of 1 micron- significantly thinner than a paramecium (which you already knew).

In truth, the depth of focus not only depends on the objective, but also on the illumination NA- typically, the condenser is larger than the objective NA, and so the objective NA determines the depth of focus. If you can stop down the condenser- not the field stop but the aperture stop- you can increase the depth of focus at the cost of a slight decrease in the small details you can image (which you may also already know).

Lastly, the front focal length is 160/40 = 4 mm, but this is unrelated to the working distance.

Does any of this help?

 

[PAllen]

Thanks, very informative. As to stopping down the condenser, what control do I use? I have two available: 1) the Iris diaphragm 2) a knob that raises and lowers the condenser. Are either of these relevant, or is my microscope missing the control I need?

 

[PAllen]

My condenser has an NA of 1.25, as does the 100x objective. All the objectives are labeled 160/.17.

The oil I use is labeled 1.515 rather than 1.518. It is made by Richard Allan Scientific (the oil, that is).

The microscope originally came with cedar oil, but I was advised not to use it, instead get a modern oil like the one I bought.

I note that my cover slips are labeled .13-.17 mm. Should I get better ones with smaller thickness tolerance?

There definitely seems no way to control the aperture of the condenser. Lowering it only spreads the light out; the iris comes before condenser in the light path. I would guess an aperture stop would come in front of the condenser or part of the condenser??

 

[Andy Resnick]

Thanks, very informative. As to stopping down the condenser, what control do I use? I have two available: 1) the Iris diaphragm 2) a knob that raises and lowers the condenser. Are either of these relevant, or is my microscope missing the control I need?

Glad to hear it. First, I erred earlier- 0.17 is a #1 1/2 (1.5) coverslip, not a #1.

Stopping down the condenser- first, make sure the condenser is aligned (centered and focused; Kohler illumination)- the goal is to focus the condenser and the objective onto the same sample plane- that's what raising and lowering the condenser does. The iris diaphragm could be either the field or the aperture diaphragm, I can't say which without more information. If the condenser is focused on the sample and the iris is focused onto the sample (closing the iris results in a circle of illuminated area stopping down), then it's the field diaphragm. The aperture diaphragm, when the condenser is properly aligned, will apparently control the brightness of the illumination. It's possible that diaphragm is not located on the condenser itself, but in the body of the scope. OTOH, you may not have control over both.

My condenser has an NA of 1.25, as does the 100x objective. All the objectives are labeled 160/.17.

The oil I use is labeled 1.515 rather than 1.518. It is made by Richard Allan Scientific (the oil, that is).

The microscope originally came with cedar oil, but I was advised not to use it, instead get a modern oil like the one I bought.

I note that my cover slips are labeled .13-.17 mm. Should I get better ones with smaller thickness tolerance?

There definitely seems no way to control the aperture of the condenser. Lowering it only spreads the light out; the iris comes before condenser in the light path. I would guess an aperture stop would come in front of the condenser or part of the condenser??

You're correct on not using cedar oil- it dries out and turns into glue. The 'modern' oil is what you want, but be aware that there are nasty chemicals in it, and that contact dermatitis is a common side-effect.

The coverslip tolerance you have seems a little sloppy- I just get ones specified as #1.5. Some folks get really anal retentive and measure each coverslip with a micrometer on their own and only use ones with the exactly correct thickness. I think that's a bit much, but whatever...

If you're condenser is specified as NA 1.25, are you using immersion oil on it? That is definitely an immersion condenser. Be careful- I try not to use immersion condensers because it means I have much less room for error on the objective side- much easier to break the sample.

What's the model scope?

 

[PAllen]

Glad to hear it. First, I erred earlier- 0.17 is a #1 1/2 (1.5) coverslip, not a #1.

Stopping down the condenser- first, make sure the condenser is aligned (centered and focused; Kohler illumination)- the goal is to focus the condenser and the objective onto the same sample plane- that's what raising and lowering the condenser does. The iris diaphragm could be either the field or the aperture diaphragm, I can't say which without more information. If the condenser is focused on the sample and the iris is focused onto the sample (closing the iris results in a circle of illuminated area stopping down), then it's the field diaphragm. The aperture diaphragm, when the condenser is properly aligned, will apparently control the brightness of the illumination. It's possible that diaphragm is not located on the condenser itself, but in the body of the scope. OTOH, you may not have control over both.

It is clear I don't have control over both.

You're correct on not using cedar oil- it dries out and turns into glue. The 'modern' oil is what you want, but be aware that there are nasty chemicals in it, and that contact dermatitis is a common side-effect.

I use it a couple of times a year - shouldn't be a problem. (I use the microscope more, but rarely bother with immersion). The whole thing is just one of several secondary hobbies.

The coverslip tolerance you have seems a little sloppy- I just get ones specified as #1.5. Some folks get really anal retentive and measure each coverslip with a micrometer on their own and only use ones with the exactly correct thickness. I think that's a bit much, but whatever...

.
So I'll get new ones. Thanks.

If you're condenser is specified as NA 1.25, are you using immersion oil on it? That is definitely an immersion condenser. Be careful- I try not to use immersion condensers because it means I have much less room for error on the objective side- much easier to break the sample.

What's the model scope?

I had no idea to use oil on the condenser. When I use the non-oil objectives, I assume I don't want oil on the condenser(?) So I have to clean it after use ? Minor issue for me, since I clean the immersion objective after use anyway, because I let it sit a long time between uses. The condenser seems to work ok without oil with the 100x objective (with oil joining cover slip and objective) if I raise it (the condenser) all the way so it touches the bottom of the slide.

Not so worried about breakage - I'm pretty careful, plus the tip of the objective is spring loaded, so I would have work to break the cover slip.

The scope is not by typical microscope brand - it was made by Celestron (discontinued, I think).

 

[Andy Resnick]

The iris most likely controls the aperture- at least that's what I guess based on celestron's website:

http://www.celestron.com/c3/category.php?CatID=79

You can use the objective as dry, but the NA can't be 1.25- I was surprised that this is not mentioned in the manual.

Another wrinkle- your stage mechanism is probably not fine enough to control the focal plane at high NA. Room vibrations can also cause a lot of problems- putting the scope on a sheet of sorbothane (or a few hockey pucks) can make a huge difference. Using the immersion objective, the spring will help prevent damage to the objective but you are still squeezing the sample- this can be very frustrating if your objects can freely move, because they won't stay still while you are focusing.

Try this- put a drop of oil on the condenser, and carefully raise it until it contacts the slide. Then try viewing with the 40X objective and see if it looks better than what you have been experiencing. Once you get a decent view, try playing with the iris to see if you can improve the depth of focus.