@conference{
author = "Pajić, Tanja and Todorović, Nataša and Živić, Miroslav and Nikolić, Stanko N and Rabasović, Mihailo D and Clayton, Andrew HA and Krmpot, Aleksandar J",
year = "2023",
abstract = "Introduction
Studies of lipid droplet (LD) physiology in fungi are still in their infancy but their quantitation
has relevance to issues in biomedicine, agriculture and industrial waste. Third Harmonic
Generation (THG) microscopy is non-invasive, produces inherently confocal images and
doesn’t require fixation or external labeling, which make it suitable for in vivo LD imaging [1,
2]. We present in vivo and label-free imaging of LD in individual fungal cells by THG
microscopy to assess the effects of nitrogen starvation. The LD quantification was performed
by two image analysis techniques.
Methods
THG microscopy was applied for the first time to a filamentous fungus and our choice was the
oleaginous fungus Phycomyces blakesleeanus. To observe the changes in LD number, the
22h old hyphae culture was divided into control and nitrogen starved groups (N-starved). A
home built nonlienar microscope with Yb:KGW laser at 1040 nm (200 fs pulses, 83 MHz
repetition rate) was used for THG imaging of live unstained hyphae [3]. THG signal was
detected by PMT in the transmission arm after passing through a Hoya glass UV filter with the
peak at 340 nm. 2D THG images of LDs (Fig. 1a) were analyzed by Image Correlation
Spectroscopy (ICS) measuring spatially-correlated fluctuations [4] and software particle
counting – Particle Size Analysis (PSA).
Results/Discussion
The small volume of hyphae suspension was placed between two coverslips of 170 μm
thickness in order to meet the criteria for the best numerical aperture of the objective lens and
for better transmission of THG signal. The high resolution of the microscopic system, the
hyphae thickness (ca 10 μm) and medium transparency made it possible for the whole
hyphae to be optically sectioned and a 3D model to be reconstructed (Fig. 1b and video).
Since ICS was primarily developed for fluorescent images and was not used to analyze THG
images, we have tested it by comparing the results to the PSA. Nitrogen starvation as
expected [5] increased LD number compared to control which was confirmed by both methods and obtained results are in good agreement. The overall increase of LDs during
growth without available nitrogen is found to be between 3 and 4.5 h time point, followed with
the loss of population of larger-than-average LDs during prolonged starvation.
Conclusions
THG microscopy is suitable for imaging and quantification of changes in lipid droplet number,
brought upon by complete removal of nitrogen, from such low density/diameter baseline. In
addition, we demonstrate that the ICA is suitable for THG images, although it is primarily
developed and have been mostly used for fluorescence signals so far.",
publisher = "European Society for Molecular Imaging",
journal = "European Molecular Imaging Meeting: 18th Annual Meeting of the European Society for Molecular Imaging: EMIM 2023; 2023 Mar 14-17; Saltzburg, Austria",
title = "Third harmonic generation imaging of live fungal cells – quantifying lipid droplets dynamics during nitrogen starvation",
pages = "1093",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_6288"
}
