Modern Fluorescence Microscopy – Tool to See the Nano-Details of Life

  • Rinalds Seržants (Speaker)
  • Irina Česnokova (Co-author)
  • Dambrova, M. (Co-author)
  • Antons Sizovs (Co-author)

Activity: Talk or presentation types › Oral presentation

Description

Modern fluorescent microscopy can overcome resolution limitations imposed by the diffraction barrier and can produce spatially super-resolved, time-resolved, and 3D images of biological samples. In this report we demonstrate the microscopy capabilities available at the Latvian Institute of Organic Synthesis/Baltic Biomaterials Centre of Excellence.. Images and data sets of fluorochromes were acquired on confocal Leica STELLARIS SP8 Super-resolution STED microscope using HC PL APO CS2 86x/1.40 water immersion objective. Excitation was ensured by supercontinuum white light laser with adjustable excitation wavelength in the range of 440-790nm, pinhole was set to 1AU. Emission depletion was accomplished with 775nm secondary laser to achieve lateral resolution below 30nm. Mitochondria of live A2058 human melanoma cells were stained with Mitotracker Deep Red dye. Fluorescence Correlation Spectroscopy (FCS) enabled by time-correlated single photon counter (TC-SPC) was used to determine diffusion coefficients of nanoparticles labeled with AlexaFluor750 in mouse serum. Fluorescence-Lifetime Imaging Microscopy (FLIM) was used to separate signals from lipophilic dye DiI and Abberior Live Mitochondria Orange dye with overlapping emission spectrums. . By using STED laser we were able to capture and distinguish separate mitochondrial tubular structures in live cells. Diffusion coefficients and the extent of nanoparticles interaction with serum proteins were determined in optically dense environment using FCS. Lack of nonspecific interactions between nanoparticles and proteins was observed. FLIM separated signal from live cells stained with two fluorophores simultaneously, with faster emission from mitochondria being 0.8ns and slower one - less than 1.0ns emission coming from membranes and other lipophilic structures. . The super resolution STED microscope with time-correlated photon counter is a versatile tool for a wide range of multidisciplinary projects. It can deliver high accuracy images and data sets for a variety of applications such as biomaterials, histology, protein tracking, and protein-protein interactions, study diffusion in live cells, and much more.
Period29 Mar 2023
Event titleRSU International Research Conference 2023: Knowledge for Use in Practice
Event typeConference
OrganiserRīga Stradiņš University
LocationRiga, LatviaShow on map
Degree of RecognitionInternational

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