Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 2

determining quality and application of fi nal microscopic sections.
Sectioning of fresh (not fi xed) and/or not embedded samples are
valuable alternatives to consider.

Fixation is commonly the initial step of the sequence. Choice of
proper fi xation is of great consequence for purpose of the prepara-
tion and its subsequent processing. We include here only the two
very common basic procedures using FAA and buffered formalde-
hyde. FAA (formalin-acetic acid-alcohol) penetrates rapidly and is
suitable for general anatomical or morphological work. However,
preservation of cytological details is far less satisfactory comparing
to formaldehyde. Fixation of samples with Clark's and Carnoy's
fl uids, alcohols, glutaraldehyde, acrolein, carbodiimides, chilled
methanol or acetone, and others should be considered as alterna-
tives according to goal of the preparation [ 1 - 4 ].
Process of fi xation includes both penetration of the fi xative
into the tissue and its action within the tissue. While alcohol-
elicited coagulation is a rapid process, saturation of the chemical
linkages within the tissue by formaldehyde takes 1-2 days [ 5 ].
Diffusibility of fi xative (distance that the fi xative diffuse per 1 h
within the object) varies strongly among tissues and fi xatives, being
about 25× higher for ethanol then for formaldehyde solutions [ 6 ].
This fact should be considered during sampling as size and charac-
ter of the object strongly infl uence penetration of the fi xative. Most
of the experimental tests of fi xative penetration use animal tissues
( see , e.g., refs. [ 5 , 7 ]), and only little data is available from plant
tissues [ 8 , 9 ]. The reasonable expectation of formaldehyde pene-
tration rate does not exceed more than few mm per hour in plant
tissues. It is diffi cult to get a coherent rule for estimation of fi xation
time regarding the size and character of the object. Low-pressure
(“vacuum”) infi ltration of the tissue might be required to facilitate
penetration of aqueous fi xative with considerable surface tension
into air-fi lled intercellular spaces. On the other hand, once fi lled
with fi xative, such intercellular spaces might provide important
entrance pathway into more voluminous samples.

1.1 Fixation

1.2 Cleared Whole-
Mount Preparations

Cleared whole-mount preparations allow for focusing through the
minute objects (usually not more than few hundreds of m deep)

and gain information on their inner arrangement. In fact there are
several attitudes to clear the object. Removal of pigments, inclu-
sions, and most cellular content decreases optical density of the
object and improves transparency of the tissue and thus enables
access to its inner structure [ 3 , 10 ]. Treatment using sodium chlo-
ride [ 11 ], hydrogen peroxide [ 12 ], strong alkali or acids [ 13 , 14 ],
phenol [ 15 ], lactic acid [ 16 - 18 ], chloral hydrate [ 19 - 21 ], and
their combinations are commonly used. Alternative saturation of
the object with compounds of high refractive index decreases light
dispersion and increases transparency of the tissue [ 22 - 24 ]. Various

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