Chapter 19
Light Microscopy and Raman Imaging of Carotenoids
in Plant Cells In Situ and in Released Carotene Crystals
Tomasz Oleszkiewicz, Marta Z. Pacia, Ewa Grzebelus, and Rafal Baranski
Abstract
Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for
carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations,
carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by
using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman
spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used
to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted
carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are
nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell
samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for
subsequent observations using light microscopy and for analysis using Raman spectroscopy.
Key words Carrot, Chromoplasts, Bright-field microscopy, Nondestructive measurement, Protoplast, Raman mapping, Spectroscopy
1 Introduction
Carotenoid biosynthesis, accumulation and metabolism vary
among species, plant organs and tissues. The content and composition of carotenoids is additionally modulated during ontogenesis
and affected by environmental factors [1–3]. Carotenoids are components of chloroplasts and are also sequestered in nongreen plant
tissues in chromoplasts. Four main types of chromoplasts are identified in plant tissues, that is, crystalloid, globular, tubular, and
membranous [4]. One or more types of chromoplasts may occur
in the same cell or tissue which is related to plastid differentiation
and the transition between different plastid types [5]. Crystalline
sub-structures may have different shapes, that is, needle-like, broad
ribbon-like, and tube-like. They can be identified by their orangered color and regular shape. Globular chromoplasts contain lipidManuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_19,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
245
Light Microscopy and Raman Imaging of Carotenoids
in Plant Cells In Situ and in Released Carotene Crystals
Tomasz Oleszkiewicz, Marta Z. Pacia, Ewa Grzebelus, and Rafal Baranski
Abstract
Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for
carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations,
carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by
using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman
spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used
to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted
carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are
nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell
samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for
subsequent observations using light microscopy and for analysis using Raman spectroscopy.
Key words Carrot, Chromoplasts, Bright-field microscopy, Nondestructive measurement, Protoplast, Raman mapping, Spectroscopy
1 Introduction
Carotenoid biosynthesis, accumulation and metabolism vary
among species, plant organs and tissues. The content and composition of carotenoids is additionally modulated during ontogenesis
and affected by environmental factors [1–3]. Carotenoids are components of chloroplasts and are also sequestered in nongreen plant
tissues in chromoplasts. Four main types of chromoplasts are identified in plant tissues, that is, crystalloid, globular, tubular, and
membranous [4]. One or more types of chromoplasts may occur
in the same cell or tissue which is related to plastid differentiation
and the transition between different plastid types [5]. Crystalline
sub-structures may have different shapes, that is, needle-like, broad
ribbon-like, and tube-like. They can be identified by their orangered color and regular shape. Globular chromoplasts contain lipidManuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_19,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
245