Label-free imaging of fibroblast membrane interfaces and protein signatures with vibrational infrared photothermal and phase signals

Abstract

Label-free vibrational imaging of biological samples has attracted significant interest due to its integration of structural and chemical information. Vibrational infrared photothermal amplitude and phase signal (VIPPS) imaging provide label-free chemical identification by targeting the characteristic resonances of biological compounds that are present in the mid-infrared fingerprint region (3 µm - 12 µm). High contrast imaging of subcellular features and chemical identification of protein secondary structures in unlabeled and labeled fibroblast cells embedded in a collagen-rich extracellular matrix is demonstrated by combining contrast from absorption signatures (amplitude signals) with sensitive detection of different heat properties (lock-in phase signals). We present that the detectability of nano-sized cell membranes is enhanced to well below the optical diffraction limit since the membranes are found to act as thermal barriers. VIPPS offers a novel combination of chemical imaging and thermal diffusion characterization that paves the way towards label-free imaging of cell models and tissues as well as the study of intracellular heat dynamics.

Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering

“…In principle, lock-in-based photothermal heterodyne imaging (PHI) can reveal thermal diffusivity of the medium through phase detection in the lock-in amplifier. This method 25 , 26 has enabled various applications, including observing superconducting transition 27 , tissue differentiation 26 , 28 , and revealing membrane interfaces 29 . However, lock-in demodulation at the fundamental modulation frequency loses the temporal resolution and all the higher-order harmonics of the photothermal signal.…”

Imaging approaches for monitoring three‐dimensional cell and tissue culture systems

“…PTI has been shown advantageous for a range of different biomaterial/bio-tissue based studies including in vivo imaging in C. Elegans [189], imaging of cellular dynamics for living neurons and oligodendrocytes [211], ultrafast chemical imaging [193,212], label-free PTI of fixed mouse melanoma [27], label-free in-vivo imaging of cellular organelles, such as the mitochondria, and heme proteins such as haemoglobin and cytochromes [202,213], PTI atomic force microscopies (AFM-IR) for studying eukaryotic cells [214], and selectively targeting molecular bonds for identification of cellular membranes [29]. However, some common limitations for PTI studies on tissues, especially quantitative biochemical PTI studies, include: the overlap of the water signal in the mid-IR region often resulting in low signal to noise values [189,215], and heat-induced photo-chemical reactions, which do not produce heat but rather may produce a new chemical species which may alter the photothermal properties of the sample [185].…”

“…However, in PTI thermal energy evolves through non-radiative heat processes, such that the spectroscopic thermal lens effect can be used to detect non-fluorescent molecules in a contact-and label-free approach, with yoctomole concentration resolution 10-24 mol [193,194]. One main advantage of photothermal imaging is the ability to perform photothermal micro-spectrometry chemical/biochemical composition mapping of proteins, lipids and nucleic acids, by raster scanning the excitation and probe beams across the sample [29,191]. Intracellular organelles have been found to experience endogenous thermogenesis temperature rises up to 1 K between the nucleus and the cytoplasm [195].…”

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

“…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. Samolis et al ( 60 ) showed improved MIP image contrast with the readout from lock-in amplifier phase channels and imaged protein distribution inside fibroblast cells embedded in collagen matrix ( 61 ). Shi et al ( 31 ) recently demonstrated mid-IR imaging of tissue with a spatial resolution of 260 nm by using ultraviolet (UV)–excited photoacoustic as detection method.…”