Molecular understanding of label-free second harmonic imaging of microtubules

Abstract

Microtubules are a vital component of the cell’s cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging

“…The majority of label-free, vital NLO microscopy techniques in biology are applied in vitro using cell culture models that allow to reproduce and to control complex phenomena such as lineage differentiation of stem cells ( Quinn et al, 2013 ; Meleshina et al, 2016 , 2017 ), cell response to drugs ( Fu et al, 2014 ; De Bortoli et al, 2018 ), synthesis or inhibition of single cellular structures ( Zhang et al, 2012 ; Van Steenbergen et al, 2019 ). Table 2 summarizes a selection of representative biological studies on vital cultured cells through NLO microscopy published during the last decade.…”

“… Saarinen et al (2017) applied CARS microscopy to a transwell culture configuration on an intestinal epithelium model of Caco-2 cells to evaluate drug permeability throughout the cell layer over 21 days of culture monitoring lipid formation. Another interesting study at the cell scale is from Van Steenbergen et al (2019) where SHG microscopy is used to observe microtubular structures, a cytoskeletal component involved in protein intracellular transport and mitosis, in neural cells axons derived from different regions of the nervous system. This unusual application of SHG was aimed to validate label-free microscopy as a promising alternative to immunofluorescence staining, since microtubules can be imaged in their native and dynamic configuration.…”

Nano-positioning and tubuline conformation determine transport of mitochondria along microtubules

“…A Zeiss LSM 780 confocal laser scanning microscope (Zeiss) fitted with an Argon laser (488 nm) and solid state lasers (561, 633 nm) was used for mitochondrial imaging (Mitotracker red, 75 nM, 10 min incubation, Thermo Fisher Scientific) in combination with an LD LCI Plan-Apochromat 25x/0.8 Imm Corr DIC M27 water-immersion objective. In-house Igor pro (Wavemetrics, OR, USA) code was used to generate kymographs and analyze mitochondrial transport time lapse recordings as described previously 35 .…”

“…The 10-minute long transport recordings consisted of stacks taken every 2 seconds and 10 micrometer thick to account for wing movement. Mitochondrial time lapse recordings were registered using the StackReg plugin (ImageJ) and in-house Igor pro code was used to generate kymographs and analyze mitochondrial transport time lapse recordings as described previously 35,38,39 .…”

Ionizing Radiation Effects on Hs 578Bst Microtubules

“…The homeostasis of the MT lattice, alternatively defined "dynamic instability," is thus controlled by the balance between the processes of adding and removing αβ-tubulin heterodimers which determines the phases of growth and shortening of the tubules. The phase when the balance of the process tips in favor of a rapid depolymerization is called "catastrophe," whereas the opposite phase, where protofilaments recover, is called "rescue" [2,3]. Since protofilaments are oriented sequences of polar αβ-tubulin heterodimers their concatenation results in the construction of protofilaments with polar ends that exhibit different behavior with respect to the processes of polymerization and depolymerization, the first taking place prevalently at the "plus end" of the filament where β-tubulin is exposed.…”