Overtone photothermal microscopy for high-resolution and high-sensitivity vibrational imaging.pdf
Article https://doi.org/10.1038/s41467-024-49691-2 Overtone photothermal microscopy for high-resolution and high-sensitivity vibrational imaging Le Wang1,4, Haonan Lin 1,4, Yifan Zhu2, Xiaowei Ge 1, Mingsheng Li1, Jianing Liu1, Fukai Chen3, Meng Zhang1 & Ji-Xin Cheng 1,2,3 Photothermal microscopy is a highly sensitive pump-probe method for map- ping nanostructures and molecules through the detection of local thermal gradients. While visible photothermal microscopy and mid-infrared photo- thermal microscopy techniques have been developed, they possess inherent limitations. These techniques either lack chemical speciﬁcity or encounter signiﬁcant light attenuation caused by water absorption. Here, we present an overtone photothermal (OPT) microscopy technique that offers high chemical speciﬁcity, detection sensitivity, and spatial resolution by employing a visible probe for local heat detection in the C-H overtone region. We demonstrate its capability for high-ﬁdelity chemical imaging of polymer nanostructures, depth-resolved intracellular chemical mapping of cancer cells, and imaging of multicellular C. elegans organisms and highly scattering brain tissues. By bridging the gap between visible and mid-infrared photothermal microscopy, OPT establishes a new modality for high-resolution and high-sensitivity che- mical imaging. This advancement complements large-scale shortwave infrared imaging approaches, facilitating multiscale structural and chemical investi- gations of materials and biological metabolism. The shortwave infrared (SWIR) window, typically spanning from 900 nm to 2 µm in the electromagnetic spectrum, offers distinct advantages for bioimaging. Compared to the mid-infrared window, the SWIR window has an ~20–5000 times (wavelength-dependent) smaller water absorption coefﬁcient1. Furthermore, SWIR imaging allows for unprecedentedly large penetration depth attributed to much-reduced light scattering2,3. This depth is wavelength-dependent and can extend to the millimeter level4,5. With the rich chemical information based on overtone absorption, SWIR spectroscopy emerges as an appealing analytical tool6. Several SWIR imaging methods have been developed, including hyperspectral reﬂectance/transmittance imaging7–9, diffuse optical spectroscopic imaging (DOSI)10–12, and photoacoustic micro- scopy (PAM)13,14. SWIR hyperspectral imaging, which measures reﬂec- tance or transmittance, primarily serves in the qualitative spectral characterization of samples at the macroscale, focusing on properties related to absorption and scattering. To probe macroscopic sample areas for improved representative sampling, this technique generally sacriﬁces spatial resolution in favor of a larger ﬁeld-of-view (FOV) due to the space-bandwidth product limit, thereby leading to resolutions on the order of ten micrometers. Diffraction-limited spectroscopic imaging can be attained by magnifying the FOV onto the camera or implementing a laser point-scanning design15,16. DOSI, adopting a wide- ﬁeld approach, measures the combined effects of broadband optical absorption and scattering. It has proven successful in the study of thick tissues, such as functional information of bone sarcomas and breast tumor hemodynamic responses. This method offers a sub-millimeter lateral resolution and a penetration depth of generally a few milli- meters. PAM utilizes an objective lens as the focusing element and Received: 30 June 2023 Accepted: 11 June 2024 Check for updates 1Department of Electrical and Computer Engineering, Boston University, Boston, MA 02215, USA. 2Department of Chemistry, Boston University, Boston, MA 02215, USA. 3Department of Biology, Boston University, Boston, MA 02215, USA. 4These authors contributed equally: Le Wang, Haonan Lin. e-mail: jxcheng@bu.edu Nature Communications|        (2024) 15:5374  1 1234567890():,; 1234567890():,; collects acoustic waves with an ultrasonic transducer to achieve opti- cal resolution. SWIR PAM was ﬁrst developed for imaging lipids based on the 2nd overtone of the C-H bond14 and more recently imaging water at 1930 nm17, whereas the sensitivity is heavily reliant on the quality of the ultrasonic transducer employed18. Here, we introduce a technique named overtone photothermal (OPT) microscopy, which excites the sample in the SWIR window and employs a visible probe to detect the thermal lensing effect caused by overtone vibrational absorption. Speciﬁcally, we utilized a fem- tosecond laser tunable in the C-H 2nd overtone window and a 520 nm probe beam through the second harmonic generation of a 1040 nm femtosecond laser. Both pulses were chirped to picose- cond to minimize photodamage. Compared to existing SWIR ima- ging methods, OPT microscopy provides simultaneous high resolution and high sensitivity. Spectroscopic OPT imaging was successfully demonstrated on polymer nanostructures. In conjunc- tion with spectral unmixing algorithms, depth-resolved OPT map- ping of protein and fatty acids in cancer cells, C. elegans, and brain tissues was performed at intracellular and multicellular levels. These results underscore the potential of OPT microscopy as a valuable technique for investigating chemical structures at high resolution in biological systems and materials. Results OPT microscope setup and signal extraction Figure 1a illustrates a lab-built OPT microscope. The speciﬁc optical components and signal extraction methods are detailed in the Meth- ods Section. In brief, the OPT microscope utilizes a pulsed SWIR pump beam, tunable in the range of 1080 nm to 1280 nm, and a 520 nm probe beam generated from the second harmonic generation of the 1040 nm output. Both pump and probe beams are chirped to picose- cond duration by glass rods. The beams are treated as pseudo- continuous waves with a repetition rate of 80 MHz, and the excitation beam is modulated at desirable frequency (hundreds of kHz to 1 MHz) and duty cycle (10% to 50%) based on the sample’s thermal decay properties. The two beams are delivered collinearly to a laser-scanning upright microscope and focused onto the sample plane using a water immersion objective with a numerical aperture (NA) of 1.2. Upon the SWIR illumination, the sample’s selective absorption within the focal volume leads to a local temperature rise and a thermal gradient, inducing a subtle decrease in the local refractive index at the pump beam focus. To efﬁciently generate photothermal signals, an axial focus displacement between the two beams is implemented, as depicted in Fig. 1b19. In this scenario, the induced thermal lens modiﬁes the propagation of the 520 nm probe beam, causing it to diverge or converge depending on its focal position relative to the pump focus. When there is no axial offset, the thermal lens forms precisely at the focal position of the probe beam. Therefore, the ray locus of the probe beam will not be modiﬁed, and the photothermal signal vanishes. By incorporating an iris with an adjustable NA on the collection condenser, the intensity modulation of the probe beam, correspond- ing to the selective absorption, can be read out by a photodiode and demodulated using a lock-in ampliﬁer. As shown in Fig. 1c, the green curve represents the transient probe beam intensity change as a function of periodic heating and cooling processes. The direction of the probe intensity change is determined by the direction of the ver- tical focus displacement of the excitation and probe beams. For the provided thermodynamic trace of dimethyl sulfoxide (DMSO) shown in Fig. 1c, the focus of the probe beam is positioned above that of the excitation beam, resulting in an equivalently divergent lens and a decreased probe intensity on the photodiode. The OPT signal at the modulation frequency is extracted by a lock-in ampliﬁer (Fig. 1d). Spectroscopic OPT imaging is performed by tuning the SWIR wave- length and recording OPT images through laser scanning facilitated by 2D galvo mirrors. Fig. 1 | Operational scheme of overtone photothermal (OPT) microscopy. a Schematics and optical components of the setup. HWP half-wave plate, PBS polarization beam splitter, AOM acousto-optic modulator, LBO lithium triborate crystal, DBS dichroic beam splitter, OBJ objective, PD photodiode. The inset is a vibrational potential energy diagram. b Beam propagation in OPT microscopy. The pulsed SWIR excitation beam and the continuous 520 nm probe beam are colli- nearly propagated and tightly focused onto the sample plane. The axial foci of the two beams have an offset of ΔZ f . c The waveforms of probe intensity as a function of periodic heating and cooling processes. The sign of the probe intensity change is determined by the sign of the axial offset between the excitation and probe foci. The shown thermodynamic trace was measured on DMSO. d Signal extraction in OPT microscopy. The probe intensity change was captured by a photodiode and converted into an electronic voltage signal. After passing through electronic ﬁlters and ampliﬁers, the periodic signal was demodulated by a lock-in ampliﬁer and then registered as OPT signals per imaging pixel. Article https://doi.org/10.1038/s41467-024-49691-2 Nature Communications|        (2024) 15:5374  2 ≥96%, Sigma-Aldrich), triglyceride (TAG, purity ≥97%, Sigma-Aldrich), and deionized water were collected, serving as the pure spectral references for protein, fatty acids, and water, respectively. Collection of NIR absorption spectra via UV-Vis-NIR spectrophotometer Standard NIR absorption spectra for both polystyrene and ethylene glycol were acquired utilizing the Cary 5000 UV-Vis-NIR Spectro- photometer (Agilent) following established protocols. Polystyrene (Mw = 35,000, Sigma-Aldrich) was dissolved in toluene, and 3 mL of the resulting solution was transferred into a quartz cuvette for inser- tion into the spectrophotometer. An absorption spectrum of toluene was acquired and subsequently utilized for baseline correction. Similarly, 3 mL ethylene glycol (purity 99.8%, Sigma-Aldrich) was introduced into a separate quartz cuvette, with ambient air as the reference baseline. The measurement parameters include a spectral bandwidth of 2 nm, an integration time of 0.1 s, and a wavelength interval of 1 nm. Preparation of phase separation patterns of PS and PMMA blends To prepare the polymer stock solution, 51.4 mg PS (Mw = 35,000, Sigma-Aldrich) and 15.4 mg PMMA (Mw = 35,000, Sigma-Aldrich) were dissolved in 2 mL toluene and allowed to settle. To observe the phase separation pattern of PS-PMMA blends, a coverslip substrate with a thickness of 130 µm was cleaned with acetone and isopropanol, fol- lowed by drying using nitrogen gas. A 40 µL mixture solution was spin- coated onto the coverslip substrate using a spinner (Headway Research, CB-15 & PWM32) at 500 revolutions per minute (rpm) for 10 seconds, followed by 1000 rpm for 50 seconds. Fabrication of PMMA striped structures The fabrication of PMMA stripes was achieved using electron-beam lithography (EBL) on a 130 µm thick coverslip substrate. Prior to fab- rication, the substrate underwent a thorough cleaning process invol- ving acetone and isopropanol, nitrogen drying, and baking at 110 °C. A 950-PMMA 6% in anisole solution was spin-coated onto the substrate to achieve a PMMA thickness of 600 nm as per the recipe used. The sample was subjected to a hard bake at 180 °C before a conduction layer of Au nanoparticles was sputtered onto the top surface. EBL was performed at 30 keV after a dose test to pattern the sample. The fab- rication process was completed by soaking the PMMA in a solution of methyl isobutyl ketone/isopropanol (MIBK/IPA) 1:3 for 70 s, followed by rinsing with IPA and a wet etch to remove the sputtered Au. OVCAR-5 cancer cells The OVCAR-5 cells used in this experiment were a generous gift from Dr. Daniela Matei of Northwestern University. The cells were cultured in RPMI 1640 media (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-Glutamine, and 1% penicillin-streptomycin. The cell line was incubated at 37 °C with 5% CO2. For imaging purposes, the cells were seeded at a density of 0.2 million per dish on glass-bottom dishes and ﬁxed in 10% neutral buffered formalin (Sigma-Aldrich) after 24 h of culturing. Prior to imaging, the cells were washed three times with either H2O PBS or D2O PBS. OPT images were obtained by directly immersing the objective into the medium in the glass-bottom dish. C. elegans preparation C. elegans N2 wild-type isolates were purchased from the Cae- norhabditis Genetics Center (CGC). The worms were maintained in a 20 °C incubator and propagated on Nematode Growth Medium (NGM) agar plates supplemented with the auxotrophic Escherichia coli mutant strain OP50. To immobilize worms for imaging, worms were harvested and washed using phosphate-buffered saline (PBS) solution (Gibco) and then ﬁxed by 10% neutral buffered formalin solution (Sigma- Aldrich). The C. elegans worms were washed using D2O PBS or H2O PBS three times and then carefully sandwiched between two cover glasses for imaging. Throughout the measurement process, PBS media was gently added along the edge of the top coverslip to maintain hydration. Mouse brain tissue preparation The brain tissue utilized in this work was from an adult C57BL/6 J mouse. The mouse was sacriﬁced and subsequently perfused with PBS solution (1X, Thermo Fisher Scientiﬁc). The brain was carefully extracted and immersed in 10% formalin solution for a ﬁxation period of 48 hours. Following ﬁxation, an oscillating tissue slicer (OTS-4500, Electron Microscopy Sciences) was employed to section the brain into 200-µm-thick coronal slices. The brain slices were washed three times with PBS solution before measurement. For OPT imaging, the brain tissue was submerged in PBS and sandwiched between two cover glasses, while for mid-IR imaging, it was positioned between a cover- slip and a CaF2 substate. MIP imaging of brain slices A tunable quantum-cascade laser (QCL, MIRcat-2400, Daylight Solutions) was utilized to effectively excite the vibrational modes within the sample. Simultaneously, a continuous-wave laser of 532 nm (Samba 532 nm, Cobolt) was used to probe the local thermal changes. The mid-IR beam operated at a repetition rate of 200 kHz, with a pulse width of 500 ns. The average mid-IR power applied to the sample was approximately 3 mW, while the probe power was set at 45 mW. The two beams were collinearly focused onto the sample plane through a reﬂective objective, and an objective Z-piezo stage controlled the axial position to acquire multi-depth images. The forward propagating probe photons were detected by a biased photodiode (DET100A2, Thorlabs). The photocurrent generated by the photodiode underwent ampliﬁcation through a low-noise ampliﬁer (SA-251F6, NF Corporation) and was subsequently direc- ted to a lock-in ampliﬁer (HF2LI, Zurich Instruments) for signal demodulation. A multichannel data acquisition card registers the data for real-time signal processing. Data availability All data supporting the ﬁndings of this study are available within the main article and the Supplementary Information ﬁle. The raw data are available from the corresponding author upon request. 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L.W. prepared the manuscript with input from the authors. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-024-49691-2. Correspondence and requests for materials should be addressed to Ji-Xin Cheng. Peer review information Nature Communications thanks Mikhail Berezin, Garth Simpson and the other, anonymous, reviewer(s) for their con- tribution to the peer review of this work. A peer review ﬁle is available. Reprints and permissions information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. 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Title: Overtone photothermal microscopy for high-resolution and high-sensitivity vibrational imaging ​
Authors: Le Wang, Haonan Lin, Yifan Zhu, Xiaowei Ge, Mingsheng Li, Jianing Liu, Fukai Chen, Meng Zhang, Ji-Xin Cheng
Publisher: Nature Communications
Date: 2024-06-25 00:00:00
Abstract: Photothermal microscopy is a highly sensitive pump-probe method for mapping nanostructures and molecules through the detection of local thermal gradients. ​ While visible photothermal microscopy and mid-infrared photothermal microscopy techniques have been developed, they possess inherent limitations. These techniques either lack chemical specificity or encounter significant light attenuation caused by water absorption. ​ Here, we present an overtone photothermal (OPT) microscopy technique that offers high chemical specificity, detection sensitivity, and spatial resolution by employing a visible probe for local heat detection in the C-H overtone region. ​ We demonstrate its capability for high-fidelity chemical imaging of polymer nanostructures, depth-resolved intracellular chemical mapping of cancer cells, and imaging of multicellular C. elegans organisms and highly scattering brain tissues. ​ By bridging the gap between visible and mid-infrared photothermal microscopy, OPT establishes a new modality for high-resolution and high-sensitivity chemical imaging. ​ This advancement complements large-scale shortwave infrared imaging approaches, facilitating multiscale structural and chemical investigations of materials and biological metabolism.