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3D Confocal Imaging of Pollen

Authors:
  • University of Texas Rio Grande Valley, Brownsville-Edinburg-Harlingen, United States

Abstract and Figures

The study of pollen is important in paleoecology, paleontology, archeology, and forensics. The outer shell of a pollen grain bears characteristic features that help in determining the species or genus of origin. To view the pollen grain from several angles in a light microscope, the sample is usually mounted in glycerol and rolled on the microscopic slide by pushing the coverslip. For teaching, training, and also for establishing databases of pollen images, it is desirable to obtain three-dimensional datasets that allow rotation and viewing from any angle and reslicing in the computer.
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26 ww w.microscopy-today.com2010 March
mismatch between the immersion oil and the specimen, or
local dierences in RI within the specimen, cause distortion
and a loss of both resolution and uorescence signal intensity.
Pollen grains are a challenging type of specimen because
the cellular content of raw pollen is highly autouorescent and
optically inhomogeneous, which leads to light absorption,
scattering, and spherical aberration. Typically, only the half of
the pollen grain closest to the objective can be imaged at good
resolution in single-photon uorescence microscopy. We have
employed sample preparation and mounting techniques that
allow single-photon 3D confocal imaging of even relatively
large grains (more than 50 mm in diameter) without excessive
loss of resolution toward the bottom of the 3D dataset. Two key
elements of our technique are pollen extraction and refractive
index matching.
Introduction
e study of pollen is important in paleoecology, paleon-
tology, archeology, and forensics. e outer shell of a pollen
grain bears characteristic features that help in determining
the species or genus of origin. To view the pollen grain from
several angles in a light microscope, the sample is usually
mounted in glycerol and rolled on the microscopic slide by
pushing the coverslip. For teaching, training, and also for
establishing databases of pollen images, it is desirable to obtain
three-dimensional datasets that allow rotation and viewing
from any angle and reslicing in the computer.
Although the scanning electron microscopy provides
sucient depth of eld and high resolution of surface features
[1], without tilting or rotating the specimen, images from the
SEM show only one side of the pollen grain at a time (Figure 1).
In contrast, confocal uorescence microscopy permits optical
sectioning and 3D reconstruction of a single pollen grain and
allows imaging of not only the surface, but also the internal
structure of the pollen grain, albeit at resolution limited by
diraction, that is, typically ~ 0.2 µm in XY and ~ 0.5µm in Z.
To achieve this resolution, oil immersion objectives with high
numerical aperture must be used. Oil immersion objectives
achieve their nominal resolution only when used for
specimens with a refractive index (RI) that is homogeneous
and equal to that of the immersion oil, that is, η=1.515. An RI
3D Confocal Imaging of Pollen
Stanislav Vitha* 1, Vaughn M. Bryant2, Amen Zwa3, and Andreas Holzenburg1, 4
1Microscopy and Imaging Center, Texas A&M University, College Station, TX 77843
2Department of Anthropology, Texas A&M University, College Station, TX 77843
3Gannon Technologies Group, 1000 North Payne Street, Alexandria, VA 22314
4Department of Biology & Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843
* vitha@mic.tamu.edu
doi: 10.1017/S1551929510000039
Figure 1: Scanning electron micrograph of Artemisia tride ntata poll en. Scale
bar = 5 µm. Image courtesy of Gretchen D. Jones, USDA-ARS, APMRU.
Figure 2: Confocal optical s ections of Celtis laeviga ta pollen grain. Raw and
deconvolved data a re shown in the left and right panels, respective ly. Top: XY
view, a single optica l section from the z-stack. Middle: XZ section through the
z-stack. The dot ted recta ngle indicates an ar ea shown e nlarged in the bottom
panels. S cale bars = 5 µm.
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3D Confocal Imaging of Pollen
Results and Discussion
e sample preparation protocol allowed confocal 3D
imaging of the entire pollen grain, without substantial loss of
resolution or signal intensity on the far side of the specimen.
Deconvolution of the 3D image stacks provided noticeable
enhancement in resolution (Figure 2). Confocal images
allowed to assess the internal structure of the pollen grain wall
(Figures 2 and 3A) and also enabled realistic visualization of
the pollen grain surface (Figures 3B, C, D). e pollen grains
shown in these gures are 25 to 30 µm in diameter. We have
imaged pollen grains more than three times that size with
similar results except that the datasets for the largest pollen
grains, almost 2.5 GB in size, could not be deconvolved using
our computers, because total memory requirements exceeded
the limits of the 32-bit operating system. Further gain in
resolution is possible by a slight increase in oversampling,
because the current XY pixel size (65 nm) is somewhat larger
than the sampling rate recommended by some calculations (for
example, the Nyquist Calculator at http://support.svi.nl/wiki/
NyquistCalculator suggests voxels 43 × 43 × 130 nm). Axial
resolution could be increased by further closing the confocal
aperture at the expense of signal intensity or imaging speed. With
confocal aperture set to 50% Airy (80 µm), the axial resolution
of the particular 100×/1.4 objective was previously measured
as 330 nm (FWHM) using a mirror slide test specimen [4].
However, much slower scanning may be impractical because
even now acquisition times are 30 minutes for Artemisia and
Celtis and over four hours for very large pollen grains, for
example for Pinus nigra.
References
[1] GD Jones and VMJ Bryant, Grana 46 (2007) 20–33.
[2] G Erdtman, Svensk Botanisk Tidskri 54 (1960) 561–564.
[3] T Staudt, MC Lang, R Medda, J Engelhardt, and SW Hell,
Microsc Res Tech 70(1) (2007) 1–9.
[4] RM Zucker, Methods Mol Biol 319 (2006) 77–135.
Preparation
Pollen extraction. Fresh or dried pollen was processed
using a modified acetolysis method according to Erdtman
[2] in order to remove the cellular contents and debris and
to isolate exine, the acetolysis-resistant outer shell. The
exine has sufficient autof luorescence to permit confocal
microscopy. The step-by-step protocol is available on the
MIC web site http://microscopy.tamu.edu/lab-protocols/
light-microscopy-protocols.html
Refractive index matching. In order to achieve RI optimal
for imaging with oil immersion objectives, the acetolysis-
processed pollen suspended in glacial acetic acid was gradually
rehydrated by adding increasing amounts of water. A total of
10 steps were performed. In order to accelerate the diusion
process, microwave irradiation in a Pelco Biowave (Ted Pella
Inc., Redding, CA) laboratory microwave processor for
1 minute at 230W was used in each step. Pollen was then
collected by centrifugation at 1000 × g for 1 minute, resuspended
in phosphate buered saline (PBS; 140 mM NaCl, 3 mM KCl,
10 mM Na2HPO4, 2 mM KH2PO4) and microwaved as above.
Subsequently, the pollen was inltrated with 2,2’-thiodiethanol
(TDE), a mounting medium for high-resolution microscopy
[3], using 10%, 25%, 50% v/v TDE/PBS mixture, and nally
three times in 97% v/v TDE/PBS, using microwave irradiation
in each step. e pollen was then spun down and the pellet
resuspended and stored in 97% TDE/PBS in microcentrtifuge
tubes. e RI of 97% TDE is 1.515, that is, the same as RI of
immersion oil [3].
Confocal Microscopy
e Olympus FV1000 laser scanning confocal microscope
with a UPLSAPO 100×/1.4 oil immersion objective was used in
photon counting mode with the confocal aperture set to 0.75 Airy
(120 µm on the FV1000 system). Excitation wavelength was
488 nm, and emission bandpass was set to 500–600 nm. e
laser output was programmed to increase with imaging depth
to maintain image brightness, typically from 7% to 12% of
maximum output. e voxel size was set to 65 × 65 × 130 nm. e
resolution of the objective under these conditions was previously
estimated as 196 ± 12 nm in XY and 418 ± 43 nm in Z (FWHM;
full width at half maximum) using 100 nm uorescent beads.
Deconvolution with Huygens Pro soware (SVI, Hilversum,
Netherlands) used 100 iteration of Classic Maximum Likelihood
Estimation method. Surface rendering was performed using
Osirix freeware (http://www.osirix-viewer.com).
Figure 3: Artemisia dr acunculu s pollen grain. A, a si ngle optical section ( XY plane ) from a co nfocal microscope, showing the outer surface a s well as the internal
structure of the wall. B-D, th ree differe nt surface rendering views generated from the same confocal datase t. Scale bar = 5 µm.
... The constraints of EM limit the number of modern and fossil specimens that can be analyzed in a given study (Traverse, 2008;Zavialova et al., 2018). As a result, confocal microscopy has become an alternative in palynological analysis ( Vitha et al., 2009;Gavrilova et al., 2018). Conventional confocal can capture some of the fine external sculpture of pollen specimens and occasionally their internal structure, but it is diffraction-limited and cannot replicate the high resolution of EM ( Vitha et al., 2009;Sivaguru et al., 2018;Gavrilova et al., 2018). ...
... As a result, confocal microscopy has become an alternative in palynological analysis ( Vitha et al., 2009;Gavrilova et al., 2018). Conventional confocal can capture some of the fine external sculpture of pollen specimens and occasionally their internal structure, but it is diffraction-limited and cannot replicate the high resolution of EM ( Vitha et al., 2009;Sivaguru et al., 2018;Gavrilova et al., 2018). ...
... This allows the analysis of the internal and external structure without the destruction of material ( Vitha et al., 2009;Sivaguru et al., 2018;Gavrilova et al., 2018). Airyscan confocal superresolution microscopy also produces an axial stack of a pollen grain, but at higher resolution. ...
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