Diatom frustule in phase contrast

[Hobbyst46]

I tried stacking phase contrast images of a 3-month old batch of marine diatoms that I had collected (cleaned with 30% H2O2 and HCl). They are mounted in PS-CBO. All photos taken with the 100X1.3 Planapo oil immersion objective. Condenser oiled to the slide as well. Stacks of between 2 and 7-9 images. Illumination was LED light, filtered through either a common green filter or a thin dichroic blue filter (reflects amber, yellow, etc, passes blue).

The resolution, judged from the photos, is 0.2-0.3micron, of the correct order of magnitude considering the NA and wavelength (although the common resolution formula apply to single images, not stacks).

I tried both green and blue light. Green should be optimal for phase contrast, whereas blue is a short wavelength that is better for resolution, but my purpose anyway was to narrow the wavelength range and thus eliminate chromatic aberrations and get a crisp photo. I do not see any difference between the green and blue, with respect to the quality of the photos.

The identification of diatom genus refers to the diatom in the center of the photo and is a pure guess based on some web sources, so I added a () mark to each name. "mm" is micrometer.

I feel that these photos give a correct geometrical representation of the diatom. They lack some "sparkle" - I hope to find out why. Would the 63x1.4 Zeiss Planapo yield a better performance?

Will appreciate comments and criticism. Thank you.

[Hobbyst46]

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[Hobbyst46]

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[IanW]

These are very good photographs showing excellent definition. I too can see little difference between blue and green in terms of detail - the blue is more pleasing to the eye.

I'd be interested to learn your processes of for mounting your slides.

thanks

[MicroBob]

Hi Doron,

nice images!

I think that all the resolution is there, but the contrast could be higher. I'm not good at photo editing, but I gave it a try:
I copied one into GIMP and had a look at the histogram: It is quite cramped into a small range of light values. You might take the image in raw format and develop it differently. For small format output in the forum the images can stand a bit more unsharp masking from my point of view. This should be the last step and always depends on the final image size. When you do the unsharp masking at full resolution and scale the image down afterwards, the effect is reduced again.

[Hobbyst46]

the blue is more pleasing to the eye.

Thanks Ian - I think so too.

I'd be interested to learn your processes of for mounting your slides.

Here it goes:

I make an appropriate (very dilute, only slightly turbid; by trial and error) slurry of diatoms in distilled water. Put a drop of the slurry on a 18x18mm coverslip and place in adsorbent-paper lined petri dish for drying. Can be then incinerated if so wished (below 400 deg C I suggest - see another forum thread).

I am using PS-CBO, an acronym for a solution of PolyStyrene in Cinnamon Bark Oil, as an experimental (*) long-term temporary mounting medium. The RI of the medium is ~1.56-1.57. It does not solidify like resins, but becomes very viscous. Seems to hold OK for several months, if the slide is sealed. Only a few sealants can be used, because CBO contains very strong solvents. I seal the slides with either gel nail polish (UV/sunlight curing; NOT regular nail polish) or, better perahps, paraffin wax. With time, a slight crystallization may appear in the slides; I suspect that the crystals are of a component of the CBO, since PS does not crystallize.

I prepare the PS-CBO in a 30 ml (say) hermetically sealed glass vial (stopper with a plastic sealing layer), preferably made of dark colored glass. Pour 10ml of CBO inside, and add small (1cm) pieces of white styrofoam, the type that is used for commercial packing, looks like a gypsum dry wall or a layer of chalk. Push the PS inside the vial with the aid of a toothpick. Stopper the vial and shake to dissolve. Add more styrofoam and shake, repeat (I guess about 10-20 times) until it becomes a thick viscous clear solution, honey-like syrup. Dissolution can be slow, but do not heat it. Do all of the above outside since cinnamon odor is prominent. Protect the vial from light, and store it in a ventilated place, at ambient temperature.

Notice: CBO is odorous and might be an irritant to the skin, eyes and throat. I handle it with latex or rubber gloves and use it in a ventilated space. Also, cinnamon stem/leaf oil is NOT as good.

To mount, I dip a toothpick in the syrup and carefully lower the toothpick onto a slide, such that a small drop, diameter 4mm, is laid on the slide. Invert the slide onto the coverslip of diatoms. Place a small weight, say 20-30grams, on the slide, to spread the mountant, but try to avoid excess leaking outside the slip borderlines. Excess can be removed very carefully with a Q-tip dipped (not dripping!!) in alcohol, or carefully wiped away with tissue.

Next, I heat the slide at about 80-90 degC for 15 minutes. I do it with a thin aluminum plate (solid plate, not foil) placed on top of a small cooking pot, in which water is boiling (kitchen stove). The slides are place inverted (coverslip down) on the plate when the water has been boiling for a few minutes.
Next I seal with gel nail polish or paraffin wax and it is ready for observation.
I store the slides in a drawer, flat and inverted.

I hope to be able to mount diatoms in Pleurax, right now I have no means to heat Pleurax slides; add the high cost of Pleurax (shipping costs make it so), so I use the above home medium. Not for arranged diatoms, just strew.

(*) Q/007.

[Hobbyst46]

Thanks Bob,
I will try it.

[IanW]

Thanks very much for the in depth account of your slide mounting Doron. Much appreciated.

[Aenima]

Wow, love these - especially the 3rd one down in green - has a nice outline and like a halo effect.

[Hobbyst46]

Thanks Aneima.

[Hobbyst46]

@IanW
P.S. mounting and sealing with paraffin wax: Just found that paraffin wax is dissolved by immersion oil!! From this aspect, gel nail polish is better for sealing.

[vasselle]

Bonjour
Très belles images

[Hobbyst46]

Thanks vasselle!