My microphotography setup

Tardigrade37 · Post by **Tardigrade37** » Sun Mar 15, 2009 2:08 pm

I am a complete microscope geek and love to see pictures of people's setups, but alas, it has taken me far too long to post about mine, so here it is. I recently acquired the Zeiss Axiovert 200M, so I have been slowly selling off all of my 160mm stuff. I am very happy with the 200M, but I am having trouble adding flash to it - just today I broke the collector lens in my lamphouse due to too close of a bulb/heat absorbing glass/flash tube sandwich

. I may have to pursue the double collector route used by Franz, that is, after I replace the collector lens...

For those of you not familiar with the Axiovert 200M, here's a quick run down of some of the features...
Motorized everything - objective changer (6-place), fluorescence filter changer (5-place), focus (down to 25nm steps), beam changer (from eyepieces to front port, to side port), etc.

Mine is equipped with the following optical components:
0.55 NA LWD condenser with Ph1, Ph2, Ph3, DIC II, and DIC III
EC Plan-Neofluar 10x/0.3 Ph1
EC Plan-Neofluar 20x/0.5 DIC
EC Plan-Neofluar 40x/1.3(!) oil Ph3 DIC
Plan-Neofluar 63x/1.25 oil Ph3
Plan-Apochromat 63x/1.4 oil DIC
Plan-Apochromat 100x/1.4 oil DIC
Click here to see the glass

This scope is also fully equipped for fluorescence with an EXFO 120W mercury halide light source and multiple fluorescence cubes for DAPI, FITC, Rhodamine, Cy5, CFP/YFP FRET, etc. It also has a structured illumination device known as the Apotome which gives the user optical slices of the sample.

For color documentation, I have mostly been using a Q-Imaging Micropublisher RTV 5.0, but I have started using my D70 which fits nicely on the front port. For fluorescence, I use a Hamamatsu ORCA-AG deep cooled monochrome camera.

I know, I know, with all of this stuff, I should be taking more and better pictures - the trouble is finding the time to use it!

In the pictures, you can also see my stereomicroscope, an Olympus SZX9. It is also a fairly recent purchase. I was using a Zeiss Stemi DV4 for a while and loved it - it was incredibly portable with built-in transmitted and reflected light sources, but attaching a camera to it was not ideal, so I decided to change. I finally bought the fiber optics for the Olympus, so it too will begin to get more use.

Anyhow, feel free to ask any questions you may have about this stuff - I know I skipped over a bunch of things us technophiles love to hear about

piotr · Post by **piotr** » Sun Mar 15, 2009 8:05 pm

Wow, this is a serious piece of equipment!!!

Are you using Axiovision software with the microscope? Does it support the Apotome device? By the way, how much time does it take to acquire an image with Apotome?

I have seen the Axiovert 200M in the lab where my girlfriend works. It certainly feels like a very solid microscope and all these motorized functions are very nice.

Piotr

Ken Ramos · Post by **Ken Ramos** » Mon Mar 16, 2009 2:47 am

If this doesn't get you into microscopy, nothing will. That Oly stereo set up is really to my liking and so is the Zeiss but I will have to trudge along with my Axiostar for the time being, which is a nice scope too. Thanks for the peek at your equipment, awesome!

Tardigrade37 · Post by **Tardigrade37** » Thu Mar 19, 2009 5:55 pm

piotr wrote:Wow, this is a serious piece of equipment!!!

Are you using Axiovision software with the microscope? Does it support the Apotome device? By the way, how much time does it take to acquire an image with Apotome?

I have seen the Axiovert 200M in the lab where my girlfriend works. It certainly feels like a very solid microscope and all these motorized functions are very nice.

Piotr

Hi Pitor,
Axiovision does control the entire microscope. I have posted the details of the Apotome here
Regards,
Chris

Charles Krebs · Post by **Charles Krebs** » Thu Mar 19, 2009 7:57 pm

Chris,

Great looking set-up. You have some really nice high NA objectives, but the condenser you listed has a NA of 0.55. Don't you miss having a higher NA condenser for DIC, phase and brightfield?

Tardigrade37 · Post by **Tardigrade37** » Fri Mar 20, 2009 7:33 am

Hi Charles,
You're absolutely correct - I would like to have a high NA condenser on hand for high resolution transmitted light imaging. To run the numbers, when imaging at 550nm, for example, with a 1.4 NA objective, the following resolutions can be obtained:

0.55 NA condenser - 344nm
0.90 NA condenser - 291nm
1.40 NA condenser - 240nm

R=1.22λ/(NAobj+NAcond)

A 1.4 NA condenser is rare on inverted scopes (an I know few people who actually oil them), but 0.9 NA condensers are used from time to time. I do have a 0.9 NA condenser that I can use on this scope, but I lack a required extension tube and the DIC prisms. I have been keeping an eye out on Ebay for a used setup for I'm not sure I can justify spending a few thousand dollars on new for a mere 50nm.

On the upside of LWD condensers, it does allow me several freedoms. For example, I can image samples in petri-dishes (glass-bottomed for DIC) for hours without worrying about having to replenish the liquid. It also allows room for me to use a micromanipulator to pick up individual cells and transfer them to culture dishes, or other slides. With an inverted, gravity acts in my favor and all non-motile or quiescent objects fall very close to the coverglass.

The DIC prisms in this condenser are massive - over an inch in diameter, so the effective NA is always at its maximum of 0.55 (unless I stop it down for the 10x objective). Do you have to stop down the 0.9 NA condenser on the BHS to achieve good DIC or does it work well at maximum? If you do stop it down, do you know the approximate effective NA when this is done?

I may have access to a good test slide for these comparisons, so I will post some results when I have them. I love testing the optical principles of microscopy - it allows for a much greater understanding of physics behind the equipment. I have actually measured my point spread functions for each objective, but I have not done any tests yet on transmitted-light. I'm such a geek

.
Regards,
Chris

Charles Krebs · Post by **Charles Krebs** » Fri Mar 20, 2009 10:58 am

Chris,

The DIC with the 0.90 Olympus condenser works well "wide open". But... I have not actually checked what, if any, the NA "restriction" is when using the prisms designed to be used with the D Plan Apo's (which I use with the S Plan Apo's). I do know that the prisms made to be used with the Plan Achromats have significantly smaller "clear apertures", and definitely do reduce the workable NA of the Apo's.

That said, with the 3 dimensional subjects I tend to work with, I will close down the condenser a bit.

Charlie

Planapo · Post by **Planapo** » Tue May 05, 2009 5:59 am

Chris,
I've got a question concerning these arms that hold the fiber light guides for the Oly stereoscope (in your first picture). I have in mind to tinker something like that for my guides which are flexible too, but currently supported suboptimally.

Yours look custom made. Did you make them yourself? If so, could you expand what parts you've used?
And how are they attached to the vertical column of the Oly stand? From here it looks, as if a chunk has been cut off the vertical column. But I don't think you did that to this column

, or did you?!

Thanks in advance for your answer.

--Betty

Tardigrade37 · Post by **Tardigrade37** » Tue May 05, 2009 6:31 am

Hi Betty,
No cutting was required. When I bought the scope, it was surplussed from an industrial company that had some tool holders hanging off of it. These were constructed of what I believe is nylon - these are the white arms coming off of the post. They are composed of two pieces (each is a "C" shape) and clamp to the post by tightening two screws. I (well actually, our machine shop) added the articulated arms you see hanging off of the nylon parts. I purchased these on Ebay for $19.99 each (see here). The clamps that hold the fiber optic had to be modified to fit the diameter of the pipe. This arrangement allows me complete freedom with 5 points of adjustment including 3 ball joints on the articulated arm that can be simultaneously adjusted by loosening a single knob (seen at the "elbow").

The quality of the articulated arm is not what I would call precision engineering, but the cost is right compared to other indicator stands. I can't take full credit for this setup as I copied it from a Zeiss Discovery V12 we have in the lab. The Zeiss setup uses high quality articulated arms made by MPTec, but I think they have a semi-exclusive agreement because I couldn't find these arms for sale anywhere.

I'll try and post some more pictures to aid in the description.

Regards,
Chris

g4lab · Post by **g4lab** » Tue May 05, 2009 8:27 am

Those arms are are available from an Israeli company called Noga.

I have after years of trying to lay my hands on some , landed a couple which came from Zeiss USA which I am fortunate enough to have some involvement in their surplus stream.

For those of you who work for universities or other non profit or similar organizations you can go to: http://www.winwarehouse.org/catalog/Mic ... cessories/ and see some of the Zeiss stuff we get for redistribution. If you don't work for a non profit
PM me and I will see if I can help. Follow the links to microscopes.
There are also industrial tools.

Soon I am going to be putting up a killer AxioImager outfit. It keeps growing and growing as I find more parts to stick onto it. Someone is going to get a really killer deal. Wish I could afford it.

Gene

Ecki · Post by **Ecki** » Tue May 05, 2009 10:14 am

Chris,

congrats to the mother of all inverted scopes

Your choice of gear is not so far away from mine (SZX12 + Axioscope A.1 with EC PN & DIC)!

regards
Ecki

rjlittlefield · Post by **rjlittlefield** » Tue May 05, 2009 12:15 pm

g4lab wrote:For those of you who work for universities or other non profit or similar organizations you can go to: http://www.winwarehouse.org/catalog/Mic ... cessories/ and see some of the Zeiss stuff we get for redistribution. If you don't work for a non profit PM me and I will see if I can help.

Just to be clear here, the Admin team's understanding is that winwarehouse.org is a non-profit organization that redistributes surplus and various donations to other non-profits. If it were a commercial outfit, this post would sound too much like advertising. Carry on...

--Rik

g4lab · Post by **g4lab** » Tue May 05, 2009 7:26 pm

Winwarehouse.org is a 501(3)c non profit organization which accepts surplus from large companies and redistributes it to other non profits. When you look at the website there are no prices unless you register, during which process they confirm that you are part of a non profit or university or governmental or non governmental organization. Religious charities would probably also qualify. They will only ship to the address of a non profit.

They charge a management fee which is typically 25-33% of the list price of the item which covers their overhead. Prices can be negotiable for worthy causes. (ie. medical missions)

I am their resident microscope tech.

Today I started setting up a Zeiss AxioImager. Motorized focus that moves to a particular set point when you change objectives (which is also motorized.) Big touch screen to control it as well as knobs and buttons.
Has a fitted vibration isolation platform. Somebody is gonna be very happy with this scope. I wish it would be me but that is not in the cards.