Hybrid photoacoustic and optical imaging of pigments in vegetative tissues

• Published in: Journal of microscopy (Oxford) Vol. 263; no. 3; pp. 300 - 306
• Main Authors: TSEREVELAKIS, GEORGE J., TSAGKARAKI, MARGARITA, ZACHARAKIS, GIANNIS
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.09.2016
• Subjects: anthocyanins; chlorophylls; imaging; microscopy; Optical Imaging; photoacoustic; pigments; Pigments, Biological; Plants; photoacoustic; pigments; anthocyanins; plants; chlorophylls; microscopy; imaging
• ISSN: 0022-2720; 1365-2818; 1365-2818
• DOI: 10.1111/jmi.12396

Summary Pigments in vegetative tissues have been a subject of intense research during the previous decades, since they play an active role in several molecular mechanisms regarding plants’ physiology and function. Towards this direction, the imaging modality that has been extensively employed and represents the state of the art for mapping pigments’ distribution is confocal microscopy. Despite the advantage of a high spatial resolution however, confocal microscopy provides a rather limited imaging depth and requires necessarily strong fluorescence properties from the specimen under observation. To overcome such limitations, we propose a hybrid, photoacoustic and optical imaging methodology for the delineation of various vegetative pigments, such as chlorophylls, anthocyanins and betalains in different plant species. The superior sensitivity and the high contrast complementarity of the hybrid technique, render it a powerful alternative to the conventional fluorescence imaging modalities, significantly expanding the current state of the art. Lay description The vast variety of vivid colours met in the plant kingdom is generally attributed to four principal pigment groups: the chlorophylls, the anthocyanins, the carotenoids and the betalains. However, apart from providing the magnificent coloration of nature, these chemical compounds present high research interest because they characterize the maturation and senescence processes of several plants. Furthermore, it has been suggested that anthocyanins accumulation changes in the case of extreme environmental conditions such as low temperatures, dehydration, increased sugar intake etc., constituting thus a reliable stress indicator of the vegetative tissue. Since several of the pigments found in fruits or leaves are known to present pronounced autofluorescence properties, the typical modality that has been extensively employed so far to map their respective distribution is confocal microscopy. Despite the fact that such an approach provides a high spatiotemporal resolution and three‐dimensional capabilities, it also presents limitations, mostly related to the shallow imaging depth (typically up to 200 μm) and the necessity of a high quantum yield regarding the emitted fluorescence, in order to acquire label‐free images of an appreciable quality. To overcome these limitations, we propose a hybrid photoacoustic and optical imaging methodology for the investigation of pigments in several kinds of vegetative tissues, significantly expanding the current state of the art. More specifically, by incorporating photoacoustic and wide‐field fluorescence microscopy modalities into a single instrument, it is demonstrated that the distribution of several pigments can be successfully mapped at optical diffraction limited lateral resolutions (∼3 μm) and at depths of up to 1 mm. The extended imaging depth and the intrinsic three dimensionality of photoacoustic microscopy, as well as the quantitative capability, the excellent contrast complementarity with conventional fluorescence imaging techniques and the simplicity of this alternative approach are anticipated to shed light into several molecular mechanisms involving common pigments in plants.