Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.
We present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed by fingerprinting a single bacterium. A wide-field fluorescence-detected mid-infrared photothermal microscope developed allows video-rate bond-selective imaging of biological specimens.
Mid-infrared photothermal (MIP) imaging is an emerging technique where a visible beam is deployed to sense the photothermal lensing effect induced by infrared absorption of molecules. This technology provides sub-micron spatial resolution defined by the visible probe beam. Yet, the photothermal lensing effect is a weak effect for most materials. For instance, the diffraction coefficient of poly (methyl methacrylate) changes around 0.1% per Kelvin. Thus, highly sensitive probes are desired to improve the detection sensitivity. Here, we report a fluorescence-enhanced mid-infrared photothermal (FE-MIP) microscope with high sensitivity. MIP microscopy uses a pump-probe strategy in which a mid-infrared light vibrationally excites a sample and a visible light probes the thermal effect. Instead of measuring the scattering modulated by mid-infrared absorption, we deploy thermal sensitive fluorescent dyes as the probe, and measure the modulated fluorescence intensity in both confocal mode and wide field mode. We demonstrate high imaging sensitivity and component specificity through fluorescence labeling.
KEYWORDS: In vivo imaging, Raman scattering, Endoscopy, Endoscopes, Microscopy, Chemical analysis, Tissues, Hyperspectral imaging, Raman spectroscopy, Single mode fibers
Compared with conventional histology, Stimulated Raman scattering (SRS) microscopy provides high specificity, fast speed and label-free histopathological analysis of the lesions by mapping their chemical compositions. However, benchtop SRS microscopy is limited to its bulky size to access the tissues of interest in-vivo inside the human body. To enable SRS in-vivo label-free histology, here, we develop an implantable fiber-scanning SRS endoscope. The endoscope is capable of providing hyperspectral Raman images at C-H and C-D regions. We use a double-clad single-mode fiber to deliver the pump and Stokes femtosecond pulses through the core and collect back-scattering signals through the outer cladding. To remove the nonlinear background induced by the pulse interactions in the fiber, we temporally separate the two pulses by tuning a delay line. We custom-design a micro-objective made of high-dispersive ZnSe glass which enables a simultaneously focusing and recombining the two pulses at spatial and time domains on the sample for excitation. A piezo actuator is designed to resonantly scan the fiber cantilever with spiral patterns. By establishing this technology, we expect the SRS endoscope to have great potential in medical applications such as label-free image-based diagnosis and surgical guidance.
KEYWORDS: Image resolution, In vivo imaging, Fiber optics, Ultrasonography, Mirrors, Imaging systems, Real time imaging, Photoacoustic imaging, Absorption, Image processing, High speed imaging
Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipidladen plaques by providing simultaneous morphological and lipid-specific chemical information of an artery wall. The clinical utility of IVPA-US technology requires real-time imaging and display at speed of video-rate level. Here, we demonstrate a compact and portable IVPA-US system capable of imaging at up to 25 frames per second in real-time display mode. This unprecedented imaging speed was achieved by concurrent innovations in excitation laser source, rotary joint assembly, 1 mm IVPA-US catheter, differentiated A-line strategy, and real-time image processing and display algorithms. By imaging pulsatile motion at different imaging speeds, 16 frames per second was deemed to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. Our lateral resolution results further verified the number of A-lines used for a cross-sectional IVPA image reconstruction. The translational capability of this system for the detection of lipid-laden plaques was validated by ex vivo imaging of an atherosclerotic human coronary artery at 16 frames per second, which showed strong correlation to gold-standard histopathology.
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