Figure 1.
Point spread functions and resolution.
Empirical point spread functions of ∼0.175 µm beads using widefield, apotome, confocal and two-photon microscopy techniques for a pair (two objectives each one for widefield-apotome pair and confocal-two photon pair) of Zeiss 63× plan apochromat 1.4NA lenses. Widefield and apotome images were acquired with a rhodamine filter (546/10 excitation filter). Images show a single bead in X-Y dimensions (upper tier) and X-Z dimensions (lower tier). Graphs show line profiles of pixel intensities across the centre of each bead in X-Y dimensions. FWHM = full width at half maximum. The point spread function at FWHM represents a measure of the resolution of a microscope [30], [32].
Figure 2.
Maximum intensity projections of Croton hirtus, Mabea occidentalis and Agropyron repens viewed using widefield, apotome, confocal (405 nm and 561 nm) and two-photon fluorescence microscopy. Raw data are shown next to images produced following blind deconvolution. Note that only raw data are shown for apotome microscopy. Images displayed in the X-Y plane show the surface texture of each pollen type; images displayed in the X-Z plane show how the microscopy techniques differ in their ability to capture information on the shape of each pollen type. For each pollen type, images from widefield and apotome microscopy show the same pollen grain. Images from confocal 561 nm and two photon 780 nm microscopy show the same pollen grain. A unique pollen grain was used for confocal 405 nm. An orientation key is provided in the lower left-hand corner of the raw widefield data of C. hirtus, which applies to all other specimens shown (note the orientation of pollen grains imaged using apotome microscopy). The approximate size (diameter) of each pollen grain shown is as follows: C. hirtus ∼50 µm; M. occidentalis ∼50 µm; A. repens ∼40 µm.
Table 1.
General imaging parameters.
Table 2.
Performance comparison of all tested microscopy techniques.
Figure 3.
The surface texture of pollen grains.
Optical sections of the pollen grains Croton hirtus, Mabea occidentalis and Agropyron repens viewed using reflected light (widefield, apotome, confocal and two-photon fluorescence microscopy) and transmitted light (widefield DIC, 405 nm and 561 nm DIC, 780 nm DIC and brightfield). Images from all reflected light techniques were deconvolved except apotome microscopy, which is represented by raw data. Images of surface planes are shown next to deep planes to highlight the depth of penetration of each technique. In order to acquire an image of C. hirtus and M. occidentalis using 405 nm DIC, the master gain was increased during acquisition. For each pollen type, images from widefield and apotome microscopy show the same pollen grain. Images from confocal 561 nm and two photon 780 nm microscopy show the same pollen grain. A unique pollen grain was used for confocal 405 nm. Images are displayed in min/max intensity profile. Pollen sizes as for Figure 2.
Figure 4.
Cropped and zoomed-in views of the surface texture of Croton hirtus, Mabea occidentalis and Agropyron repens.
Pollen grains imaged using confocal microscopy (405 nm and 561 nm), two-photon microscopy (780 nm) and DIC microscopy (405 nm, 561 nm and 780 nm). Images highlight the effect of wavelength, resolution, absorption and detectablility on the recovery of textural information from pollen grains. In order to acquire an image of C. hirtus and M. occidentalis using 405 nm DIC, the master gain was increased during acquisition. All images are raw data, measure 5 µm in the X-Y direction and are displayed in linear intensity profiles.
Figure 5.
Cropped and zoomed-in images and intensity profiles on the surface texture of Croton hirtus and Agropyron repens, showing differences in the signal-to-noise ratio of each technique and the impact of blind deconvolution. CF = confocal; 2P = two-photon; WF = apotome; SNR = signal-to-noise ratio; Decon. = deconvolved. SNR calculated as follows: x−y/x+y where x = the average of six maximum pixel intensity values from the peaks of the line profile, and y = the average of six minimum pixel intensity values from the troughs of the line profile (both areas randomly selected). In the case of C. hirtus, line profiles were traced on a single full Croton structure on the same pollen grain. In the case of A. repens, line profiles were traced on randomly selected regions of A. repens specimens. Data from widefield and apotome microscopy is from a single pollen grain. Data from confocal (405 nm and 561 nm) and two-photon microscopy is from a single pollen grain, but not the same specimen analysed used widefield and apotome microscopy. Intensity profiles were not drawn on precisely the same pixels in X-Y positions owing to the movement of pollen grains in the mounting media during the movement of slides between microscopes. Detailed images measure 5 µm in the X-Y direction and are displayed in linear intensity profiles.
Figure 6.
Orthogonal projections to highlight the depth penetration of each optical microscopy technique investigated here.
All images show single optical plane approximately from the centre of the Z-stack. In merged reflected and DIC planes, data from reflected light techniques has been pseudocolored according to the excitation wavelength. 405 nm = blue; 561 nm = green; 780 nm = red. The widefield images are red psuedocolored to reflect the rhodamine (546/10) excitation filter. Images from all reflected light techniques were deconvolved except apotome microscopy, which is represented by raw data. Images are displayed in min/max intensity profile. Pollen sizes as for Figure 2. Projections were constructed using the Autoquant slice viewer rendering algorithm.
Figure 7.
Absorption of light by the pollen exine.
Showing measurements of the percentage of light absorbed by the exine of the three pollen types investigated here, together with brightfield images (focused to the middle plane) of these pollen grains. Absorption was calculated by measuring the difference between the intensity of light passing through the mounting media and the intensity of light passing through a pollen grain. Three absorption measurements were made from three specimens per pollen type. Error bars represent maximum and minimum values. Where no error bars are visible, the range of data was less than the size of the symbol.
Figure 8.
Scanning electron microscopy and super-resolution-SIM images of Agropyron repens.
Note the surface texture of the pollen grain, which comprises a dense covering of elements that measure ∼250 nm in all directions (granules; [16]). A: showing the characteristic sub-circular profile and single pore of A. repens; ×2K magnification; scale bar represents 10 µm. B: showing details of the surface texture and pore that lacks a prominent annulus; ×6K magnification; scale bar represents 2 µm. C–F: showing details of the surface texture of A. repens at ×12K (C) (scale bar represents 1 µm), ×20K (D) (scale bar represents 1 µm) and ×30K (E & F) (scale bars represent 500 nm). E shows an oblique view of the surface texture of A. repens and F shows a vertical view of the surface texture of A. repens. G and H show widefield and SR-SIM images from the Zeiss Elyra super resolution system. Note that the granules (∼250 nm in all directions) on the surface of A. repens are well resolved in H.
Table 3.
Simplified guide to the imaging of pollen grains using optical microscopy.