Two-photon microscopy in plant research

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Benedict Francis
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1 Femto2D MULTIPHOTON LASER SCANNING MICROSCOPE Two-photon microscopy in plant research Femtonics Kft. Tűzoltó u. 59. H-1094 Budapest Hungary

Advantages of two-photon to confocal microscopy Due to its high spatial resolution and improved signal

and better image quality. Sunflower pollen grain.

Colorful pollen grains measured with three channels using the Femto2D two-photon microscope Due to its larger

Images can be recorded approximately 4 times deeper than with confocal microscopes.

Twophoton microscopy allows perfect discrimination and contrast up the 10 th cell layer whereas confocal

2 Advantages of two-photon to confocal microscopy Due to its high spatial resolution and improved signal collection efficiency, two-photon laser scanning microscopy (2PLSM) offers much better contrast, crispness and better image quality. Sunflower pollen grain. Much more detail is seen by a twophoton microscope especially on the distal and proximal faces. Colorful pollen grains measured with three channels using the Femto2D two-photon microscope Due to its larger penetration depth, image acquisition is possible from thicktissue sections. Images can be recorded approximately 4 times deeper than with confocal microscopes. For example, studying roots definitely needs this unique feature. Whole living root application. Twophoton microscopy allows perfect discrimination and contrast up the 10 th cell layer whereas confocal microscope resolves no better than 5 cell layers. Photobleaching and phototoxicity is reduced in out-of focus regions as only molecules in focus are excited. This allows long time structural and functional measurements without harming or altering the living specimen.

Under two-photon excitation, however, the anthers show more transparency, allowing section close to 200 µm, that is, 6-7 cell layers.

3 Unique possibilities by two-photon microscopy Imaging opaque specimens 2PLSM makes the studying of living anther and ovary possible. In confocal microscopy the autofluorescence of the inner cell layers makes it impossible to image deeper than 2 or 3 cells. Under two-photon excitation, however, the anthers show more transparency, allowing section close to 200 µm, that is, 6-7 cell layers. Similar problems arise with the imaging of the highly opaque ovary in confocal microscopy. However, with 2PLSM even a pollen tube progressing along the micropyle can be shown. Imaging thick tissues Due to the high performance of the external nondescanned detectors, the problem of light scattering in thick slices can be solved. With an external detector, a deeper penetration can be achieved, and organelles in cells far away from the surface show a more detailed morphology. Wall and plastid autofluorescence in a living Arabidopsis leaf. In many confocal images, wall autofluorescence is hardly visible while chloroplast autofluorescence is saturated. By contrast, two-photon images captured with external PMTs show more information. The number of small fluorescent inclusions are visible with the same gain needed to acquire both the chloroplasts and cell walls.

Unique possibilities with two-photon microscopy Vital imaging, cell viability Two-photon microscopy provides the best approach to study primary metabolism in vivo.

The growth rate was not affected by the two-photon excitation. Growing root.

4 Unique possibilities with two-photon microscopy Vital imaging, cell viability Two-photon microscopy provides the best approach to study primary metabolism in vivo. The irradiation does not impair the tissue: e.g. ovules can set viable seeds while continuously scanning with 2PLSM. Growing pollen tube system. The growth rate was not affected by the two-photon excitation. Growing root. Given the weak level of fluorescence, the signal faded before imaging could be done with standard confocal microscopy. Imaging could be done by 2PLSM and the radiation did not affect either the division cell cycle or the root growth. Images are adopted from: Imaging plant cells by two-photon excitation. Feijó JA, Moreno N. Protoplasma Mar;223(1):1-32.

Second and third harmonic generation Second harmonic generation (SHG) is triggered by tightly focused short pulse laser beam and

SHG is an intrinsic coherent process which happens in many structures especially the ones having repetitive interface changes.

physiological conditions. A B C Forward (a) and backward (b) SHG and two-photon excitation fluorescence (c) images of a leaf.

A B THG C 2PLSM The third harmonic generation (THG) signal mostly arises from lipid bodies.

(b) Simultaneous THG (left) and 2PLSM (right) images.

SHG is due to starch granules, whereas the THG image reveals the presence of a lipid-rich cell layer at the peripheral region.

5 Second and third harmonic generation Second harmonic generation (SHG) is triggered by tightly focused short pulse laser beam and is collected by the objective and condenser yielding forward and backward images. SHG is an intrinsic coherent process which happens in many structures especially the ones having repetitive interface changes. Imaging of intrinsic signals avoids the complications of slicing and labeling and samples can be investigated under physiological conditions. A B C Forward (a) and backward (b) SHG and two-photon excitation fluorescence (c) images of a leaf. The signal of the second harmonic imaging in the forward and backward directions depends on the thickness of the chloroplast. A B THG C 2PLSM The third harmonic generation (THG) signal mostly arises from lipid bodies. THG imaging of lipid bodies in a plant seed. (a) THG image of an intact unstained seed. (b) Simultaneous THG (left) and 2PLSM (right) images. (c) Simultaneous THG (purple) and SHG (green) images recorded from an unstained seed section. SHG is due to starch granules, whereas the THG image reveals the presence of a lipid-rich cell layer at the peripheral region. Images are adopted from: Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy. Débarre D, Supatto W, Pena AM, Fabre A, Tordjmann T, Combettes L, Schanne-Klein MC, Beaurepaire E. Nat Methods Jan;3(1): and Second harmonic generation from thick leaves using the two-photon laser scanning microscope. Reshak AK Micron Jun;40(4):

A simple introduction to multiphoton microscopy

Journal of Microscopy, Vol. 243, Pt 3 2011, pp. 221 226 Received 29 April 2011; accepted 28 June 2011 doi: 10.1111/j.1365-2818.2011.03532.x A simple introduction to multiphoton microscopy A. USTIONE &