Single-Molecule Imaging in Living Plant Cells: A Methodological Review

Abstract

Single-molecule imaging is emerging as a revolutionary approach to studying fundamental questions in plants. However, compared with its use in animals, the application of single-molecule imaging in plants is still underexplored. Here, we review the applications, advantages, and challenges of single-molecule fluorescence imaging in plant systems from the perspective of methodology. Firstly, we provide a general overview of single-molecule imaging methods and their principles. Next, we summarize the unprecedented quantitative details that can be obtained using single-molecule techniques compared to bulk assays. Finally, we discuss the main problems encountered at this stage and provide possible solutions.

Fluorescence Microscopy Methods for the Analysis and Characterization of Lignin

“…Similar to Epi, CLSM uses a focused laser at a defined point and at a specific depth and performs a transverse and axial scan to collect all the emitted fluorescence information by a point detector consisting of a pinhole. The pinhole eliminates most of the light outside the focal plane, so better-quality images can be obtained compared to Epi [ 65 ] ( Figure 3 ).…”

“…TIRF uses specific optics to produce illumination light only in the range of 50 to 100 nm at the interface [ 56 ], which drastically reduces the light from the bulb and improves the ability to detect fluorescent molecules only on the surface of the sample. TIRF uses the evanescent wave generated when the incident light undergoes total internal reflection or a highly inclined laminated optical sheet (HILO) to illuminate only a partial volume smaller than 200 nm, obtaining the dynamic behavior of a single fluorescent molecule [ 65 ] unlike CLSM, which detects the fluorescence emission of the sample from the inner part of the sample. Due to better resolution images, TIRF can be used in conjunction with epifluorescence to characterize lignified fibers and cellular structures [ 108 ].…”

“…Similar to Epi, CLSM uses a focused laser at a defined point and at a specific depth and performs a transverse and axial scan to collect all the emitted fluorescence information by a point detector consisting of a pinhole. The pinhole eliminates most of the light outside the focal plane, so better-quality images can be obtained compared to Epi [65] (Figure 3). The use of this method precedes and is the basis of other techniques because the image is first taken of the area to be analyzed, and later, analyses are carried out with techniques such as FRET [66,67].…”

Macrophage Identification In Situ

“…Fluorescence molecules are genetically encoded proteins. For in vivo or in vitro expression analysis, a fusion between a target protein and a fluorophore was used [62]. In 1962, the wild-type green fluorescent protein (GFP) has generated as a marker for gene expression and localization from the jellyfish Aequorea victoria [63]; soon after, two GFP mutants with brighter fluorescence, S65T and EGFP (F64L/S65T) were discovered.…”

“…In living cells, fluorescent proteins are the most common fluorescent molecules. However, their big size is a disadvantage that can affect the properties of the target protein [62].…”