questions on photography with a microscope

kirispupis · Post by **kirispupis** » Wed Jan 07, 2009 10:06 am

Hello! (this being my first post)

I have been involved with macro photography for some time - currently using an MP-E 65 and 180mm and a Canon 5D Mark II - and I greatly enjoy taking pictures of insects and other small invertebrates.

I am now looking at expanding into microscopy. I suspect my primary targets will be small water organisms but in truth I will photograph anything interesting. I am of course interested in identifying and knowing some info about what I see, but my interests are more on the photography side than the scientific side. My hope is to also sell some of my work as fine art or for other uses - similar to what I do for macro today.

I have been reading up on microscopy for some time but there are some questions that I cannot find definitive answers to. I would very much appreciate it if anyone could take the time to answer these questions.

First, in terms of a scope I think the types of lighting that will be most helpful are phase contrast, brightfield, and darkfield. Polarizing light is also interesting, but for my purposes I think it will be less useful. My primary concern is finding a microscope with enough resolution for a 21MP camera.

1) I am currently thinking about buying the Meiji 5310 (http://www.microscopeworld-professional ... spx?ID=173). Is this a good scope/manufacturer? Is it capable of resolving 21 megapixels?

2) I called the store and they said that it can do phase contrast, brightfield, and darkfield out of the box. I had thought phase contrast and brightfield require different objectives. Is it possible to do brightfield with phase contrast objectives?

3) I would like to use my 5D Mark II for photographs. From researching there appear to be two main methods of connecting the camera. The first is to use tubes that attach to a trinocular head and are sold by the manufacturer. The second is to place the camera directly over the trinocular head but not physically connect them - the idea being that the moving shutter will cause the image to be blurry. Is the second method truly necessary? The mirror can be taken care of through mirror lock but does the shutter generate enough force to shake a 20 lb microscope and cause the image to be blurry?

4) For moving subjects, it sounds like the light source from the microscope is not sufficient, so flash is used. I have read a number of articles of people taking flashes apart to make their own lighting devices but I hesitate at doing this since I have absolutely no electronics skills. I would prefer to buy something already made and from the description it sounds like this device should work - http://www.novoflex.com/english/html/fr_ebl4.htm (B&H also sells them) Basically I can use a 580EX flash that a fire with a Pocketwizard and is piped through the three flexible tubes at the subject. Do you think this will work? Are there better solutions that don't require any electronics skills?

5) Which would provide better optical quality - buying a used phase contrast microscope and outfitting it with a trinocular head (not sure where to buy?) or buying the new Meiji microscope listed above?

Thank you for your time,

'Joe

Charles Krebs · Post by **Charles Krebs** » Wed Jan 07, 2009 12:33 pm

Greetings neighbor! (I'm in Issaquah)....

Whew... the answers to your questions could fill a small book! But here are a few thoughts.

First, in terms of a scope I think the types of lighting that will be most helpful are phase contrast, brightfield, and darkfield. Polarizing light is also interesting, but for my purposes I think it will be less useful.

If you really get into photomicrography in a big way you will want to add DIC to that list. But it is expensive to set up, so, unless funds are of no concern, it may be best to see if the microscope remains an ongoing interest. Personally I find phase contrast wonderful for viewing, but prefer other methods for photography. Look through the images posted in the forum to get an idea of how the different lighting methods compare.

To your specific questions:

1) Meiji is a good solid manufacturer. (I have a soft spot for them because I started with a used Meiji). Their stereo scopes are highly regarded. Their compound microscopes are also nice, but their line is nowhere near as extensive as the "big four". From a practical point of view this means you will be fine for brightfield, darkfield and phase but will not have DIC as an option. If you eventually desire really high-end "glass" (like Plan Fluor's or Plan Apo's) they do not manufacture them. The model you mentioned is one of their newer "infinity" corrected scopes. This is the type of optics everyone uses now. It has some benefits, but means you are locked into a specific manufacturers line of optics. (You can't access the pool of nice used 160mm tube length objectives that is out there).

No light microscope can really exploit a 21Mp DSLR. Diffraction limits the resolution of even the finest objectives. Somewhat counter intuitively, the most demanding objectives are low magnification, high numerical aperture Plan Apo's like a 4/0.20 or a 10/0.40. After that, even the best (light) microscope optics would not even tax an old 6 Mp Canon 10D.

2) My experience has been that I do not care for the brightfield images obtained with the phase contrast objectives I have tried. I emphasize that because there is a wide variety of opinions on this. I've become convinced that part of the reason for this is that there is great variability between objectives. Some apparently work very well in brightfield (with perhaps a slight loss in contrast), others don't. As with other objectives, the big manufacturers offer more than one "quality level" of phase objectives.

3) Yes, the shutter alone can be a problem. However... the Canon 40D, 50D and 5DII all have a very desirable feature when used in the "live view, silent mode". They utilize an electronic first shutter curtain, and a "conventional" mechanical rear shutter curtain. This should effectively eliminate vibration as a problem below 1/60 second exposures. (Some testing will be needed to reveal how much it helps at shutter speeds above 1/60. I have not tried it yet. Unless there is some hack, or work-around I'm not aware of, this "mode" can't be used with flash).

4) For live subjects electronic flash is wonderful (I would argue essential). There are some very effective methods that do not require disassembling flash units. Perhaps the easiest and most effective is placing a semi-mirror (or piece of glass) at 45 degrees under the condenser and firing the flash into that.

Keep in mind that the using Novoflex unit as you mention would require sufficient distance between the front of the objective and the subject. Except for (certain) low power objectives, or specialized "long working distance objectives" this will not be the case on a compound microscope.

If the magnifications you desire do not go much higher than about 20X, and such "surface" lighting is desired you might want to consider a "table-top" setup using certain microscope objectives on bellows. the "Technical and Studio" section here is a great resource for this.

If you want really high magnification "surface" illumination, you will need to look into a microscope with a vertical ("epi") illuminator.

5) The new Meiji is good. The only real downside would be the inability to "expand" to higher grade optics (if needed) and DIC illumination.

This should give you a few things to think about. I suggest you look through both the "Microscope" and "Technical" sections, and ask a bunch more questions before you make the big decision.

Charlie

kirispupis · Post by **kirispupis** » Wed Jan 07, 2009 1:39 pm

Thank you for the responses Charles! In fact your work (particularly in the Nikon competitions) is what inspired me to make the move to micro.

I know that micro is something that I will spend a lot of time with and I want to put the budget into it that will do the job. One of the things I love the most about macro photography is I don't need to leave my yard to take great photos - which goes even more for micro. I plan to make a purchase in December 2009 and am taking between now and then to determine exactly what I need (and save the money).

As Meiji cannot do DIC I am taking a look at the Nikon Eclipse 80i - http://nikon.com/products/instruments/l ... /index.htm

It's a bit pricy and I have a phone call into Nikon Instruments to find out how pricy it is, though when I gave my budget they did say I was in the right ballpark. Would this microscope work well, or are there better ones when one starts talking that kind of money?

Tardigrade37 · Post by **Tardigrade37** » Thu Jan 08, 2009 7:06 pm

Just to add my 2 cents...

In my experience, the Nikon 80i is a spectacular microscope with one of the nicest DIC setups I have used (the shear is adjusted by the polarizer, so no fiddling with the wollaston prisms). Of course, my experience may be biased as this scope is outfitted with the best of the best optics (all Plan-Apos including a gorgeous pair of 40x/1.3 and 60x/1.4) on the C1si laser scanning confocal.

To follow up with with Charles' comments on what objectives require the highest camera resolution, I've always found the following chart from a Zeiss Axiocam brochure helpful...

Charles Krebs · Post by **Charles Krebs** » Fri Jan 09, 2009 9:57 am

Joe,

Hmmm, a Nikon 80i.... yeah, that would probably be OK, I guess ...

(actually...

$\:D/$

... there are not sufficient "superlative" emoticons to show approval!)

I've had some brief encounters with it's predecessor, the Eclipse 800, and it's great. Truth is, when you look at that level of current hardware from any of the "big four" you can't go wrong. (Like anything else I'm sure people can argue between them, but it becomes a Lexus/Mercedes/BMW type of argument.)

It also sounds like you are going about researching your purchase wisely.

The table Tardigrade37 provided really reinforces the fact that the higher magnification objectives do not require huge pixel counts. It also shows how critical N.A. is to resolution (compare 4+5 and 9+10 in the chart). And it illustrates how sensor "requirements" will vary with the relay optics used (1.0X and 0.63X for this chart). Keep in mind that this chart is for a camera that has a 2/3" sensor (8.5x6.4mm). So it will be recording FN's (field numbers) of about 11mm with the 1X, and 17.5mm with the 0.63. If you do use a 5DII on the microscope your sensor will be 24x36mm. To record comparable field sizes on the 24x36mm sensor you would use a 2.5X relay (recording a FN of 17.3) and a 4x relay (FN of 10.8mm).

The high-end Zeiss cameras use pixel shifting to sample each pixel in red, green, and blue. (Great for stationary subjects, but it means no high-speed electronic flash for "stopping" moving cilia and other such rapidly moving subjects.) With a conventional Bayer pattern sensor it's a good idea, if possible, to sample details at a higher rate (more pixels) than in cameras that sample RGB values at each sensor site.

kirispupis · Post by **kirispupis** » Fri Jan 09, 2009 1:57 pm

Thanks! What I find most interesting about this chart is that it seems to quantify the difference between the objectives. I have seen several sources state that I need "such and such" objectives - but nothing about how much better they are optically.

I really would like to buy the Nikon 80i setup for DIC illumination but I'm having a very difficult time determining what they actually cost. I found a used Olympus online at $13500 - http://www.labx.com/v2/adsearch/detail3 ... umb=376379

The problem is that this was the budget I was planning to spend for a new one.

Therefore it seems like I will need to temper my goals a bit and buy a used scope - though I'll need to do a lot more research because I'm not sure what the differences are between all of the different models - though it seems to make sense to buy from one of the 'Big 4'.

However, it does seem like when it gets down to it - it's just optics which don't improve that much year in and out. Are the new scopes much better than the older ones in that regard? For instance, would there be a huge difference between the Nikon 80i and the Olympus BX41 mentioned above?

Charles Krebs · Post by **Charles Krebs** » Sat Jan 10, 2009 11:56 am

Joe,
In your first post you said:

my primary targets will be small water organisms

With this in mind the Olympus BX41 you referenced (on LabX) is not set up properly. That one is configured primarily for reflected light (episcopic) observation. It has BD (Brightfield/Darkfield) MPlan objectives. These are designed to view subjects without coverslips. Generally these are used for opaque subjects with the vertical illuminator. The DIC appears to be only for reflected light. Transmitted light DIC (what you want for small water critters) requires different (and expensive) components.

Absolutely nothing wrong with Olympus BX series microscopes... they are excellent. This one has simply been put together for a different purpose than you desire.

Hairyduck · Post by **Hairyduck** » Sat Jan 10, 2009 1:50 pm

Well spotted charles, that'd need a universal condenser and prisms, which if you were to buy new from Olympus would set you back around $7000, DIC definately isn't cheap!

kirispupis · Post by **kirispupis** » Sat Jan 10, 2009 8:17 pm

Thank you for pointing that out! I think my next step will be to order some books on microscopy and do a bit of research before I ask any more questions. However, it does seem like DIC is a rather advanced topic. Do you know of a good book that covers it?

The main thing I would like to figure out right now though is the approximate cost I am aiming for. I have the feeling that the Nikon Eclipse 80i will cost around $25,000 - which is quite a bit above my budget. Does this sound correct? What is the approximate price for aan extremely good used DIC microscope (in other words not just DIC but very good optics too)?

Hairyduck · Post by **Hairyduck** » Sun Jan 11, 2009 2:13 am

Cost depends on how lucky you are really, there are lots of industrial auctions around where you can pick up very good scopes for $3-4k the lenses that come with them aren't usually great though.

I bought my first decent scope from ebay,an olympus BHA with flourescence, cost was £400, however the lenses were rubbish so I bought a full set of Nikon plan apos in mint condition over a period of time, cost for these was around £2000

I then got the urge to included DIC in my setup so I bought another olympus scope off ebay that included the dic condenser and slider for £800 (purchased after a crafty bid outside fo ebay)

So that equates to a total of £3200 or roughly $5000 for something that is optically fantastic so as you can see if you're prepared to put a bit of time and effort into sourcing the parts there are huge savings to be made, I'd never contemplate buying a new high end scope.

Charles Krebs · Post by **Charles Krebs** » Sun Jan 11, 2009 5:16 pm

Buying new is very expensive. As Hairyduck mentioned, used equipment and patience can save a lot of money.

You can, on occasion, come across used (or "demo") current models for considerably less than new.

The other option is to try an find one of the last 160mm finite models made by Olympus or Nikon. I just don't see much used Leitz available in the USA. Zeiss is great equipment, but you really need a Zeiss guru to find your way through huge variety of gear they made. (And while 160mm Zeiss Plan Apo's are superb, care must be made when purchasing them. Unfortunately internal element "separation" or "delamination" is fairly common.)

Depending on how lucky and patient you are, it should be possible to come up with a nice 160mm tube length scope, with DIC and excellent glass for between $7-10k. (And a sizable chunk of that... close to half... is in the DIC components!)

I would recommend talking with some reputable dealers of used equipment, telling them what you want, and let them see if they can assemble a scope for you. (It may take them some time to locate all the components as well). It's tricky enough on eBay even if you have some experience with the hardware.... positively daunting if it is all new to you.

kirispupis · Post by **kirispupis** » Mon Jan 12, 2009 1:14 pm

Thanks for the info again. It looks like a used DIC microscope is the way to go and fits my budget. As I continue my research the following would help greatly.

1) Do you know of any good books that cover DIC?
2) I like the idea of having a reputable store find the different parts for the microscope. Are there any places you recommend?