Haematococcus under different lighting methods

[gekko]

Haematococcus. 20x objective.

1. Brightfield (with scale bar):


2. Oblique illumination:


3. DIC:


4. Phase contrast:


5. Darkfield:


5. Cross-polarized light: [should be 6.]


Thanks to Crater Eddie for the water sample containing Haematococcus.

[vasselle]

Bonjour Gekko.
Très belles démonstration des différents types éclairages au microscope.
Et les photos sont très belles.
Merci pour le partage.
Cordialement seb

[Manfred]

Hello Gekko,

all of them are grandious, my personal favorite: Image 1 and 4

Best regards
Manfred

[75RR]

Great images.
Wonderful to see such a range of techniques on a single subject.
Practically a course in illumination all on its own.
I am going to go for 1, 3 and 6

[Charles]

I like how each one brings out something different.

I like all 1-5 and 5 again.

[75RR]

1 is brightfield, 3 is DIC, and 6 is 5a?

[Charles]

Yes, I see Gekko has two 5s.

[JimT]

"Gekko has two 5's." That's because he only has five fingers on his counting hand

I vote for the DIC. Really gives a 3D effect.

[billbillt]

They are all fantastic but #3 is my favorite....

[Rodney]

# 3 really does stand out.

Rodney

[gekko]

seb, thank you very much for your always kind comments.
Manfred, thank you very much, and I hope you will show us some of your very beautiful images soon!
75RR, many thanks; I needed an excuse to post yet another Haematococcus image, so the various lighting technique provided such an excuse .
Charles, many thanks for your remark. "I like all 1-5 and 5 again": at first, the "5 again" baffled me, until I read JimT's comment about my counting hand .
JimT, Than you for your comment and sense of humor. The 3D effect of DIC, unlike that of oblique illumination, does not show a real 3D view of the object,
billbillt, thank you very much for your comment.
Rodney, many thanks, yes #3 appears to be the favorite of most people.

Again, thank you all for your comments. My conclusion is that my oblique illumination (as I had expected) needs much work.

[Crater Eddie]

Excellent set of images! Quite impossible for me to choose a favorite.
I think it is very interesting to see the same subject under the different illumination techniques.

[gekko]

Many thanks, CE. In your comment on my previous post of Haematococcus , you suggested that I do exactly that. Well, I took these images the day before you suggested that I do that

[zzffnn]

Great photos. I especially like the pol and DIC.

I think your oblique photo can be even better. Maybe try pushing the stop off center even more (for more contrast)? Did you use COL or simple oblique? If you were using COL, maybe try a bigger central stop?

You may also try that GUF/UGF filter (basically a combination of oblique and gradient darkfield, the inventor of that was awarded administrator's award at the other forum). With a 20x objective, many people think GUF can obtain image comparable to DIC.

http://www.photomacrography.net/forum/v ... hp?t=15142

http://www.photomacrography.net/forum/v ... ht=#173390

viewtopic.php?f=12&t=858&p=9280#p9280

[gekko]

Many thanks, zzffnn. I think my poor oblique images result from poor vision, poor ability to judge the effect by looking through the eyepieces or the camera's LCD (if I could view it on the computer monitor, I might be able to judge better). I used simple oblique by setting the phase condenser somewhere between the brighfield and darkfield settings. Actually I did take an image of the Haematococcus using COL (I used the PH3 setting on my Nikon phase condenser), but unfortunately at that point much of the water had evaporated and the algae were compressed by the cover glass, so I didn't post it.

I am aware of litonotus's UGF and, from reading one of your posts here, I became aware of your modification of it. I have for a long time recommended it to others on the forum based on the excellent results it produces. Unfortunately, I cannot use it because the construction of the Nikon condensers that I have does not allow access to the plane of the aperture or close to it without doing serious surgery to the body of the condenser. For the same reason I cannot use Rheinberg, etc. I did once adapt a "generic" condenser to fit the substage, but it accidentally fell to the floor and came apart, so I abandoned the exercise .

Again, thank you for all your comments and suggestions.

[zzffnn]

gekko,

I may be mistaken, but I thought that one can remove any of those circular filter inserts from your Nikon phase turret condenser and put in DIY ones? Or at least you can use that empty bright field slot (if you don't mind losing regular bright field, that is)?

With Rheinberg, you may take out one stock filter and put in your DIY circular filter.

With UGF, you may leave a filter slot empty and rotate turret to that empty slot. Then use putty to attach a glass slide holding your UGF to the bottom of your condenser, covering that open slot with UGF. Then slide that glass slide left-right and up-down to achieve desired balance of oblique and gradient darkfield.

Also for judging light effects, you may want to remove an eyepiece, then look into that empty eye tube with a naked eye (by looking at the back focal plane image of an objective).

[apochronaut]

All very excellent . Interesting, how the pol image gives the impression of a convoluted group of curves, rather than a group of distinct circles.

[gekko]

zzffnn, many thanks for your comments and excellent suggestions. There is a lot there to digest and think about, so, if I may, I will delay my reply until I've had time to think them over .

apochronaut, many thanks for your comments. You raise a very interesting point about "the impression of a convoluted group of curves". I think this is due to the close and propitious apposition of the cells with the dark X that shows up in the polarized light image. This dark X is very reminiscent of isogyres that one would expect to see in the conoscopic view, i.e. in the diffraction (back focal) plane of the objective, and not in the image plane. My decidedly uninformed, and hence rather vague, explanation (and I very much hope someone would give the real explanation) is that, Haematococcus, being essentially spherical, acts as another lens in the micoroscope's optical system, and that this "lens" focuses a conoscopic view (the isogyres) in the image plane. I don't have anything to back this up but it is the best explanation I can come up with. Next time I get this, I hope to remember to look at the objective's back focal plane to see what the conoscopic view looks like.

Here are two more polarized light examples of lens-shaped objects that demonstrate what I am guessing are isogyres showing up in the image plane:

1. Ostracod (10x objective)
http://i1070.photobucket.com/albums/u49 ... g~original

2. Haematococcus (100x oil-immersion objective)
http://i1070.photobucket.com/albums/u49 ... g~original

[billbillt]

Hi Gekko,

What method do you use to embed your scale bar?..
Thanks!
BillT

[gekko]

Hi Gekko,
What method do you use to embed your scale bar?..
Thanks!
BillT

Hi BillT,
What I do is very straightforward: I use Adobe Elements, but any image editing software with (or without) layers will do: I paste the bar in a new layer, then add the text in another layer, center the text to the bar if necessary, merge the two layers, then adjust the position of the bar-&-text as desired. After downsizing the image for upload, I merge all the layers and save as jpeg. That's it. If you don't have layers, you can do the same thing, only paste the bar and add text to the actual image (which is fine, but is less flexible).

[billbillt]

Hi Gekko,
Thanks for the info and quick reply!..
BillT

[charlie g]

All these images make me hungry, gekko! A scoop of these on a cracker , and a dry champaigne! The colors are delicious...and that rich antioxidant pigment (astazanthine) is used by so many diverse organisms (flamingo birds, crusteaceans, etc..). thanks for this eye candy...charlie guevara

[gekko]

Thank you very much, Charley, for your kind comments and lovely, evocative imagery. I too like the beautiful reds that Haematococcus displays. And thank you for the interesting tidbits of information: who would have guessed that flamingos use the same coloring ingredient?

[gekko]

zzffnn, many thanks for all your comments and suggestions.

I may be mistaken, but I thought that one can remove any of those circular filter inserts from your Nikon phase turret condenser and put in DIY ones? Or at least you can use that empty bright field slot (if you don't mind losing regular bright field, that is)?

You are absolutely right that one can replace the inserts with some other filter. But I do need those and I don't want to lose a phase annulus or the DF stop, nor can I lose the BF position, for obvious reasons.

With Rheinberg, you may take out one stock filter and put in your DIY circular filter.

Again, I'd rather have phase than Rheinberg. I tried Rheinberg using another condenser that I adapted, but I think I lost interest in pursuing it further:
viewtopic.php?t=619
viewtopic.php?f=6&t=704&p=4381&hilit=rh ... +2nd#p4381

With UGF, you may leave a filter slot empty and rotate turret to that empty slot. Then use putty to attach a glass slide holding your UGF to the bottom of your condenser, covering that open slot with UGF. Then slide that glass slide left-right and up-down to achieve desired balance of oblique and gradient darkfield.

Thank you for the suggestion, but this is too complicated for me. If I didn't have DIC, I would certainly have tried UGF, but with the adapted condenser (which I now need to glue together after I dropped it ).

Also for judging light effects, you may want to remove an eyepiece, then look into that empty eye tube with a naked eye (by looking at the back focal plane image of an objective).

I always check the back focal plane of the objective (I have a little lens that I place over the eyepiece-- much more convenient that removing the eyepiece or using a centering telesecope). I do that to set the starting point of condenser aperture (then tweak it a little if necessary), and to set the starting position for oblique illumination, again followed by slight tweaking. The problem is in judging when the best oblique angle and aperture are obtained (I usually err on the side of "too much", I think).

One interesting note: I was pleasantly surprised that I could get a DF image (using the DF position of the phase condenser) with my 20x/0.75 objective. I usually have to go to 10x/0.30 or 40x/0.70 for DF even if I'd been using the 20x for BF, etc.

Finally, I would like to again thank you for taking the time and effort to think through and write up all those excellent suggestions. I will keep them in mind in case a situation arises where I need to use one of them.

[zzffnn]

Sure gekko,

If I have DIC, I would have made the same decision that you made. What scope is your DIC on? Is it a Nikon Optiphot?

Bright field is useful for its flat surface resolution - very deep stack of bright field may actually offer more surface details than DIC.

I mentioned switching between bright field and UGF, as on my modified scope, I can switch between the two within 2 seconds. But I understand each scope/user is unique.

Which 20x 0.75 do you have? I have an old Zeiss Jena 20x 0.65 and love its center resolution.

[gekko]

What scope is your DIC on? Is it a Nikon Optiphot?

Yes.

Bright field is useful for its flat surface resolution - very deep stack of bright field may actually offer more surface details than DIC.

Focus stacks are not very friendly towards me, so I avoid them whenever possible .

Which 20x 0.75 do you have? I have an old Zeiss Jena 20x 0.65 and love its center resolution.

Nikon Fluor 20 Ph3DL.