Microscopy

[flaxton]

Hi all you high powered Microscopists.
I examine my specimens using a standard microscope and usually congo red or Meltzer stains but someone I spoke to recently said he uses Interference phase contrast. I didn't get the opportunity to ask him so can anyone explain the difference and if it involves a special type of microscope or just fittings for a standard scope.
Thanks

[FungiJohn]

Hi Mal

Phase contrast is very useful and preferable to 'bright field microscopy' in viewing specimens which have little colour associated with them. This is especially so when viewed at higher magnifications.

In simple terms it exaggerates contrast of colourless objects. As light travels through the specimen it creates a 'phase shift' in the transmitted light path. This is due to the varying density of the specimen.

On passing through a 'phase plate' (alters wavelength by a 1/4 or 1/2 normally) it creates light of different shades and intensity. It is this effect which produces the resulting image. Without this plate you wouldn't see any difference!

A very useful feature of PCM is that the specimen remains intact because there is no reaction as there would be when using a chemical stain.

Biological microscopes for example are all Phase Contrast in order to study cells without interaction.

You can sometimes adapt a microscope to allow PC but if you use it frequently it's well worth the investment.

I hope it makes sense. It's much better explained by a demo on the scope.

John

[Chris Yeates]

Originally Posted by flaxton

Hi all you high powered Microscopists.
I examine my specimens using a standard microscope and usually congo red or Meltzer stains but someone I spoke to recently said he uses Interference phase contrast. I didn't get the opportunity to ask him so can anyone explain the difference and if it involves a special type of microscope or just fittings for a standard scope.
Thanks

hi

I'm a bit confused as to whether the person you spoke to was using Phase Contrast or Differential Interference Contrast, as you used the word 'interference'

depending on your 'scope it could be adaptable to PC, but DIC (often referred to as "Nomarski Illumination") is a specialist matter: a friend of mine in New Zealand bought a microscope with Nomarski illumination some years back (he still refuses to say how much it cost - perhaps he sold a kidney); an Olympus technician spent a couple of days with him setting it up! so I think that is a bit beyond most WABbers . . . lovely images (though not of fungi) using a range of lighting techniques can be seen at:
Spike's Gallery. A selection of aquatic life.

best

Chris

[FungiJohn]

Originally Posted by Chris Yeates

hi

I'm a bit confused as to whether the person you spoke to was using Phase Contrast or Differential Interference Contrast, as you used the word 'interference'

depending on your 'scope it could be adaptable to PC, but DIC (often referred to as "Nomarski Illumination") is a specialist matter: a friend of mine in New Zealand bought a microscope with Nomarski illumination some years back (he still refuses to say how much it cost - perhaps he sold a kidney); an Olympus technician spent a couple of days with him setting it up! so I think that is a bit beyond most WABbers . . . lovely images (though not of fungi) using a range of lighting techniques can be seen at:
Spike's Gallery. A selection of aquatic life.

best

Chris

This is quite a jump up from 'normal' phase contrast.

A similar? technique using cross polars and further enhanced contrast but costly too. However the 'halo' effect is quite stunning.

John

[flaxton]

Hi Chris
It was Ern Emmett up in Scotland. In an email he said he used "Interference phase contrast". When I looked this up it did seem to be a mixture of differing techniques.
Those photos from "Spike" are stunning and probably "just" outside my budget Have either of you two used Phase contrast or do you find staining good enough.

Mal

[FungiJohn]

Originally Posted by flaxton

Hi Chris
It was Ern Emmett up in Scotland. In an email he said he used "Interference phase contrast". When I looked this up it did seem to be a mixture of differing techniques.
Those photos from "Spike" are stunning and probably "just" outside my budget Have either of you two used Phase contrast or do you find staining good enough.

Mal

Hi Mal

I used Phase Contrast techniques when I was involved in asbestos analysis but my current home setup is non PC

I have considered upgrading my scope but I never seem to get round to doing it

Considering the problems in obtaining some chemicals and stains I would now suggest that people thinking of buying a scope for the first time seriously think about this method ... for fungi anyway!

John

[mollisia]

Hallo,

I have a different sight concerning phase contrast and think, that it gives nice looking pictures and so on, but that it is not very useful for microscoping fungi. At least for measurements you can not use it, because of the coloured "halo" around the object. It gives the impression of a more threedimensional picture, but in my eyes you don't see anything more or better what you would see with the normal light microscop.
I'm selling microscopes too and I usually advice the customers not to take phase contrats. It is expensive and of not much help in my eyes.

best regards,
Andreas

[Chris Yeates]

Originally Posted by mollisia

Hallo,

I have a different sight concerning phase contrast and think, that it gives nice looking pictures and so on, but that it is not very useful for microscoping fungi. At least for measurements you can not use it, because of the coloured "halo" around the object. It gives the impression of a more threedimensional picture, but in my eyes you don't see anything more or better what you would see with the normal light microscop.
I'm selling microscopes too and I usually advice the customers not to take phase contrats. It is expensive and of not much help in my eyes.

best regards,
Andreas

These are very interesting comments; when I bought my current 'scope from Brunel I also got a phase set, and to date I haven't got them out of the box (after several years!); I envisage that when I have more time I shall experiment, but with fungi I haven't felt the need (I'm glad that I recently got a 'normal' x60 objective though); I may well get into PC if I look at other things when I get more time - algae for example, but what it gives you often is 'artefacts'

One problem is that so many people do not know how to set up their
microscopes to achieve maximum resolution - resolution can be more important than a higher magnification at times: how many people have set-up the substage condenser at its correct focus? how many people when they change magnifications take out the eyepiece and adjust the diaphragm for the new objective? believe me it can make a lot of difference particularly when photographing down the 'scope

(none of the above I would say about Nomarski (i.e. DIC) illumination which can be stunning, and is very useful when looking at things like the 'hyphomycetes', where details of how the conidia are formed are of great taxonomic importance)

best wishes

Chris

[flaxton]

Originally Posted by Chris Yeates

One problem is that so many people do not know how to set up their
microscopes to achieve maximum resolution - resolution can be more important than a higher magnification at times: how many people have set-up the substage condenser at its correct focus? how many people when they change magnifications take out the eyepiece and adjust the diaphragm for the new objective? believe me it can make a lot of difference particularly when photographing down the 'scope



best wishes

Chris

Having been on the microscope course that you organized in Scarborough I don't have a problem getting the best out of my scope

Mal

[Chris Yeates]

Originally Posted by flaxton

Having been on the microscope course that you organized in Scarborough I don't have a problem getting the best out of my scope

Mal

Ah! so you are Malcolm *******; now I can put a face to a WABber

I'm glad you found it useful (I certainly did!)

best wishes

Chris