I have a zoom microscope that I would like to use to determine the size of pollen. I assumed that I could find inexpensive slides with internal calibration lines, but I have not found anything below costs of hundreds of dollars. Does anyone have any suggestions for a simple, low cost method?
Do you have an eyepiece to your microscope with an internal scale (little hash marks)? That has been very helpful when using the scope I used to use. If you can borrow a slide with the internal micrometer calibration lines then you can calibrate your eyepiece markings for your various zoom settings with your particular objective. You can then measure pollen as having so many of your hash mark distances and then use whatever multiplication factor to get the distance in micrometers. If you don’t have an eyepiece with a built in scale I suppose you would want to then own your own slide with the internal calibration marks to always be comparing against and referencing which can become tricky because you wouldn’t be able to have your sample and the scale in a field of vision at once unless you try to put pollen and a coverslip over the slide with the internal scale. It may be hard to get both in focus at once because they would be on different planes.
If you have an eyepiece with a scale that you can calibrate under your different zoom settings I think that would probably be best. Just borrowing a slide with the internal calbrations should hopefully be possible from a college teaching intro biology or botany. I was able to borrow one from those coordinating the intro biology classes at the U of MN when I needed one. I now have access to a microscope connected to a camera and computer. I was able to calibrate distances within the computer program using that particular scope and slide with the calibration marks and can drag and click the mouse over digital images to determine length.
Sincerely,
David
Dave, unfortunately, there is not an internal scale. The zoom range is continuious from 100X to 500X.
Even glass calibration spheres are in the several hundred dollar (or more) ballpark.
I wonder if it would be possible to dye a diploid rose pollen and then mix some of it with the unknown chromosome pollen and do a relative comparison. What I would need to first determine is the amount (if any) that dying would permanently swell the diploid pollen. I could do this by calibrating the dyed pollen against the same type of “not dyed” pollen.
Comments or other suggestions?
The following student experiment instruction sheet appears to be useful for those interested in determining ploidy levels:
http://onion.nmsu.edu/Publications/PDF/HortTech99.pdf
I am particularily interested in the simple stomata experiment. Does anyone have any experience with it and roses?
There’s a different approach you might take.
There is industrial screening that allows only certain sizes to pass through. If you can find some with a 10 micron or a 20 micron size and use it on a microscope slide, it would be similar to using graph paper as background on a scope with lesser magnification.
I don’t have the name of the company I used on my no-longer-in use oil industry rolodex, but the screening was significantly cheaper and sold by the yard. The link below might help
Thanks Ann, I should have some flat wire gasoline/water filter disks in the garage. Maybe I can calibrate them.
I checked. The wire gasoline/water filter disks are too coarse.
That’s a great idea Ann. There are some papers out on the use of sieves (nylon mesh of known pore size) to screen and save pollen of certain sizes. The papers are on potato and tulip to separate 2n pollen from n pollen.
Stomate size or density can be useful when considering a limited rose germplasm base like with chromosome doubling experiments. One can compare the potentially doubled plants with diploid controls. I did it for R. chinensis minima and others have done so for R. multiflora chromosome doubling experiments. However, when considering roses in general and trying to find something predictive of ploidy, stomate size is all over the board and does not work well. It is too variable. It varies across species and modern hybrids are all over the place. Perhaps something with stomate density may still be worth looking into, but I doubt it would be very useful as a general ploidy predictor either. I’ll be trying to publish some data on rose stomate sizes and ploidy hopefully in the near future. Nailpolish works as they recommend. However, superglue works great too and seems to be easier. Just put a drop on a slide and press your leaf into it. After a half hour or so pull your leaf off and you have a great imprint. It adheres to the slide and can be stored for years.
How much money would it cost Henry to get a new eyepiece with an internal scale that you can hopefully calibrate with a borrowed slide with a micrometer scale? Having a slide with internal calibration for reference or using mesh of known size to put your pollen over or into for comparison, or somehow preferentially staining pollen of known ploidy all seem to be procedurally challenging and adds extra steps each time you look at a sample. If you can get a new eyepiece then you don’t have to worry about such details and may be able to get more accurate measurements as well.
What do you think?
Sincerely,
David
A micrometer eyepiece isn’t going to be useful with a general zoom microscope unless the zoom part has some clicks to indicate certain magnifications.
On a non-zoom, micrometer eyepieces work, because we know if we are using objectives of 10x, 25x or 40x (often by the scale of what we are looking at, as well as by the feel of the objective as we rotate them.) But with a zoom, only the two ends of the zoom cycle could be calibrated…and often the usefulness of the zoom is in between.
The calibrated spheres that Henry mentioned could be used with a calibrated eyepiece for measuring things they are mixed with. But “good” calibrated spheres are pricey. So is there something else in nature that could substitute? I wonder if the fungal spores off of “everyday” grocery store mushrooms could be used? They would be smaller than rose pollen, but would be constant. All you really want is a dependable constant…for multiplication factors, you might not even need to know the size of that spore, because you are really just wanting to measure the size of the rose pollen versus other rose pollen and if you can compare numbers in terms of spore diameters, it doesn’t really matter what the multiplier is. (Am I making any sense here?)
(I spent years as a palynologist.)
Individual fungal hyphae could also be used.
Spider webs are too thin and hard to manuver.
Unfortunately my Bausch and Lomb does not have click stops.
Ann and Dave, thank you both for your experienced comments. I am now thinking of mixing “not dyed” geranium pollen (see, my link given earlier in this thread).
I orginally proposed mixing the “to be determined” pollen with dyed diploid rose pollen as an internal calibration. The reason for dying was to be sure I knew which pollen grains were which. I hope/assume that geranium pollen will have enough of a different appearance so that I can skip the dying step and remove the possibility of swelling complications. If the geranium pollen are not that different, I will look at impatiens pollen as my wife grows many plants of both so I should have a ready supply of both at all times during the growing season. If neither prove practical I will try Ann’s suggestion of mushroom fungul spores.
Henry,
There’s a procedure called acetolysis used in palynology to process modern pollen. It strips off the exine (outer layer) and also imparts a brown color to the pollen. This would give you something with a color that would be distinctive in transmitted light.
A problem with a simple die job is that we see the color, but when it gets on the microscope, with the intensity of the transmitted light …everything looks translucent.
A lot of angiosperm pollen looks superficially alike. Whether something is colpate or colporate or porate is hard to tell.
But gymnosperm pollen is very different, as are fern spores, fungal spores, etc. That’s why I’d go to another class or order of plants for my scale.
Ann
I just looked up lycopodium spores dimensions (google search) and found that they are 25-35 um diameter. Duke scientific sells a whole range of pollens to use for calibration of processes. they didn’t show prices on the page I looked at but they do have a price list further along. There are pollens for every size ranging from 2 to 100 um.
Dear Henry,
Maybe you can use a haemacytometer (counting chamber with grid). I used it a lot for fungal spore counts, maybe it is usefull for pollen sizes as there is a callibrated grid in it. You can find them on e-bay for 8dollar in Europe, probably also in te US (the adress mentioned can also send it):
best regards
leen
The following link sells an inexpensive 10 micrometer calibrated slide:
https://wardsci.com/product.asp_Q_pn_E_IG0011880_A_Ruled+Stage+Micrometer
I expect that using this slide along with a 640 X 480 web cam attached to my microscope will allow me to easily measure pollen diameters and the lengths of stomata guard cells.
The following discusses a free program for digital microscope measurements:
Yesterday I purchased this $9.99 web cam at a local Radio Shack store. It works with my XP operating system (a driver apparently can be downloaded for Vista):
http://rsk.imageg.net/graphics/uc/rsk/Support/ProductManuals/2500157_PM_EN.pdf
I removed the web cam’s lens (it simply unscrews) and the microscope’s eyepiece (the ocular). This is done because the wide angle lens used in a web cam can cause some vignetting and because the X 10 magnification of the eyepiece is no longer needed as the web cam provides more than enough magnification. I used the lowest setting on my microscope (X 100) for the following picture of a red rugosa’s pollen:
http://picasaweb.google.com/HAKuska/MicroscopePictures/photo?authkey=t7Y4J3vWxJA#5107093179143069170
The web cam was attached to a short piece of PVC water pipe (I just used tape, but will probably change to super glue plus tape later) which just fitted into the barrel of the telescope. A layer of plastic tape was added to the outside of the pipe to make a tighter fit. The resolution for a still picture is 640 X 480 pixels. The web cam uses a CMOS sensor.
The web cam can be simply pulled out and the eye piece put back in to restore the microscope to its original condition.
Pretty nice henry! What a creative idea!!! Perhaps you can get some pretty accurate measurements by calibrating your microscope pictures with a cultivar that has pretty uniform pollen size.
For instance using acetocarmine and the cultivar Therese Bugnet (mean pollen diameter (30 grains) of 31.2 microns with a standard deviation of 1.3). Using your camera in the same setup for additional photos you can know how far ~31.2 microns is and extrapolate from there. You should be able to see some nice chromosome counts with your microscope and its magnification too and hopefully some good pictures as well if you can adjust the intensity of the light a little.
Thanks for sharing your idea and success Henry!
Sincerely,
David
The Ward’s glass slide “Stage Micrometer” arrived today. I calibrated the X 100 position on the microscope and then used the Micam program to measure some pollen that I had. See:
http://picasaweb.google.com/HAKuska/MicroscopePictures?authkey=t7Y4J3vWxJA
Eileen White Erlanson in Botanical Gazette, volumn 91, pages 55-64, (1931) in an article titled "A Group Of Tetraploid Roses In Central Oregon: gave the following pollen ranges in her Table II, page 60 (numbers in micro meters):
Diploid:
Oval 30-39.4
Rounded 20-25
Tetraploid:
Oval 38.3-46.6
Rounded 23.3-26.6
Hexaploid:
Oval 45-51.6
Rounded 25-30 (limited data)
Apparently another source of pollen length data is the 1957 Ph. D. thesis of W.H. Lewis, University of Virginia. I do not have a copy of that thesis. Can anyone suggest other sources of data?
Link: picasaweb.google.com/HAKuska/MicroscopePictures?authkey=t7Y4J3vWxJA
For pollen size it is very important to be using the same staining and preparation protocol as others use if you are interested in making comparisons with the data of others. Stains and how hydrated pollen is can affect the observed size. That is why I said I used acetocarmine before giving you my data for Therese Bugnet. The data from Eileen has been odd from what I have gotten and the pollen data presented in much of the rose chrosomosome doubling work and haploid characterization of roses reported. She does not talk about hydrating the pollen and that may be why her sizes are smaller and more variable than those of others.
I suggest that you decide how you will treat your pollen before observation and then look at pollen of roses you have of known ploidy and set your standards that way. If you are going to use acetocarmine I could give you my standards and that should work. I suggest using some liquid of some sort (water, acetocarmine, some other stain) to hydrate the pollen because I believe there is more uniformity in size and it is easier to make measurements with spheres than shrunken, oddly shaped, dry pollen.
Sincerely,
David
Thanks Dave, please present your staining method as I would like to make my measurements as useful as possible.
Of course I may decide not to use your method if I feel that it is not suitable for “safe” home use.
Some background material for others considering using such a system.
Two commercial U.S.B. digital eyepieces are available (for example) from the following link:
The 1/2 inch CMOS 1.3 megapixel unit is probably a step up from my home build unit which I assume is a 1/3 inch CMOS 0.35 megapixel unit. There are now 1.3 megapixel web cams available, but I decided for my next project to jump one size even higher. I have on order a unit that has a 2 megapixel CMOS sensor (costs $19.99 refurbished). The web page does not allow one to link directly to the product. The product is: “PocketCam X”; the web page “front door” is: http://www.aiptek.com/
This may not be as easy to convert as it has a fixed focus lens so it may be glued in instead of having a threaded mount.
The same company also sell inexpensive microscopes:
On the internet there are also links on how to build your own digital microscope with a webcam and the lens (or lenses?) from CD/DVD players/recorders.