Cytoplasmic Protein Imaging with Mid-Infrared Photothermal

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Cytoplasmic Protein Imaging with Mid-Infrared Photothermal Microscopy: Cellular Dynamics of Live Neurons and Oligodendrocytes Jong Min Lim, Chanjong Park, Jin-Sung Park, Changho Kim, Bonghwan Chon, and Minhaeng Cho J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b00616 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry Letters

Cytoplasmic Protein Imaging with Mid-Infrared Photothermal Microscopy: Cellular Dynamics of Live Neurons and Oligodendrocytes Jong Min Lim,1,‡ Chanjong Park,1,2,‡ Jin-Sung Park,1 Changho Kim,1 Bonghwan Chon,1 and Minhaeng Cho1,2* 1Center

for Molecular Spectroscopy and Dynamics, Institute for Basic Science, Seoul 02841,

Korea. 2Department of Chemistry, Korea University, Seoul 02841, Republic of Korea AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACS Paragon Plus Environment

1

The Journal of Physical Chemistry Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 18

ABSTRACT

Mid-infrared photothermal microscopy has been suggested as an alternative to conventional infrared microscopy because in addition to the inherent chemical contrast available upon vibrational excitation, it can feasibly achieve spatial resolution at the submicrometer level. Furthermore, it has substantial potential for real-time bioimaging to observe cellular dynamics without photodamage or photo-bleaching of fluorescent labels. We performed real-time imaging of oligodendrocytes to investigate cellular dynamics throughout a life cycle of cell, revealing details of the cell division and apoptosis, as well as cellular migration. In the case of live neurons, we observed a photothermal contrast associated with travelling protein complexes on an axon, which correspond to transport vesicles from the cell body to the dendritic branches of the neuron through the cytoskeleton. We anticipate that mid-IR photothermal imaging will be of great use for obtaining insights in the field of the biophysical science, especially regarding cellular dynamics and functions.

TOC GRAPHICS


2

Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Live-cell imaging to investigate cellular dynamics such as changes in shapes and microstructures and the time-dependent redistribution of biomolecules such as proteins and lipids in space within and outside of the cells has extended our fundamental understanding of cellular processes such as stereotypical features in developmental and altered states when subjected to pathogenic or infectious agents.1-7 However, several inherent issues in conventional microscopic techniques must be addressed to be applicable to long-time monitoring studies of cellular dynamics. In the fluorescence microscopy, the photobleaching of organic fluorophore labels have hampered the long-time observation of cellular dynamics and evolution, where live cells are prone to photodamage upon the laser excitation of fluorophores.8-10 The application of coherent Raman scattering microscopy for live-cell imaging is restricted with the comparatively small Raman scattering cross-sections of biomolecules in the fingerprint region (1100-1700 cm-1).11,12 The practical application of infrared (IR) microscopy that is a label-free vibrational imaging technique complementary to spontaneous Raman microscopy also remain restricted due to fundamental limitations associated with both its spatial and depth resolutions despite of its virtues of chemical selectivity, nondestructiveness, and label-free modality.13,14


3


Page 4 of 18

Figure 1. Experimental scheme of mid-IR photothermal (MIP) microscopy, MIP image and spectra. (a) An experimental scheme of MIP microscopy (inset: comparison of probe beam propagations upon photothermal perturbation with or without mid-IR excitation). OAPM: off-axis parabolic mirror, M: mirror, L: lens, DM: dichroic mirror, OBJ: objective, PD: photodiode. (b) MIP image (top) and intensity profile (bottom) for 1 μm (actual size: 0.92 μm) PS beads spincoated on a CaF2 cover glass upon mid-IR excitation frequency of 1600 cm-1. The peak-to-peak distance is estimated to 0.95 μm. The laser powers are set to 20 and 25 mW for mid-IR pump and visible probe, respectively. Scale bar. 1 μm. (c) A comparison of FT-IR and MIP spectra for PS film and bead. As an alternative imaging tool to address these issues, recently, an IR imaging technique utilizing an IR excitation-induced photo-thermal effect, known as mid-IR photothermal (MIP) microscopy, has been suggested, in which a pump-probe paradigm is used to achieve the aforementioned chemical sensitivity using mid-IR excitation and to attain submicrometer spatial resolution with a visible laser probe.15-19 Excitation of molecular vibrations with mid-IR radiation induces a local temperature increase, which changes the refractive index of the surrounding medium. As a result, the probe beam is deflected by the transient spatial gradient of the refractive index in the vicinity


4

Page 5 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of the IR-absorbing molecule or material. The extent of the deflected probe beam intensity is then proportional to the concentration of IR-active molecules at the focal point. Since a visible light source is applied as a probe, submicrometer lateral resolution is easily achieved when using a reflective objective with a high numerical aperture (NA) in the reverse Cassegrain scheme.17,18 Moreover, unlike nonlinear optical experiments with ultrashort laser pulses, MIP microscopy can be performed with a relatively weak excitation laser power, thereby avoiding severe photo-damage of biological sample assays.8,9 Zhang et al. experimentally demonstrated an IR photothermal imaging microscopy that is readily extendable and applicable to live-cell imaging studies with exceptional chemical sensitivity.18 These authors were able to monitor lipid droplets in living cancer (PC-3 prostate) cells with the selective excitation of C=O bond vibrations at 1750 cm-1. In the present work, we demonstrate for the first time an MIP imaging technique that is capable of monitoring cytoplasmic protein distributions, thereby providing information on the cellular dynamics of live brain cells such as oligodendrocytes and neurons. The entire life cycle of an oligodendrocyte from its cell division process to the migration of its two daughter cells and then to cell death was investigated. We also show that the axonal transport of biomolecules through the cytoskeletal filaments without any exogenous labels can be studied with the MIP imaging technique. It should be noted that exogenous fluorescence labels, e.g., GFP, often affect cellular dynamics involving small and light biological systems. From these experimental results, we believe that MIP microscopy can provide important insights regarding long-term cellular dynamics with an unprecedented chemical contrast and without the need to rely on any exogenous perturbation for enhanced imaging contrast. The schematic presentation of our MIP microscopy is depicted in Fig. 1a, which is similar to that developed and reported by Zhang et al.18 Two mid-IR pump and visible probe are utilized;


5


Page 6 of 18

while a pulsed mid-IR quantum cascade laser (QCL) induces photothermal contrast at the focal spot, resulting refractive index change is probed by a continuous wave visible (640 nm) laser. The deflected probe beam is collected by a variable aperture condenser and is then directed to the photodiode detector (see Supplementary information Fig. S1,2). The spatial resolution is found to be close to the diffraction limit, by analyzing the point spread functions of polystyrene (PS) beads with various diameters upon excitation of 1600 cm-1 C=C stretching vibrations (Figs. 1b, 1c, and S3-5). Two adjacent PS beads of 1-μm diameter show a clear spatial separation in the MIP image. To examine the detection sensitivity, we obtained MIP contrasts for the C=O stretching modes of ascorbic acid, bovine serum albumin (BAS), and N-methylacetamide (NMA) (Fig. S6). The detection limits were found to be 2 mM for ascorbic acid, 5.1 mM for NMA, and 15 μM for BSA. Then, to maintain the optimal conditions for the live cell environment and MIP imaging study, an incubation chamber was placed on the scanning piezo-stage. Experimental details are presented in Supplementary information.


6

Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Microscopy images of oligodendrocytes. (a,b) Bright-field and MIP images of oligodendrocytes. Scale bar. 25 μm. (c) Visualization of the time course of cell division monitored every 5 minutes for the white box region in b. The mid-IR and visible prove laser powers are 7.5 and 25 mW, respectively. Scale bar. 10 μm. (d) The profile of total integrated MIP signal in the oligodendrocyte body region is plotted with respect to time during the cell division. (e) Representative MIP images of cells in normal, metaphase, and cytokinesis states at -20, 25, 50 minutes, respectively, in the experimental time. Scale bar. 10 μm. The mid-IR excitation frequency is 1650 cm-1 (rep. rate, 50 kHz) to selectively excite the protein amide I band. Pixel dwell time, 5 ms. Total number of pixels, 251×251 pixels. Here, the time zero (0 min.) is arbitrarily chosen. The bright-field and IR photothermal images of cultivated oligodendrocytes in the incubation chamber are shown in Figs. 2a and 2b, respectively, for the sake of direct comparison. The MIP contrast is generated by mid-IR (1650 cm-1) excitation of the amide I band that mainly originates from peptide C=O stretching vibrations. In the bright-field image, a typical branched structure of the cell body appears as relatively bright vesicle-like spots (Fig. 2a). In contrast, in the MIP image, the photothermal contrast inside each cell results from concentrated regions of protein; spots of strong contrast in the cell body mainly correspond to the nucleoli in the nucleus (Fig. 2b). To confirm that the MIP contrast is produced by IR excitation of the protein amide I vibrations, we


7


Page 8 of 18

carried out the measurement without mid-IR excitation (IR-off), which shows that background scattering contribution is negligible in the MIP image (see Fig. S7 in Supplementary Information). To investigate the cellular dynamics of live brain cells throughout their entire life cycle, we acquired a series of IR photothermal images at a time interval of 5 minutes (Fig. 2). A real-time cell division process is shown in Fig. 2c, where a metaphase cell progression through anaphase and telophase to cytokinesis can be clearly seen.3,4 Compared to the normal state of the cell (Fig. 2b), the oligodendrocyte during cell division process (Fig. 2c) shows notably strong MIP signals that are fairly localized in the cell body region with rim-like structures, which indicates breakdown of the nuclear envelope followed by a debranching or a branch shrinking process to grow into a round cell structure with an increase in size (1.5 times, from 154 to 232 μm2) and signal intensity. Then, strong MIP contrast (an increase in protein concentration) appears at the equatorial plane, which might in turn be related to the tight coordination of protein complexes such as condensin and cohesion as well as histones in the condensed chromosomes,3,4,20 corresponding to metaphase. After that, the localized signals are gradually separated into two parts from the equatorial plane before the cytokinesis, resulting in a separation of the two daughter cells. In each daughter cell, new branched structures are formed, and the two daughter cells then migrate toward neighboring oligodendrocytes. The series of photothermal images provides immensely important information about protein concentration and the distribution of protein inside each cell. Thus, we analyzed the spatiotemporal dynamics of MIP signals during the cell division process (Figs. 2d and 2e). The cell body regions are selected by excluding axons and dendritic structures to monitor the changes in size as well as local protein distributions during the cell division process. The integrated (total) MIP signal intensity was analyzed with respect to time spanning the entire cell division process (Figs. 2d and


8

Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2e). Compared to the total signal intensity of normal state, it is observed that the MIP intensity of the metaphase is about 1.37 times strong and that of two daughter cells is 0.82 times weak. Interestingly, the integrated MIP signal was observed to plateau in the middle of the process, from 20 to 50 minutes, for approximately thirty minutes after the constitution of the rim-like structure.3,4,21 In addition to the oligodendrocyte cell division, we also studied cellular dynamics, such as the migration and apoptosis of cells in real time (Figs. S8 and S9). One of the branches of an oligodendrocyte appears in the view field of our MIP microscopy and then slides (migrates) to the center of the observation window for 30 minutes. Interestingly, the shape of the cell during migration has an asymmetric morphology (front-to-back polarity), indicating that the front side of the cell to the migration direction is relatively narrow but its backside is wide. Within 40 minutes, the cell relocates and regains a relatively symmetric structure like those of other neighboring oligodendrocytes. Although most oligodendrocytes that are subject in this study exhibit very high photostability under our experimental conditions, after long times (>10 hours), one particular cell was found to undergo apoptosis with characteristic morphological changes including cell shrinking, blebbing, and chromatic condensation (Fig. S9).


9


Page 10 of 18

Figure 3. Live-cell microscopy images of neurons (day 5 and 7). (a,b) Bright-field and MIP images for neuron 1 (on day 2). MIP measurements are conducted for the white box (125×125 μm2) region in the bright-field image. (c) Superposed image of consecutive photothermal contrast images at the region of orange box in b. Time step: 20 seconds from position S to M (80 seconds) and 60 seconds from M to F (120 seconds). Scale bar. 5 μm. (d,e) Bright-field and MIP images for neuron 2 (on day 7). Scale bar. 20 μm. (f) Traces of each photothermal contrast (particle 1,2,3,4) in e. All contrast spots move through the dendrite structures from position S to M1, M2 and F. Scale bar. 10 μm. Total measurement time is 4 hours with a frame-to-frame time interval of 340 seconds. Rep. rate, 50 kHz. Pixel dwell time, 5 ms. Total number of pixels, 251×251 and 61×61 pixels for b,e and c, respectively. During the live-cell MIP measurement (see section 2.2 in Supporting Information for a detailed discussion on phototoxicity), the mid-IR pump and visible probe laser powers are set to 7.5 and 25 mW, respectively. Another important application of the label-free MIP microscopy demonstrated here is to track vesicle transport processes on the axons of live neuron. In Fig. 3a, the bright-field image of the characteristic neuronal structures of the axon and axon hillock is shown.22 The photothermal image in Fig. 3b reveals a high concentration of proteins at the soma and axon regions and a relatively blurred and weak signal from the dendrite structures. Compared to the active cellular dynamics of the oligodendrocyte, neurons exhibit relatively static behaviors under our live-cell imaging


10

Page 11 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conditions. However, we were able to observe a strong photothermal contrast signal appearing anterograde transport, from cell body region to its axon terminal, along one of the axons (Fig. 3c). It is well known that cytoskeletal filaments along the axon serve as a route for material transport between the axon and its termini and the cell body.22 To illustrate this protein-rich vesicle transport process, a series of MIP images are superposed in Fig. 3c, where the vesicle moves from S to F with an average velocity of 0.14 μm/s (Fig. S10). From this observation, we found that there is no spatial extension of or change in the axon diameter, which indicates that such an internal vesicle transport process occurs through the axon. We also found the same trend for dendrite structures in neurons obtained (Figs. 3d and 3e). A long-term (4 hours; 340 seconds time interval) MIP imaging allowed us to track four distinctive IR photothermal signals associated with axonal protein transports along the dendrite branches, which exhibited bidirectional and relatively slower movements. Their maximum speeds reach up to 11.219.1 nm/s, but the average speed of those vesicles is about 4.4 nm/s (Fig. S11 and Table S1). Such slow transport processes are consistent with the previous findings, and they are associated with cytoskeletal proteins such as microtubules, neurofilaments, and microfilaments.22,23,24 These experimental results again clearly show the advantage of the label-free MIP imaging technique for studying long-term cellular dynamics associated with protein production, aggregation, and/or transport processes in live cells. In summary, we have experimentally demonstrated mid-IR photothermal imaging microscopy as a feasible tool to observe the entire life cycle of cellular processes without incurring severe photo-damage or the need for exogenous labels. Focusing on the protein amide I absorption band at 1650 cm-1, we showed that the IR photothermal contrast of neural cells with characteristic cellular structures is consistent with that of bright-field images. Moreover, our cytoplasmic protein imaging technique enabled us to monitor a variety of cellular dynamics in real time, such as cellular


11


Page 12 of 18

migration, apoptosis, and cell division processes, as well as an inner-cellular protein-rich vesicle transportation from the cell body to the dendritic termini and vice versa. Combining the chemical sensitivity via IR excitation of imaging target vibrational modes and enhanced spatial resolution via diffraction measurement with a visible beam, we demonstrated that MIP microscopy can be of exceptional use to shed new insights into cellular dynamics and structures of live cells in real time. We thus anticipate that this method will be of use for scrutinizing cellular dynamics under active and programmed control parameters when it is combined with advanced techniques developed in molecular biology and biophysics.

ASSOCIATED CONTENT Supporting Information. Experimental details (experimental set-up, sample preparation, sample images and intensity profiles, detection limit, and dark-field image) and photothermal images and analysis (intensity sum and cell volume comparison, cell migration, apoptosis, time trajectory of vesicle transport, and phototoxicity). Movies of the cell division of oligodendrocyte and vesicle transport process on the axons of a neuron (file type: mov). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Jong Min Lim: 0000-0003-0311-1032


12

Page 13 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Minhaeng Cho: 0000-0003-1618-1056 Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was supported by IBS-R023-D1. We thank Prof. S. H. Shim (IBS CMSD, Korea) for stimulating discussion and for suggesting us to consider BSA for testing the quantitative sensitivity limit of our MIP microscopy and to examine time-dependent integrated MIP signal in Figure. 2d. REFERENCES (1) Stender, A. S.; Marchuk, K.; Liu, C.; Sander, S.; Meyer, M. W.; Smith, E. A.; Neupane, B.; Wang, G.; Li, J.; Cheng, J.-X.; Huang, B.; Fang, N. Single cell optical imaging and spectroscopy. Chem. Rev. 2013, 113, 2469-2527. (2) Welsher, K.; Yang, H. Multi-resolution 3D visualization of the early stages of cellular uptake of peptide-coated nanoparticles. Nat. Nanotechnol. 2014, 9,198-203. (3) Cai, Y.; Hossain, M. J.; Hériché, J. K.; Politi, A. Z.; Walther, N.; Koch, B.; Wachsmuth, M.; Nijmeijer, B.; Kueblbeck, M.; Martinic-Kavur, M.; Ladurner, R.; Alexander, S.; Peters, J. M.; Ellenberg, J. Experimental and computational framework for a dynamic protein atlas of human cell division. Nature 2018, 561, 411-415. (4) Walther, N.; Hossain, M. J.; Politi, A. Z.; Koch, B.; Kueblbeck, M.; Ødegård-Fougner, Ø.; Lampe, M.; Ellenberg. J. A quantitative map of human Condensins provides new insights into mitotic chromosome architecture. J. Cell Biol. 2018, 217, 2309-2328. (5) Park, J.-S.; Lee, I.-B.; Moon, H.-M.; Joo, J.-H.; Kim, K.-H.; Hong, S.-C.; Cho, M. Labelfree and live cell imaging by interferometric scattering microscopy. Chem. Sci. 2018, 9, 2690-2697.


13


Page 14 of 18

(6) de Wit, G.; Albrecht, D.; Ewers, H.; Kukura, P. Revealing Compartmentalized Diffusion in Living Cells with Interferometric Scattering Microscopy. Biophys. J. 2018, 114, 29452950. (7) McDonald, M. P.; Gemeinhardt, A.; König, K.; Piliarik, M.; Schaffer, S.; Völkl, S.; Aigner, M.; Mackensen, A.; Sandoghdar, V. Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity. Nano Lett. 2018, 18, 513-519. (8) Magidson, V.; Khodjakov, A. Circumventing photodamage in live-cell microscopy. Methods Cell Biol. 2013, 114, 545-560. (9) Ettinger, A.; Wittmann, T. Fluorescence Live Cell Imaging. Methods Cell Biol. 2014, 123, 77-94. (10) Contu, D. L.; Schroeder, T. Probing cellular processes by long-term live imaging – historic problems and current solutions. J. Cell Sci. 2013, 126, 3805-3815. (11) Cheng, J. X.; Xie, X. S. Eds. Coherent Raman scattering microscopy. (CRC Press, 2016). (12) Camp, C. H. Jr.; Cicerone, M. T. Chemically sensitive bioimaging with coherent Raman scattering. Nat. Photonics 2015, 9, 295-305. (13) Wetzel, D. L.; LeVine, S. M. Imaging molecular chemistry with infrared microscopy. Science 1999, 285, 1224-1225. (14) Fernandez, D. C.; Bhargava, R.; Hewitt, S. M.; Levin, I. W. Infrared spectroscopic imaging for histopathologic recognition. Nat. Biotechnol. 2005, 23, 469-474. (15) Lee, E. S.; Lee, J. Y. Nonlinear optical infrared microscopy with chemical specificity. Appl. Phys. Lett. 2009, 94, 261101-3. (16) Mërtiri, A.; Jeys, T.; Liberman, V.; Hong, M. K.; Mertz, J.; Altug, H.; Erramilli, S. Mid-infrared photothermal heterodyne spectroscopy in a liquid crystal using a quantum cascade laser. Appl. Phys. Lett. 2012, 101, 044101-4.


14

Page 15 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17) Furstenberg, R.; Kendziora, C. A.; Papantonakis, M. R.; Nguyen, V.; McGil, R. A. Chemical imaging using infrared photothermal microspectroscopy. Proc. SPIE 2012, 8374, 837411. (18) Zhang, D.; Li, C.; Zhang, C.; Slipchenko, M. N.; Eakins, G.; Cheng, J.-X. Depthresolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution. Sci. Adv. 2016, 2, e1600521. (19) Li, Z.; Aleshire, K.; Kuno, M.; Hartland, G. V. Super-Resolution Far-Field Infrared Imaging by Photothermal Heterodyne Imaging. J. Phys. Chem. B 2017, 121, 8838-8846. (20) Dick, A. E.; Gerlich, D. W. Kinetic framework of spindle assembly checkpoint signalling. Nat. Cell Biol. 2013, 15, 1370-1377. (21) Gookin, S.; Min, M.; Phadke, H.; Chung, M.; Moser, J.; Miller, I.; Carter, D.; Spencer, S. L. A map of protein dynamics during cell-cycle progression and cell-cycle exit. PLOS Biol. 2017, 15, e2003268. (22) Maday, S.; Twelvetrees, A. E.; Moughamian, A. J.; Holzbaur, E. L. F. Axonal transport: cargo-specific mechanisms of motility and regulation. Neuron 2014, 84, 292309. (23) Lasek, R. K.; Garner, J. A.; Brady, S. T. Axonal transport of the cytoplasmic matrix. J. Cell Biol. 1984, 99, 212s-221s. (24) Conde, C.; Cáceres, A. Microtubule assembly, organization and dynamics in axons and dendrites. Nat. Rev. Neurosci. 2009, 10, 319-332.


15

a

b

PD

mid-IR ON PD

Page 16 of 18

Condenser

iris sample

XYZ piezostage OBJ

OAPM

mid-IR pulse

DM L

visible CW

c

1 �m M

FT-IR MIP

data fitting curve

0.20

intensity

0.08

intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mid-IR OFF


0.04

0.00

0.95 μm

0.15 0.10 0.05 0.00

1500

1550

1600

1650

-1

wavenumber (cm )

1700

-4 ACS Paragon Plus Environment

-2

0

width (μm)

2

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b


d intensity (a.u.)

Page 17 of 18

50 40 30

25��m

c

-20 0

20 40 60 80 100 120

time (min.)

e ��m

0 min.

+ 30 min.

+ 35 min.

+ 40 min.

+ 50 min. - 20 min.

��m

+ 45 min.

+ 50 min.

+ 80 min.


+ 110 min.

+ 25 min.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b

c


Page 18 of 18

F M

S 5��m

d

e

f

particle 1

F

particle 2

F S 5��m

5��m

4

F

1 2

particle 3


particle 4 S

3 20��m

S

M2 10��m

S

5��m

F M1

Cytoplasmic Protein Imaging with Mid-Infrared Photothermal

Recommend Documents