Phase Imaging in Plant Cells and Tissues - Biomedical Optical Phase Microscopy and Nanoscopy - page 63

250 3 400 µ m 2 detail of an area from this image. The yellow scale bar is 250 µ m and the
color bar shows optical path length in nanometers.
SLIM microscopy has been extended recently to super resolving microscopy by [22] .
4.9 Second Harmonic Imaging Microscopy
SHIM was originated with confocal microscopy in transmission. Double photon absorption
by orientated structures such as cell walls, starch grains, and chloroplasts is remitted in a
shorter wavelength. The fluorescence that can result from this is always a longer
wavelength than the remitted light which is at a shorter wavelength than the irradiation with
pulsed wavelength.
This kind of microscope obtains the half wavelength of the light source using the nonlinear
optical effect (i.e., second-harmonic generation) caused by the noncentrosymmetric
structure of the specimen as a contrasting method to the image of the specimen. Such
method is equivalent to the way that a conventional optical microscope obtaining contrast
of specimen by detecting variations in optical density, path length, and/or the refractive
index of the specimen. Campagnola and Leow [22] specify the advantages of this
microscopy as unsusceptible to phototoxicity or photo-bleaching and also molecules with
exogenous probes not necessarily to be labeled since many of the biological structures can
produce strong second-harmonic generation signals, and hence will not affect the
functioning of these biological systems.
This is commercially available from Till photonics and Leica. In the latter, only the light
emitted forward is imaged, but in the Till microscope one collects both directions and these
give different information. A conventional laser-scanning double-photon microscope can
also be simply upgraded to achieve the SHIM's capability with minor modification [23] .
The paper by Reshak et al. [24] shows the case in chloroplasts of a moss leaf.
4.10 Magnetic Resonance Imaging of Plants Showing the Flow
Component
After early work with Sir Paul Callaghan and Xia, MRI was used by Sarafis and
Campbell ( 25 ) to demonstrate flow by imaging the xylem and phloem of plants.
Figures 4.12 and 4.13 show the power of this technology which examines the flow of the
fluids in these channels and is unlike Doppler methods in optical microscopy [26] .
The Doppler methodologies are not commercial whereas the MRI is done on commercial
machines which are not commercial; these latter depend on the carrying of particulates in
the flow streams, but the MRI imaging is utterly independent of these components.