Gregory Eakins

Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons

Vibrational imaging reveals vitamin E distribution on mouse skin in vivo and captures human breast cancer tissues in situ. In vivo vibrational spectroscopic imaging is inhibited by relatively slow spectral acquisition on the second scale and low photon collection efficiency for a highly scattering system. Recently developed multiplex coherent anti-Stokes Raman scattering and stimulated Raman scattering techniques have improved the spectral acquisition time down to microsecond scale. These methods using a spectrometer setting are not suitable for turbid systems in which nearly all photons are scattered. We demonstrate vibrational imaging by spatial frequency multiplexing of incident photons and single photodiode detection of a stimulated Raman spectrum within 60 μs. Compared to the spectrometer setting, our method improved the photon collection efficiency by two orders of magnitude for highly scattering specimens. We demonstrated in vivo imaging of vitamin E distribution on mouse skin and in situ imaging of human breast cancerous tissues. The reported work opens new opportunities for spectroscopic imaging in a surgical room and for development of deep-tissue Raman spectroscopy toward molecular level diagnosis.

Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

Photothermal measurement enabled infrared spectroscopic imaging of live cells and organisms with submicrometer resolution. Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption–induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins in Caenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

Stimulated Raman spectroscopic imaging by microsecond delay-line tuning

Stimulated Raman scattering (SRS) microscopy is an emerging platform for vibrational imaging of living systems. We present microsecond-scale SRS spectroscopic imaging by temporally tuning two spectrally focused pulses through a resonant delay line. Our platform is able to acquire an SRS spectrum in 42 μs and form a spectral image of 40,000 pixels within 3.3 s. Spectroscopic identification of single bacteria and fungi in blood and 4-D imaging (x–y–z–λ) of intracellular organelles in live C. elegans are demonstrated.

Real-Time intravascular photoacoustic-ultrasound imaging of lipid-laden plaque in human coronary artery at 16 frames per second

Intravascular photoacoustic-ultrasound (IVPA-US) imaging is an emerging hybrid modality for the detection of lipid-laden plaques, as it provides simultaneous morphological and lipid-specific chemical information of an artery wall. Real-time imaging and display at video-rate speed are critical for clinical utility of the IVPA-US imaging technology. Here, we demonstrate a 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 size, differentiated A-line strategy, and real-time image processing and display algorithms. Spatial resolution, chemical specificity, and capability for imaging highly dynamic objects were evaluated by phantoms to characterize system performance. An imaging speed of 16 frames per second was determined to be adequate to suppress motion artifacts from cardiac pulsation for in vivo applications. 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. Thus, this high-speed IVPA-US imaging system presents significant advances in the translational intravascular and other endoscopic applications.

Stimulated Raman Spectroscopic Imaging by Microsecond Delay-line Tuning

We report microsecond-scale acquisition of simulated Raman spectra by resonant delay-line tuning. Our scheme improved the spectral acquisition speed by 100 times compared to previous works by motorized translational-stage tuning. 4-D images (

High-Speed Spectroscopic Transient Absorption Imaging of Defects in Graphene

\mathrm{x}-\mathrm{y}-\mathrm{z}-\lambda

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

) from highly dynamic organelles in live C. elegans was demonstrated.

Triboluminescence from Pharmaceutical Formulations

Graphene grain boundaries (GBs) and other nanodefects can deteriorate electronic properties. Here, using transient absorption (TA) microscopy we directly visualized GBs by TA intensity increase due to change in density of state. We also observed a faster decay due to defect-accelerated carrier relaxation in the GB area. By line-illumination and parallel detection, we increased the TA intensity imaging speed to 1000 frames per second, which is 6 orders of magnitude faster than Raman microscopy. Combined with a resonant optical delay tuner which scans a 5.3 ps temporal delay within 92 μs, our system enabled spectroscopic TA imaging, at a speed of 50 stacks per second, to probe and characterize graphene nanodefects based on the TA decay rate. Finally, we demonstrate real-time nondestructive characterization of graphene at a rolling speed of 0.3 m/min, which matches the fastest roll-to-roll manufacturing process reported.

Spatial-spectral multiplexing for hyperspectral multiphoton fluorescence imaging

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam’s position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

Stimulated Raman scattering flow cytometry for label-free single-particle analysis

Triboluminescence (TL) is shown to enable selective detection of trace crystallinity within nominally amorphous solid dispersions (ASDs). ASDs are increasingly used for the preparation of pharmaceutical formulations, the physical stability of which can be negatively impacted by trace crystallinity introduced during manufacturing or storage. In the present study, TL measurements of a model ASD consisting of griseofulvin in polyethylene glycol produced limits of detection of 140 ppm. Separate studies of the particle size dependence of sucrose crystals and the dependence on polymorphism in clopidogrel bisulfate particles are both consistent with a mechanism for TL closely linked to the piezoelectric response of the crystalline fraction. Whereas disordered polymeric materials cannot support piezoelectric activity, molecular crystals produced from homochiral molecules adopt crystal structures that are overwhelmingly symmetry-allowed for piezoelectricity. Consequently, TL may provide a broadly applicable and simple experimental route for sensitive detection of trace crystallinity within nominally amorphous materials.