For amino acid test of pollen u dont have to bother about the anthers. Just collect the anthers and dry them at room temperature and after that make them into powder form, filter through the mesh of 200, 300, 400 um mesh to find out the percentage of pollen, a 80% pollen mass is best and effective. From that you can do the HPLC analysis

I recommend that you dry the anthers at 37 celsiums degrees for a few hours to expedite the process and prevent microbial degradation of the product. Bacteria and fungi may grow very quickly on a moist product if left at room temperature.

As you know, the "dry and sieve technique" is extensively used to clean large quantities of pollen, but not a few milligrams of it because you would loose a lot of material after each cleaning step. Many times pollen derived from insect-pollinated plants are obtained from insect bodies, resulting in a pure product.

Pollen collected directly from the anthers can also be in various stages of dehydration and needs to be hydrated under controlled conditions to allow the pollen to swell to its natural hydrated form. A well defined hydration routine is especially important if building up a meaningful collection of pollen types to allow 'like to be compared with like' and for comparison with pollen databases etc.

A 50% water/glycerine solution is one recommended way to hydrate the pollen and which has the benefit of being compatible with one of the recommended permanent mountants for pollen i.e. glycerine jelly.

Certainly much of the pollen I collected direct from the flowers and examined dry were difficult to interpret and looked shrivelled. The difference between the untreated and treated pollen is quite striking. (Although it is worth studying pollen from fresh anthers before treatment, especially to see the pollen colour).

It's worthwhile viewing the washed grains (still in a little alcohol) under a stereo microscope (ca 40X or greater for larger pollen types) as the 50% glycerine is added; the hydration and swelling of the grains is interesting to watch. Studying the hydrated larger pollen grains under the stereo also gives an idea of their gross shape and structure before studying at higher powers under the compound microscope.

We use static to separate pollen from anther parts. We grind up the anthers and place the mixed anther parts and pollen into a large top plastic vial. We then vortex the vial, and pollen clings to the walls. After ~2minutes of vortexing, we transfer the anther parts to another vial; the few small anther parts that cling to the walls of the vial can be removed with sharp tapping onto a flat surface. Finally we use sterile razor blades, and static free brushes to remove the pollen from the vial.

Most of the possible methods have already described above, I am not sure this will help you or not.

http://journal.ashspublications.org/content/130/3/341.full.pdf

http://pubs.acs.org/doi/abs/10.1021/jf00086a003

You can sample the strawberry pollen loads brought back to the bumblebee colonies by foragers with a pollen trap.

It's a method commonly used with honeybees by beekeepers and scientists in order to collect pollen.

This trap tool can be suitable in your case if you have bumblebee colonies as well as strawberry orchards/crops (in order to assure the strawberry origin into pollen loads collected).

Hi everyone, thank you for all your answers! Sadly Rosa is correct in that I will have very small amounts of pollen, so I wouldn't realistically be able to mesh the samples without losing a significant amount of my sample. Though I'm not sure I'd be able to heat the pollen either, as the literature suggests that the amino acids tend to degrade when you heat them. The usual method appears to be freeze drying.

So I could perhaps freeze dry the anthers, then grind them into a power. And perhaps use the vortexing technique that Jacob describes. Jacob, could you recommend a paper/procedure that uses this technique? I’d be interested to look at it in more detail.

Sadly collecting pollen from the bees themselves is not really an option because I will also be observing bee visitation choice on different cultivars of strawberries, with no way of differentiating which pollen came from which treatment. Which is further complicated as the nectar that bees include into their pollen baskets have been found to actively degrade protein structures which could influence my amino acid HPLC readings. But most papers seem to look at bee collected pollen methods so I was struggling a bit to find information on hand collection techniques.

Most of the possible methods described above are correct, and it would be work effectively with few modification as-

After collection of anthers allowed them to dehisced in oven for short time period which increase the pollen amount. Thermal degradation of amino acids occur at high temperature therefore drying of the anther must be at room temperature it may takes 24-48 hrs. Then powder filter through the mesh, 80-90% pollen purity is enough to carry biochemical analysis.

Extraction must be carried out by cold extraction method in vials by continuous vortexing and resuspend the pellet in solvent after 2 hrs, repeat it twice for complete removal of amino acids. 70% Ethanol would be the best solvent for extraction. Extract required for analysis is 5-10 micro-liter, so don't bother about quantity. Extract would be more than enough.

I have been used this protocol for analysis of amino acid from pollen. If you are not satisfied with extract quantity for HPLC, then you must go for HPTLC which require only 2-5 micro-liter extract, even you can go further for quantitative analysis. HPTLC work effectively with lower amounts samples.

https://www.researchgate.net/post/Can_anybody_suggest_me_how_to_collect_pure_powdered_pollen_of_wheat_separated_from_anthers