Vibrational Spectroscopic Detection of a Single Virus by Mid-Infrared Photothermal Microscopy

Abstract

We report a confocal interferometric mid-infrared photothermal (MIP) microscope for ultra-sensitive and spatially resolved chemical imaging of individual viruses. The interferometric scattering principle is applied to detect the very weak photothermal signal induced by infrared absorption of chemical bonds. Spectroscopic MIP detection of single vesicular stomatitis viruses (VSVs) and poxviruses is demonstrated. The single virus spectra show high consistency within the same virus type. The dominant spectral peaks are contributed by the amide I and amide II vibrations attributed to the viral proteins. The ratio of these two peaks is significantly different between VSVs and poxviruses, highlighting the potential of using interferometric MIP microscopy for label-free differentiation of viral particles. This all-optical chemical imaging method opens a new way for spectroscopic detection of biological nanoparticles in a label-free manner and may facilitate in predicting and controlling the outbreaks of emerging virus strains.

Figure 1.. Illustration and principles of interferometric MIP microscopy.

(a) Schematic. A pulsed mid-infrared pump beam is provided by a quantum cascaded laser (QCL) laser and focused into a sample by a reflective objective (RO). A continuous visible (green) probe beam is focused on the sample by a water immersion objective lens (OL). To measure the IR power, a small fraction of the IR beam is reflected by a CaF₂ glass onto a mercury cadmium telluride (MCT) detector. The interferometric signal (E_det) is collected in an epi-illumination configuration using a beam splitter (BS). A silicon photodiode (PD) detects E_det after filtering by a pinhole (PH) in a confocal configuration. The 4f system consisting of two achromatic doublets L1 and L2 provides access to the confocal conjugate plane for the filtering. (b) Illustration of the counter propagation interferometric scattering detection principle. (c) Electronic connections for system control and signal detection. PD is connected to a resonant amplifier (RA) to amplify the detected probe beam. Lock-in amplifier (LIA) demodulates the PD signal to isolate the photothermal signal. PC controls the motorized scanning stage and data acquisition. The IR intensity detected by MCT is also received by LIA for normalization of the photothermal signal by IR power at each wavenumber. The lock-in sends a reference signal to trigger the laser at 100 kHz frequency.

Figure 2.. Experimental demonstration and theoretical calculations of interferometric detection.

(a) Interferometric scattering image of 100 nm PMMA bead, (b) Interferometric MIP image of the 100 nm PMMA bead at 1729 cm⁻¹. Probe power on the sample: 40 mW; IR pump power on the sample: 8.5 mW. Scale bars: 500 nm; pixel dwell time: 20 ms; image acquisition time: 19.6 s. (c-d) Simulated contrast comparison between interferometric cross term and scattering intensity term for PMMA bead with particle size range from 50 nm to 200 nm in diameter. (d) Photothermal signal calculations corresponds to the signals in c.

Figure 3.. Performance of interferometric MIP microscopy.

(a) Interferometric scattering image of 200 nm PMMA beads, (b) (Left) MIP image in a and (Right) zoom-in region of a single PMMA bead. Probe power on the sample: 30 mW; IR pump power on the sample: 5 mW at 1729 cm⁻¹. Scale bars: 500 nm; pixel dwell time: 20 ms; image acquisition time: 127 s. (c) SNR characterization of MIP signal showing that shot-noise limit is reached. (d) (Left) Vertical and (Right) horizontal cross-section profiles across the enlarged single bead in b. The Gaussian fitted full width at half maximums (FWHM) are respectively 376 nm and 396 nm. (e) MIP spectrum obtained from a single PMMA bead and (f) FTIR acquired from a PMMA film. MIP acquisition time: 10 ms per wavenumber.

Figure 4.. Interferometric MIP imaging and spectroscopy of single poxviruses.

(a) Interferometric scattering image of a poxvirus. Probe power on the sample: 30 mW. (b) MIP image of the same poxvirus at the 1550 cm⁻¹. IR pump power on the sample: 18 mW. (c) MIP image of the same virus at the 1650 cm⁻¹. IR power: 20 mW; Scale bars: 500 nm; pixel dwell time: 20 ms; image acquisition time: 46.4 s. (d) MIP spectra of four randomly chosen poxviruses.

Figure 5.. Interferometric MIP imaging and spectroscopy of a single VSV.

(a) Interferometric scattering image of a single VSV and MIP signal with the IR laser tuned to 1650 cm⁻¹ (Amide I bond). Probe power on the sample: 30 mW; IR pump power on the sample: 20 mW. Scale bars: 500 nm; pixel dwell time: 20 ms; image acquisition time: 34 s. (c) MIP spectra of two VSV particles. (d) Each plot represents the averaged spectra of individual viruses in Figures 4d and 5c. To emphasize the spectral differences, each spectrum is normalized by the maximum MIP intensity at the amid I band.

Figure 6.. AFM topography images of VSV particles.

(a) AFM topography of VSV particles in a 3×3 μm are on a silicon chip. Scale bar is 500 nm. (b) AFM topography of a single VSV. The image is a zoomed-in view of the area marked by a white-dashed-line box in (a). Scale bar is 100 nm.