Label-free biochemical quantitative phase imaging with mid-infrared photothermal effect

Abstract

Label-free optical imaging is valuable in biology and medicine with its non-destructive property and reduced optical and chemical damages. Quantitative phase (QPI) and molecular vibrational imaging (MVI) are the two most successful label-free methods, providing morphology and biochemistry, respectively, that have pioneered numerous applications along their independent technological maturity over the past few decades. However, the distinct label-free contrasts are inherently complementary and difficult to integrate due to the use of different light-matter interactions. Here, we present a unified imaging scheme that realizes simultaneous and in-situ acquisition of MV-fingerprint contrasts of single cells in the framework of QPI utilizing the mid-infrared photothermal effect. The fully label-free and robust integration of subcellular morphology and biochemistry would have important implications, especially for studying complex and fragile biological phenomena such as drug delivery, cellular diseases and stem cell development, where long-time observation of unperturbed cells are needed under low phototoxicity.

Adaptive dynamic range shift (ADRIFT) quantitative phase imaging

“…Only ADRIFT-DH clearly visualizes the signal localization, especially at the nucleus, nucleoli and some particles indicated by the arrows in Fig. 5a, which could represent the richness of proteins 13 .…”

“…To illustrate the advantage of ADRIFT-QPI, we apply this technique to MIR photothermal QPI [10][11][12][13] . MIR photothermal QPI is a recently developed molecular vibrational imaging method, where the refractive index change of the sample caused by the absorption of MIR pump light is detected through the OPD change of visible Figure 4a shows the pump-OFF-state OPD images obtained by conventional DH and ADRIFT-DH.…”

“…Phase imaging [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] provides morphological phasecontrast of transparent samples and is widely used in various fields, especially in biological science, because morphological features of micrometre-scale specimens provide valuable information on complex biological phenomena. Quantitative phase imaging (QPI) [1][2][3][4][5][6] is the most powerful method for studying cellular morphology among various phase imaging methods, such as phase-contrast 7 and differential-interference-contrast 8 imaging, because it is able to accurately measure the optical phase delay (OPD) caused by a sample.…”

“…Since the minimum detectable OPD of conventional QPI (~10 mrad) is already small enough to clearly observe the surface roughness of a glass slide that limits the detectable sample-induced OPD, sensitivity improvement of QPI, especially of the temporal OPD sensitivity, has not been intensively explored. However, recent developments in pump-probe-type perturbative QPI techniques, such as mid-infrared (MIR) photothermal QPI [10][11][12][13] , have shown that temporal differential analysis of consecutively measured images can cancel out the substrate background and reveal small OPD changes. Thus, sensitivity improvement of QPI is highly demanded in this context.…”

Bond-selective imaging by optically sensing the mid-infrared photothermal effect

“…Pleitez et al ( 32 ) demonstrated mid-IR photoacoustic microscopy with broad biomedical applications for monitoring carbohydrates, lipids, and proteins in cells and tissues. The Ideguchi group ( 62 ) recently reported the MIP quantitative phase imaging with depth-resolved capability and extended dynamic range ( 63 ).…”