Phase Contrast and Objectives

[qawine]

Is the principle of "empty magnification" a factor when selecting between Phase Contrast magnification and PH Objectives? I have an Amscope Phase Contract Kit.

http://www.amscope.com/accessories/phas ... ion_tabbed

Phase Contrast includes 10X, 40X and 100X. Objective: PH 10/0.25, PH 40/0.65, & PH OIL 100/1.25 (160/0.17)

Can I mix and match or is it better to use the 10X Phase Contrast with the PH10 objective vs. using the 40X with the PH 10 objective?

[Charles]

For phase you need to use the appropriate phase piece (annuli) with the same phase objective...10X with 10X, 40X with 40X, etc, otherwise you won't get proper phase. Each phase ring is a different diameter and is matched to the phase ring in the objective. It would be great if one worked for all but unfortunately they don't in your case. Some makers try to make it easier by using a phase condenser with all three or four phase units in one condenser like a carousel and each phase annuli is rotated into place instead of swapping out condenser annuli like yours. Also some makers like Zeiss can use the same phase 2 annuli (Ph2) for 16X, 25X, 40X, 63X and some 100X. There are also phase condensers like the Leitz Heine and the Zeiss Pancratic condensers which uses one adjustable 'annuli' on a condenser for use with a variety of phase objectives as well as for brightfield and darkfield.

[75RR]

The principle of empty magnification applies to all objective/eyepiece sum magnification.
(Condensers do not provide magnification - in this case the numbering is a link to the correct objective)

You question on Phase however is of a different sort. Each Phase position in the condenser is designed to match a specific objective. The reason for this is that both Phase objectives and Phase condensers contain annuli (rings) that must align.

http://www.microscopy-uk.org.uk/mag/ind ... phase.html


simultaneous posting with Charles!

[gekko]

I just want to add something you didn't ask about (please overlook if you are already aware of it): you can use your phase objectives as brightfield objectives, so you can quickly switch between phase and brightfield (or darkfield, oblique).

[qawine]

Thank you both for the information. I'm so new at this and having the Beginner's corner is very helpful.

[qawine]

Gekko - I was not aware that I could use for darkfield. Do I need additional equipment?

[gekko]

For the lower NA objectives (20x and below) one way is to make a darkfield stop and place it in the filter holder of your condenser. Much information is available on this. You can search this forum as well as the main Microbe Hunter website. See, for example, http://www.microbehunter.com/darkfield-microscopy/. Darkfield condensers are available for higher NA objectives if you require them.

[Charles]

You can also use your 100X phase annuli and your 10X or lower objective to get darkfield.

[gekko]

You can also use your 100X phase annuli and your 10X or lower objective to get darkfield.

Actually that is more convenient and a better suggestion (I didn't think of it even though I use it all the time ).

[apochronaut]

Actually, DF is very good for yeast and bacteria. I would think the 2 most important uses of a microscope for you would be counts and species i.d., especially for malolactic.
DF images spore bearers quite well, as does phase but DF gives a more complete picture, with more depth of field, so species i.d. is a little easier.I don't think it is used much though for oenology but I don't know why. Maybe just unfamliarity? Most of the species i.d. is done by differential staining in winemaking.

If you did want to get into DF, you need high magnification , for your application, so you would need a D.F. oil condenser, a 100X objective with a built in iris diaphragm and very high illumination. Your microscope likely does not have enough illumination for critical high magnification DF. However, several companies built DF condensers in the past, with built in illuminators, which overcome this problem. Sometimes these show up second hand and can be retrofitted to work on other microscopes. The objective is available for around 300.00 , often less, the condenser, less than 100.00.
You could contact your microscope supplier about this but the online retail outlets, have a track record of overstating the potential of their instruments. When it comes to magnification potential , you now know this to be true, and a similar situation exists for DF.

[qawine]

Thanks again. So much to learn. Amscope offers this 100X Oil Darkfield Condenser and objective with Iris.

http://www.amscope.com/oil-darkfield-co ... copes.html

http://www.amscope.com/accessories/obje ... -iris.html

Will those work or do I still have a problem with insufficient light?

[gekko]

If I may, my personal opinion, for what it's worth, is that now you have had much useful information and advice, which I would file for future use. I would say, use the equipment you have, gain experience with it, and you will, at some point, conclude that you really would like or need this or that additional condenser or objective or what have you, and you would know exactly what it is you want. From experience, it is easy to buy all kinds of accessories that you may or may not find useful in the long run.

[qawine]

Yes, good advice. At this point, just trying to understand what would be needed for Darkfield, in the event it's worth it. Don't worry - this is probably the last thing I'll be asking about for a while.

[apochronaut]

That's right. Gekko has it down. Get some experience with the system you have. Your microscope is a 20 watt . You cannot use that kind of illumination for the type of DF you need for yeast and bacteria. The condensers that had built in illuminators will still be around, at such time as you may decide to buy one. It may take a bit of patience. Don't buy the amscope DF condenser. It will only be useful at low magnifications . The objective isn't necessary yet either.
When you make the decision that you want to pursue that course, check in again or p.m. someone. I would be willing to send you some pictures of fermenting must in DF , if that would be of interest to you.

[gekko]

Yes, good advice. At this point, just trying to understand what would be needed for Darkfield, in the event it's worth it. Don't worry - this is probably the last thing I'll be asking about for a while.

Don't worry about asking questions! members here are more than happy to answer and help out as we can. apochronaut's knowledge and expertise are invaluable to me and to many members of this group, and when he gives advice in response to members' questions, I, for one, always learn something new.

[qawine]

Thank you both! And I would be very interested in in seeing DF pictures of must, apochronaut.

[qawine]

Thank you both! And I would be very interested in in seeing DF pictures of must, apochronaut.

[qawine]

Thank you both! And I would be very interested in in seeing DF pictures of must, apochronaut.

[p3aul]

I just want to add something you didn't ask about (please overlook if you are already aware of it): you can use your phase objectives as brightfield objectives, so you can quickly switch between phase and brightfield (or darkfield, oblique).

Thanks, gekko, I'm interested in PC also and i thought I'd have to change out the objectives each time I wanted PC and I thought "To much bother!" But now I see I won't.

Paul

[apochronaut]

This is a diminishing but still active culture destined for apple cider vinegar. It hasn't been seeded with an acetobacter mother yet, so yeast still dominate the population. It was fermented on wild yeast, native to the skins.
I took a med. power image and an oil immersion image of each of BF, Phase and DF., so 6 pictures.

For this subject, in general, it seems that BF yields lots of information. Phase and DF did not capture as faithfullyto the camera as the BF did, so there is more information with those than the digital image shows. However, BF is a very good option for this subject.Phase offers little advantage.
I'll see if I can do a series of another fermentation , to include more active populations of both desirable and undesirable bacteria, such as acetobacter, lactobacillus, oenococcus or pediococcus, the more common ones you will encounter.

[apochronaut]

The last 100X DF picture.

[apochronaut]

Here are some bacterial images. Lactobacillus and acetobacter.
The Lactobacillus are the more delicate strands that look like linked sausages. The acetobacter are more robust cells that look like little pickling cucumbers. They are seldom in chains of more than 3.

[apochronaut]

One more.