Sample prep tips for 3D confocal of pollen

[cromey]

I have very close to zero experience prepping plant tissues for any type of microscopy.  I’ve been contacted by a botanist that is interested in looking at a variety of pollen types and he’s never done confocal.  He’d like to look at the 3D structures and it sounds like good 3D may very well be important.

 

I know from limited experience that the far side of a pollen grain often doesn’t look as good in a 3D rendering, presumably due to spherical aberration, scattering, and other optical effects.  I gather from the resources I have that pollen is highly autofluorescent across the entire VIS spectrum (probably why the vendors love to use it for demos).

 

Any tips, suggested review articles, web sites, etc that people could point me to for sample prep techniques?  Any microscopy caveats to be aware of?

 

Many thanks.

Doug

 

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

Douglas W. Cromey, M.S. - Assistant Scientific Investigator

Dept. of Cell Biology & Anatomy, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  AHSC 4212         email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/exppath/

Home of: "Microscopy and Imaging Resources on the WWW"

 

[Stanislav Vitha]

Hi Doug,
I have been doing some 3D pollen imaging with good success. So far I imaged
pollen grains up to 50 um in diameter, and it seems I get almost the same
resolution on the far side as I get on the side closest to the coverslip.

The sample prep was the key.

1st, the pollen I received from our pollen expert was extracted (not sure of
the exact protocol, apparently somehting the pollen researchers do routinely),
and stored in glacial acetic acid. The result is that the highly absorbent and
scattering contents of the pollen grain has been removed, and all that is left is
the pollen "shell" with its characteristic shape and features. It has enough
autofluorescence for confocal imaging without any staining.

The pollen is gradually re-hydrated and infiltrated with 2-2-thiodiethanol
mounting medium.
below is the prep protocol: The microwave just accelerates the process, you
could just do longer time each step without the MW.

100 µl of the pollen suspension was placed in a microcentrifuge tube and
gradually re-hydrated by adding increasing amounts of water (from 10 µl to
600 µl), pulse-mixing (vortexing) and irradiating in a Pelco Biowave (Ted Pella
Inc., Redding, CA) laboratory microwave processor for 1 min at 230W to
accelerate the diffusion process. Total 12 steps were performed. Pollen was
then spun down by centrifugation at 500 x g for 1 min, resuspended in
phosphate buffered saline (PBS; 140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2
mM KH2PO4) and microwaved as above.

Subsequently, the pollen was gradually infiltrated with 2,2’-thiodiethanol
(TDE), a mounting medium for high-resolution microscopy (Staudt et al.,
2007). The pollen was spun down as above before each step. The pollen pellet
was resuspended using 10%, 25%, 50% v/v TDE/PBS mixture, and finally three
times in 97% v/v TDE/PBS, In each step, 1 min microwave irradiation at 230W
was used after resuspension.  
The pollen was then spun down and the pellet resuspended and stored in 97%
TDE/PBS.

Confocal Microscopy (Inverted microscope)
For microscopy, a droplet of the pollen suspension was applied to a coverslip-
bottom imaging chamber (coverslip thickness ~175 µm) and the pollen
immobilized by placing a small coverslip on the top of the droplet, with a small
dab of dried nail polish on one side as a spacer so that the weight of the
coverslip does not cause flattening of the pollen grain. Close to the spacer,
there layer of the mounting medium is too deep, and the pollen is floating
freely and cannot be imaged. On the other end of the coverslip, there is no
spacer and the pollen is squished, deformed. So I try to find a position in
between, where the pollen is not moving, but is not visibly squished.

Imaging was done using Olympus FV1000 laser scanning confocal microscope
system attached to an Olympus IX81 inverted microscope with a 100x/1.4 oil
immersion objective (UPLSAPO 100x/1.4). Fluorescence was excited using 488
nm Argon ion laser, the fluorescence detector was set to 500-600nm
bandpass. Laser output was programmed  to increase as we go deeper from
the surface in order to compensate for the loss of signal. Typically, the laser
output would double from 8 to 16% for a Z-stack going from 0 to 50 um depth.
 
Confocal scanning was performed in the photon counting mode, scan speed
(pixel dwell time) was 20 µs/pixel or 10 µs/pixel. The confocal aperture was
set to 120 µm, which corresponds to 0.75 Airy Unit. The confocal zoom and
scan size was set to achieve 65nm pixel size in XY. The z-step was set to
130nm.
Typical acquisition time was between 20 and 50 min per stack, depending on
the size of the pollen grain and scan speed (i.e., signal intensity).

You may not need to do such a fine XYZ step, but I have to say the resulting
datasets look great;

Sincerely,

Stan Vitha
Microscopy and Imaging Center,
Texas A&M University


On Tue, 6 Jan 2009 10:47:06 -0700, [hidden email] wrote:

>I have very close to zero experience prepping plant tissues for any type of
>microscopy.  I've been contacted by a botanist that is interested in looking
>at a variety of pollen types and he's never done confocal.  He'd like to
>look at the 3D structures and it sounds like good 3D may very well be
>important.
>
>
>
>I know from limited experience that the far side of a pollen grain often
>doesn't look as good in a 3D rendering, presumably due to spherical
>aberration, scattering, and other optical effects.  I gather from the
>resources I have that pollen is highly autofluorescent across the entire VIS
>spectrum (probably why the vendors love to use it for demos).
>
>
>
>Any tips, suggested review articles, web sites, etc that people could point
>me to for sample prep techniques?  Any microscopy caveats to be aware of?
>
>
>
>Many thanks.
>
>Doug
>
>
>
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

^^^^^^^^^^^^

>
>Douglas W. Cromey, M.S. - Assistant Scientific Investigator
>
>Dept. of Cell Biology & Anatomy, University of Arizona
>
>1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
>
>
>office:  AHSC 4212         email: [hidden email]
>
>voice:  520-626-2824       fax:  [phone]
>
>
>
>http://swehsc.pharmacy.arizona.edu/exppath/
>
>Home of: "Microscopy and Imaging Resources on the WWW"
>
>
>
>