Campanella umbellaria, phase and flash

Charles Krebs · Post by **Charles Krebs** » Sat Jan 27, 2007 12:12 am

The "new" flash arrangement for my Olympus stand is much more efficient than the one I had previously used for phase contrast. This allows me to use a power setting that that provides a much shorter effective exposure time, and I thought it would be interesting to see what could be done at high magnifications with cilia that was moving extremely rapidly. The top image was made with a 40X, the lower two with a 100X.

MacroLuv · Post by **MacroLuv** » Sat Jan 27, 2007 3:31 am

Wow Charles!

Great photographs!

It looks very... very... .... cosmic!

Ken Ramos · Post by **Ken Ramos** » Sat Jan 27, 2007 3:42 am

Reall neat images Charlie, your new flash arrangement is working really well. This really brings out the three semimembranes of cilia that we don't normally see when viewing the Vorticella through the light microscope. Cool

bernhardinho · Post by **bernhardinho** » Sat Jan 27, 2007 5:42 am

Good Lord, these are fantastic!!! The whirly sense of motion is incredible. What shutter speed did you use then?

Bernhard

gpmatthews · Post by **gpmatthews** » Sat Jan 27, 2007 10:11 am

Woderful images, Charlie!

Charles Krebs · Post by **Charles Krebs** » Sat Jan 27, 2007 10:18 am

Bernhard... the camera is set to it's fastest flash sync which is 1/200 sec. But the microscope viewing light is adjusted low enough that it will not "register" any image at that setting... that is, without the flash firing the picture would be black. That means the effective exposure time is the same as the flash duration. The more efficient flash set-up means I can use a reduced power setting on the electronic flash. While I don't know for sure, I estimate that the effective exposure time (flash duration) is about 1/10000 second for the top image and about 1/5000 second for the other two.

It is always interesting to take these because you can't really see anything to focus on! The cilia are moving so fast that they are a complete blur. Just take lots of shots at slightly different focus settings and see what comes up.

Phase contrast actually gives better contrast and visibility on cilia than does DIC, but it is not as "crisp" looking, and it can be really tough with "3D" objects.

ralfwagner · Post by **ralfwagner** » Sun Jan 28, 2007 12:38 am

Hello Charles,

these are very impressive and I learned a lot about flash at the microscope. Thank you!

You mentioned, that DIC is not as good as phase contrast with this particular problem of moving cilia. I think it would be a good demonstration of the differences between these two methods, if you would show the DIC fotos, too. Thanks.

svalley · Post by **svalley** » Sun Jan 28, 2007 6:40 pm

Charles, great images. Electronic flash really opens up the possiblilties of imaging features that are completely imvisible otherwise.

Charles Krebs · Post by **Charles Krebs** » Sun Jan 28, 2007 11:40 pm

Ralf... you might want to compare these images with a posting I made of the same subject using DIC:
http://www.photomacrography.net/forum/v ... .php?t=460
It is not a direct comparison of course, but you can see some of the differences in how the cilia is rendered. My impression is that in phase contrast the cilia are presented in greater contrast from the background. When observing live creatures three things I find exceptionally visible with phase contrast are cilia, bacteria, and amoeba. These are also seen quite well with DIC where I personally find them "sharper" looking and with that "3D" look, but with less contrast from the background. For simple visibility phase-contrast is hard to beat, but for photography of 3-dimensional subjects I must say I prefer the look of DIC. The way things go out of focus has a more natural appearance than with PC. When phase contrast goes out of focus on a part of a subject it has a very "busy" look that appears unnatural to me. So when I use PC I try to work with subjects that are as thin and flat as possible... which makes it tricky to work with some larger more complex micro-animals.

If you look at this rotifer shot made with phase contrast:
http://www.photomacrography1.net/forum/ ... php?t=1482
you can see what I mean. The cilia contrast quite high, but the compare the out of focus areas to this one made with DIC:
http://www.photomacrography.net/forum/v ... php?t=1320

Each of the lighting methods has good and bad points, and each individual will have their own preferences.

Also see the pictures referenced in this post:
http://www.photomacrography.net/forum/v ... php?t=1645

And still more examples of electronic-flash/ DIC cilia can be seen here:
http://www.photomacrography.net/forum/v ... .php?t=919
http://www.photomacrography.net/forum/v ... php?t=1157
http://www.photomacrography.net/forum/v ... php?t=1153
http://www.photomacrography.net/forum/v ... php?t=1319

Charles Krebs · Post by **Charles Krebs** » Sun Jan 28, 2007 11:47 pm

Steve... yes I agree about flash! You can sometimes see the cilia on dead subjects, but there is nothing like having a look at it in action, doing what it is supposed to do!

And while you can get enough light when using brightfield to allow fast shutter speeds to be set on the camera, it would be very difficult to take even basic shots of live active subjects with some of the other lighting methods (like phase and DIC) that require much more light.

Gyrosigma · Post by **Gyrosigma** » Mon Jan 29, 2007 5:02 am

Hello Chrles,
Always excellents pictures, I agree with the others members in theire admiration...
Can you tell us what is your "new flash arrangement" cause I have somme speed problem with my own configuration....
best regards

Dominique

Charles Krebs · Post by **Charles Krebs** » Mon Jan 29, 2007 9:39 am

Dominique... good to hear from you. (You need to post more pictures

)

While I do not have a photo of the completed setup, I do have one picture I made while it was "a-work-in-progress". But this photo illustrates the important features, and it can be seen here:
http://krebsmicro.com/forumpix/BHS_flash.jpg

On the Olympus BHS, the entire bulb/condenser "unit" simply plugs into the rear of the stand. I was fortunate to locate a second complete lighting unit and make the modifications to one as seen in this picture. The picture referenced shows the "stock" BHS 100W bulb and collector on the left side. On the right is the modified unit. I positioned a flash tube at the exact position formerly occupied by bulb. The arrangement that holds the flash tube permits it to be adjusted forward and back, as well as up and down. It can be positioned very precisely to maximize the light that passes through the "collector" condenser. After this picture was taken I added a second "platform" directly behind the flash tube that holds a Luxeon K2 LED. This way I am able to fully adjust the position of the flash and LED. The LED is there to provide my viewing and focusing light.

The flash unit I used was one of my Vivitar 283's with the control "box" that was discussed in the "old" forum (There is also a page on my Krebsmicro.com site). This time, the flash tube was removed from the flash. Every time disassembling a small electronic flash unit is discussed the following is very important to remember:
Extreme caution must be used since the power stored in the capacitors of these flashes can be dangerous! The shock hazard is very real. All capacitors must be properly, and completely, discharged before doing any work.

Gerd Günther did an nice internal modification like this to a Leica stand, but the page he posted describing it does not seem to exist any longer.

This is the 4th method of incorporating flash that I have tried and it provides the most light. I was fortunate because the BHS light assembly made it fairly easy to accomplish. When I first started using the BHS I had set up a 70/30 pellicle mirror at a 45 degree angle just above the light port. The flash was directed into that... and the method actually worked very well once aligned properly. I would probably have developed that method and still be using it if it had not been so easy to make the modification to a BHS assembly.

rjlittlefield · Post by **rjlittlefield** » Mon Jan 29, 2007 9:56 am

Charles Krebs wrote:I got "server error" message when posting this... but it posted three times! Sorry 'bout that! I've removed the text, but is there any other way to remove the duplicates?

Admins can remove the duplicates. I'll do that with your extras. ... OK, they're gone now!
--Rik