Fingerprinting a Living Cell by Raman Integrated Mid-Infrared Photothermal Microscopy

Abstract

Vibrational spectroscopic imaging techniques, based on infrared absorption or Raman scattering, allow for noninvasive chemically specific visualization of biological systems. The infrared and Raman modalities with different selection rules provide complementary information. Specifically, infrared microscopy provides strong signals in the fingerprint region, but suffers from low spatial resolution. Raman microscopy provides submicrometer resolution, but requires a long acquisition time. We developed a system that combines the strengths of both techniques by integrating confocal Raman microspectroscopy to the recently developed mid-infrared photothermal microscopy. This hybrid system is capable of fast infrared photothermal imaging of living cells with submicrometer resolution to identify points of interest, followed by a full-spectrum Raman analysis of the identified objects. In addition, a fingerprint photothermal spectrum can be acquired by scanning the wavelengths of the infrared laser. Comprehensive vibrational fingerprint mapping of live cells, demonstrated in adipocytes and single bacteria, promises broad applications of this technology in biology and material science.

Optical Photothermal Infrared Microspectroscopy with Simultaneous Raman – A New Non-Contact Failure Analysis Technique for Identification of <10 μm Organic Contamination in the Hard Drive and other Electronics Industries

“…A new capability recently added to the mIRage system is simultaneous collection of O-PTIR and Raman spectra of the same sample location with the same submicrometer spatial resolution (Figure 10) [9,23]. This is possible by use of a Raman-grade laser to measure the O-PTIR response.…”

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

“…These properties allow MIP imaging of intact specimens in liquid environment including living cells, which is beyond the reach of the direct IR and AFM-IR technologies. The MIP signals can be measured by a transmission dark-field geometry ( 26 , 27 ), by reflection ( 28 , 29 ), interferometrically ( 30 ), and acoustically ( 31 , 32 ) in a stage scanning mode. Importantly, unlike AFM-IR, the MIP effect can be measured in a widefield manner through an interferometric reflectance microscope ( 33 ), a quantitative phase microscope ( 25 , 34 ), or a low-coherence interferometric microscope ( 35 ).…”

“…A MIP system with optical phase as readout was individually reported by Cheng’s and Ideguchi’s groups ( 25 , 34 , 59 ). MIP microscope integrated with a Raman spectrometer enabling the comprehensive characterization of biological samples was reported ( 29 ). Schnell et al ( 35 ) has improved the spatial resolution of IR imaging by coupling a visible light-emitting diode (LED) into the system and using low-coherence interferometry to detect the surface deformation.…”

Proof‐of‐Principle Experiment of the Pseudorandom Multiplexing of White‐Pump Light for Spectral Photothermal Microscopy

“…For instance, a diffractionlimited spatial-resolution-power of a mid-infrared Pu, ≈10 µm, was modified to ≈500 nm by a visible Pr. [11,12] Further, absorption spectroscopy with photothermal microscopy enables the identification and quantification of constituent substances with high spatial resolutions. The spectrum could be observed by scanning the wavelength of Pu.…”

“…The spectrum could be observed by scanning the wavelength of Pu. The wavelength could be scanned by modulating the light source [11][12][13][14] or by filtering the white light with a monochromator or optical filters (OFs). [15][16][17] However, the rapid wavelength scanning of the light source, at a sufficient range, is difficult.…”