Two-color infrared photothermal microscopy

Abstract

Infrared photothermal microscopy is an infrared (IR) imaging technique that enables non-invasive, non-destructive, and label-free investigations at the sub-micrometer scale. It has been applied in various research areas targeting pharmaceutical...

Monitoring the synthesis of neutral lipids in lipid droplets of living human cancer cells using two-color infrared photothermal microscopy

“…It should be noted that the IR absorption by the background water affects the IPI contrast under investigation. 20,22 To effectively distinguish the LD signal from the undesired water-related signal in the IPI measurements, we adjusted the power of both IR excitations. This adjustment was made in order to match the IP intensities at the background region of the two IP images obtained at 2193 cm −1 and 2855 cm −1 , respectively (see ESI Note 3†).…”

“…An important advantage of the two-color IPI technique is its capability to simultaneously monitor two distinct molecular species within a living cell, as demonstrated in our previous works. 22 To investigate lipid dynamics, specifically focusing on the synthesis of neutral lipids within individual LDs, we conducted a time-lapse two-color IPI on living U2OS cells treated with 250 μM PA-d 31 . Every 10 min, we simultaneously captured two-color IP images at 2193 cm −1 (50 kHz) and 2855 cm −1 (45 kHz) ( Fig.…”

“…The two-color IPM technique utilizes a pair of pulsed IR lasers and a continuous wave (CW) visible laser to examine two distinct IR-absorbing vibrational modes. 22 This approach enables microscopic investigations with chemical contrasts, offering a spatial resolution of a few hundred nanometers. In this study, we opted for an IR laser source spanning from 1980 cm −1 to 2239 cm −1 .…”

“…IR photothermal microscopy (IPM) has undergone significant methodological developments over the past decade 7–24 and has been considered a powerful tool to investigate molecular dynamics in living systems without exogenous labels. 25 This IR microscopic technique accomplished an enhanced spatial resolution of sub-micrometer level and chemical information by probing the IR pulse-induced transient photothermal dynamics with a non-resonant visible light source.…”

“…Moreover, it should be noted that the non-phototoxic characteristic of IPM also allows a long-time evolution of living systems covering the lifecycle of cells. Dark-field scattering-based IR photothermal imaging (IPI), a representative among various IPM branches, has been widely applied to investigate molecular information in biological specimens 9,16,20,22,26,27 as well as materials. 28–31 Its potential for studying the dynamics of biomolecules in living organisms was demonstrated with the visualization of LDs in living cells by targeting the ester group of lipids.…”