Total internal reflection microscopy for live imaging of cellular uptake of sub‐micron non‐fluorescent particles

Abstract

Key words. Drug delivery, endocytosis, evanescent wave microscopy, imaging systems, live-cell imaging, medical and biological imaging, microscopy, total internal reflection fluorescence. SummaryThis paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to realtime imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly-L -lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron-sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.

Fluorescence Techniques to Study Lipid Dynamics

“…Other applications were to elucidate the role of ceramide in membrane restructuring (Ira et al 2009), the organization of bacterial light harvesting complex 2 (Dewa et al 2006), the role of cholesterol in antibody binding ), EGFR activation by EGF (Sako et al 2000;Cannon et al 2005;Teramura et al 2006), and the phase preference of peptides (Choucair et al 2007). Membrane curvature, exocytosis, and endocytosis are some other topics in which TIRF is successfully applied (Merrifield et al 2002(Merrifield et al , 2005Byrne et al 2008;Nagamatsu and Ohara-Imaizumi 2008;Joselevitch and Zenisek 2009;Ohara-Imaizume et al 2009;Aoki et al 2010;Gorg et al 2010;Lam et al 2010).…”

“…Recently, it has been shown that TIRF has the capacity to show the adsorption of proteins and peptides to lipids in SLBs (Fox et al 2009;Jorgensen et al 2009). TIRF was combined with singleparticle tracking to show the enrichment of GPI-anchored proteins in sphingolipid rich regions, as proposed by lipid raft theory Membrane curvature, exocytosis, and endocytosis are some other topics in which TIRF is successfully applied (Merrifield et al 2002(Merrifield et al , 2005Byrne et al 2008;Nagamatsu and Ohara-Imaizumi 2008;Joselevitch and Zenisek 2009; Ohara-Imaizume et al 2009;Aoki et al 2010;Gorg et al 2010;Lam et al 2010). …”

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy

“…25 The penetration depth (decay) of the evanescent field varies with incident angle, ranging from approximately 180 nm when the incident beam is 2 degrees above the critical angle to about 120 nm when the incident beam angle is increased by a further 5 degrees. Similar was previously reported for variable TIRF microscopy.…”

Label‐free evanescent microscopy for membrane nano‐tomography in living cells

“…The specificity of labeling is also at the same time the main advantage and the main limitation of fluorescence imaging because it prevents whole sample visualization. In label-free evanescent microscopy (EM), configurations similar to TIRF (prism-based or through-the-objective) have been proposed [7,8]. However, in the latter case, these evanescent techniques have not spread much, with a relative preference for the prism-based configuration also called total internal reflection microscopy (TIRM) [9,10].…”