Name: a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy
Uploaded: 2022-08-31T15:00:00
Description: Watch this Scientific Journal Video about A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy at JoVE.com

We want to thank the 2019 Undergraduate Senior Design Team (Suk Chul Yoon, Ian Foxton, Louis Mazza, and James Walsh) at Binghamton University for the design, fabrication, and testing of the microscope enclosure box. We thank Scott Hancock, Olga Petrova, and Fabiola Moreno Olivas at Binghamton University&#160;for helpful discussions. This research was supported by the National Institutes of Health under Award Number R15GM140444.

1. Build the microscope environmental enclosure
NOTE: This large microscope environmental enclosure is used to control the temperature of the microscope body and the imaging environment to be stabilized at 37 &#176;C (Figure 1A).
<ol>
	<li>Mark the locations of the feet of the SRS microscope frame and the motorized stage using a marker pen on the optical table. Mount two Iris Diaphragms in front of the Galvanometer scanner of the microscope and adjust to make th...

<ol>
	<li>Mertz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Optical Microscopy. , (2019).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Methods in Cell Biology. 123, 77-94 (2014).</li><li>Park, Y., Depeursinge, C., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phase+imaging+in+biomedicine.">Quantitative phase imaging in biomedicine.</a> Nature Photonics. 12 (10), 578-589 (2018).</li><li>Hu, C. D., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nature Biotechnology. 21 (5), 539-545 (2003).</li><li>lamo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dye+labeling+changes+the+biodistribution+of+tumor-targeted+nanoparticles.">Fluorescent dye labeling changes the biodistribution of tumor-targeted nanoparticles.</a> Pharmaceutics. 12 (11), 1004 (2020).</li><li>Cheng, J. X., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrational+spectroscopic+imaging+of+living+systems:+An+emerging+platform+for+biology+and+medicine.">Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine.</a> Science. 350 (6264), aaa870 (2015).</li><li>Lu, F. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+DNA+imaging+in+vivo+with+stimulated+Raman+scattering+microscopy.">Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (37), 11624-11629 (2015).</li><li>Hill, A. H., Fu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+imaging+using+stimulated+Raman+scattering+microscopy.">Cellular imaging using stimulated Raman scattering microscopy.</a> Analytical Chemistry. 91 (15), 9333-9342 (2019).</li><li>Bu&#273;a, R., Vuku&#353;i&#263;, K., Toli&#263;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. (139), 81-101 (2017).</li><li>Chiarelli, T. J., Grieshaber, N. A., Grieshaber, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+forward+genetic+approach+to+identify+and+isolate+developmental+mutants+in+Chlamydia+trachomatis.">Live-cell forward genetic approach to identify and isolate developmental mutants in Chlamydia trachomatis.</a> Journal of Visualized Experiments. (160), e61365 (2020).</li><li>Lemon, W. C., McDole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+in+the+era+of+too+many+microscopes.">Live-cell imaging in the era of too many microscopes.</a> Current Opinion in Cell Biology. 66, 34-42 (2020).</li><li>Birk, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+oral+biofilms+by+nisin+delivery+in+adhesive+microdevices.">Management of oral biofilms by nisin delivery in adhesive microdevices.</a> European Journal of Pharmaceutics and Biopharmaceutics. 167, 83-88 (2021).</li><li>Watanabe, I., Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+on+growth+rate+of+cultured+mammalian+cells+(L5178Y).">Effects of temperature on growth rate of cultured mammalian cells (L5178Y).</a> Journal of Cell Biology. 32 (2), 309-323 (1967).</li><li>Lac, A., Lam, A. L., Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescent Microscopy. , 57-73 (2022).</li><li>Grossman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machinable+glass-ceramics+based+on+tetrasilicic+mica.">Machinable glass-ceramics based on tetrasilicic mica.</a> Journal of the American Ceramic Society. 55 (9), 446-449 (1972).</li><li>Yuan, Y., Shah, N., Almohaisin, M. I., Saha, S., Lu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+fatty+acid-induced+lipotoxicity+and+its+therapeutic+potential+in+glioblastoma+using+stimulated+Raman+microscopy.">Assessing fatty acid-induced lipotoxicity and its therapeutic potential in glioblastoma using stimulated Raman microscopy.</a> Scientific Reports. 11 (1), 1-14 (2021).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2 (1), 1-9 (2003).</li><li>Sun, M. W., Yuan, Y. H., Lu, F. K., Di Pasqua, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+factors+that+influence+the+biocompatibility+of+cationic+liposomes+and+their+ability+to+deliver+DNA+to+the+nuclei+of+ovarian+cancer+SK-OV-3+cells.">Physicochemical factors that influence the biocompatibility of cationic liposomes and their ability to deliver DNA to the nuclei of ovarian cancer SK-OV-3 cells.</a> Materials. 14 (2), 416 (2021).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Ozeki, Y., Itoh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+Raman+scattering+microscopy+for+live-cell+imaging+with+high+contrast+and+high+sensitivity.">Stimulated Raman scattering microscopy for live-cell imaging with high contrast and high sensitivity.</a> Laser Physics. 20 (5), 1114-1118 (2010).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+live-cell+imaging+of+nucleic+acids+using+stimulated+Raman+scattering+microscopy.">Label-free live-cell imaging of nucleic acids using stimulated Raman scattering microscopy.</a> ChemPhysChem. 13 (4), 1054-1059 (2012).</li><li>Stiebing, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+Raman+and+SRS+imaging+of+living+human+macrophages+reveals+cell-to-cell+heterogeneity+and+dynamics+of+lipid+uptake.">Real-time Raman and SRS imaging of living human macrophages reveals cell-to-cell heterogeneity and dynamics of lipid uptake.</a> Journal of Biophotonics. 10 (9), 1217-1226 (2017).</li><li>Miao, K., Wei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+and+quantification+of+PolyQ+aggregates+by+stimulated+Raman+scattering+of+selective+deuterium+labeling.">Live-cell imaging and quantification of PolyQ aggregates by stimulated Raman scattering of selective deuterium labeling.</a> ACS Central Science. 6 (4), 478-486 (2020).</li><li>Brzozowski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+Raman+scattering+microscopy+in+chemistry+and+life+science+-Development,+innovation,+perspectives.">Stimulated Raman scattering microscopy in chemistry and life science -Development, innovation, perspectives.</a> Biotechnology Advances. 60, 108003 (2022).</li><li>Hislop, E. W., Tipping, W. J., Faulds, K., Graham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+lipid+droplets+in+prostate+cells+using+stimulated+Raman+scattering+microscopy+and+multivariate+analysis.">Label-free imaging of lipid droplets in prostate cells using stimulated Raman scattering microscopy and multivariate analysis.</a> Analytical Chemistry. 94 (25), 8899-8908 (2022).</li><li>Chen, T., Yavuz, A., Wang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+lipid+droplet+biology+with+coherent+Raman+scattering+microscopy.">Dissecting lipid droplet biology with coherent Raman scattering microscopy.</a> Journal of Cell Science. 135 (5), jcs252353 (2022).</li><li>Cao, C., Zhou, D., Chen, T., Streets, A. M., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+digital+quantification+of+lipid+droplets+in+single+cells+by+stimulated+Raman+microscopy+on+a+microfluidic+platform.">Label-Free digital quantification of lipid droplets in single cells by stimulated Raman microscopy on a microfluidic platform.</a> Analytical Chemistry. 88 (9), 4931-4939 (2016).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+Raman+scattering+flow+cytometry+for+label-free+single-particle+analysis.">Stimulated Raman scattering flow cytometry for label-free single-particle analysis.</a> Optica. 4 (1), 103-109 (2017).</li><li>Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+chemical+imaging+flow+cytometry+by+high-speed+multicolor+stimulated+Raman+scattering.">Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (32), 15842-15848 (2019).</li><li>Gala de Pablo, J., Lindley, M., Hiramatsu, K., Goda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+Raman+flow+cytometry+and+beyond.">High-throughput Raman flow cytometry and beyond.</a> Accounts of Chemical Research. 54 (9), 2132-2143 (2021).</li><li>Cole, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging:+the+cell's+perspective.">Live-cell imaging: the cell's perspective.</a> Cell Adhesion &amp; Migration. 8 (5), 452-459 (2014).</li><li>Kreft, M., Stenovec, M., Zorec, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus-drift+correction+in+time-lapse+confocal+imaging.">Focus-drift correction in time-lapse confocal imaging.</a> Annals of the New York Academy of Sciences. 1048 (1), 321-330 (2005).</li><li>Firestone, L., Cook, K., Culp, K., Talsania, N., Preston Jr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+autofocus+methods+for+automated+microscopy.">Comparison of autofocus methods for automated microscopy.</a> Cytometry: The Journal of the International Society for Analytical Cytology. 12 (3), 195-206 (1991).</li><li>Van Helvert, S., Storm, C., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoreciprocity+in+cell+migration.">Mechanoreciprocity in cell migration.</a> Nature Cell Biology. 20 (1), 8-20 (2018).</li><li>Zong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmon-enhanced+stimulated+Raman+scattering+microscopy+with+single-molecule+detection+sensitivity.">Plasmon-enhanced stimulated Raman scattering microscopy with single-molecule detection sensitivity.</a> Nature Communications. 10 (1), 1-11 (2019).</li><li>Wei, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-multiplex+vibrational+imaging.">Super-multiplex vibrational imaging.</a> Nature. 544 (7651), 465-470 (2017).</li></ol>

Time-lapse live-cell SRS microscopy is an alternative imaging technique for molecule tracking in a label-free manner. Compared to fluorescence labeling, SRS imaging is free from photobleaching, enabling long-term monitoring of molecules. However, to date, the live cell imaging system on an upright SRS microscopy is not commercially available. In this work, a live cell imaging system with a stable thermal-insulated microscope enclosure box and a flexible inner soft chamber was developed to enable transmitted SRS time-laps...

Optical microscopy is used to observe the microstructures of samples. Optical imaging is rapid, less invasive, and less destructive than other technologies1. Live-cell imaging with optical microscopy is developed to capture the dynamics of cultured live cells over a long period2. Different types of optical contrasts provide distinct information about biological samples. For instance, optical phase microscopy shows the subtle difference in the refractive indices across the sample3. Fluorescence microscopy is widely used to image specific biomolecules or cellular organelles. However, the broadband excitation and emission spectra of fluorescence usually result in spectral overlapping when multicolor imaging is performed4. Fluorescent molecules are light-sensitive and can be bleached after long-term, periodic light exposure. In addition, fluorescence labeling may change the biodistribution of the molecules in cells5. SRS microscopy is a label-free chemical imaging technology6. The contrast of SRS relies on the vibrational transition of specific chemical bonds. The vibrational frequency of a chemical bond often exhibits a narrow spectral bandwidth, making it feasible to image multiple Raman bands in the same samples7. SRS microscopy is a unique tool for live-cell imaging, providing multiple chemical contrasts in a label-free manner8.
While SRS imaging of unstained cells has been used for many studies, long-term time-lapse SRS imaging of live cells has not been widely adopted. One reason is that commercial open chambers cannot be directly used for SRS imaging because of their large thickness9,10,11,12. These chambers with a glass lid are mostly designed for brightfield or fluorescence imaging using a single high NA objective with a backward detection scheme. However, SRS imaging prefers transmitted detection using both a high NA objective and a high NA condenser, which leaves only a very short gap (typically a few millimeters) between the objective and the condenser. To overcome this problem, we designed a flexible chamber using a soft material to enable time-lapse SRS imaging of live cells using an upright microscope frame. In this design, the water dipping objective was enclosed in the soft chamber and can freely move in three dimensions for focusing and imaging purposes.
The optimal temperature for culturing most mammalian cells is 37 &#176;C, while the room temperature is always 10&#176; lower than this. Temperature higher or lower than 37 &#176;C has a dramatic effect on cell growth rate13. Therefore, temperature control of the cell culture environment is required in a live-cell imaging system. It is known that temperature instability will lead to defocusing issues during long-term imaging14. To achieve a stable 37 &#176;C environment, we built a large enclosure chamber to cover the entire microscope frame, including a thermal insulation layer underneath the microscope (Figure 1). Within the sizeable temperature-control chamber, the small flexible chamber helps to accurately maintain the physiological humidity and pH via the regulated air flow supplemented with 5% CO2 (Figure 2). The temperature and humidity of the chambers were measured to confirm that the double-chamber design provided the optimal cell culture condition for cell growth under long-term, periodic SRS imaging (Figure 3). We then demonstrated the application of the system for time-lapse imaging and tracking lipid droplets (LDs) in SKOV3 cancer cells (Figure 4, Figure 5, and Figure 6).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A lab-built SRS microscope</td>
			<td></td>
			<td></td>
			<td>https://rdcu.be/cP6ve</td>
		</tr>
		<tr>
			<td>HF2LI 50 MHz lock-in amplifer</td>
			<td>Zurich Instruments</td>
			<td>HF2LI</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris diaphragm</td>
			<td>Thorlabs Inc</td>
			<td>SM1D12</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinematic mirror mount</td>
			<td>Thorlabs Inc</td>
			<td>KM100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope frame</td>
			<td>Nikon Inc</td>
			<td>FN-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized microscopy stage</td>
			<td>Prior Scientific</td>
			<td>Z-Deck</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil-immersion condenser (C-AA Achromat/Aplanat, NA 1.4)</td>
			<td>Nikon Inc</td>
			<td>MBL71405</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-immersion objective (CFI75 Apo 25XC W 1300)</td>
			<td>Nikon Inc</td>
			<td>MRD77225</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and parts for the microscope enclosure (31&#39;&#39; x 29&#39;&#39; x 28&#39;&#39; L x W x H)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Airtherm heater module</td>
			<td>World Precision Instruments (WPI)</td>
			<td>AIRTHERM-SAT-1W</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtherm heater controller, CO2 and humidity monitor</td>
			<td>World Precision Instruments (WPI)</td>
			<td>AIRTHERM-SMT-1W</td>
			<td></td>
		</tr>
		<tr>
			<td>Air/CO2 mixer module</td>
			<td>World Precision Instruments (WPI)</td>
			<td>ECU-HOC-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible duct hose (2-1/2&#39;&#39; ID, 2-3/4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>56675K71</td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature glass-mica ceramic, easy-to-machine (6&#39;&#39; x 6&#39;&#39;, 1/4&#39;&#39; thickness)</td>
			<td>McMaster-Carr</td>
			<td>8489K62</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate sheets (thickness 0.25&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8574K286</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber sheets (36&#39;&#39; x 36&#39;&#39;, thickness 1/8&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>5827T43</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and parts for the Flexible chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>McMaster-Carr</td>
			<td>31745K11</td>
			<td></td>
		</tr>
		<tr>
			<td>High-purity inline filter, 1/4 NPT</td>
			<td>McMaster-Carr</td>
			<td>6645T18</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw (cutting diameter 1-7/8 inch)</td>
			<td>McMaster-Carr</td>
			<td>4066A34</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw (cutting diameter 50 mm)</td>
			<td>McMaster-Carr</td>
			<td>4556A19</td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature silicone rubber tubing, soft, 2 mm ID, 5 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5054K313</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline filter (1/4 NPT, 40 micron)</td>
			<td>McMaster-Carr</td>
			<td>98385K843</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum round tube (1/8&#39;&#39; wall thickness, 4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>9056K42</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum round tube (3/4&#39;&#39; wall thickness, 3-3/4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>9056K47</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum bar (12&#39;&#39; x 12&#39;&#39;, thickness 1/4&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8975K142</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum bar (8&#39;&#39; x 8&#39;&#39;, thickness 3/8&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>9246K21</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective nosepiece (single)</td>
			<td>Nikon Inc</td>
			<td>FN-MN-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample holder (modified)</td>
			<td>Prior Scientific</td>
			<td>HZ202</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-thin natural rubber film (thickness 0.01&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8611K13</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum-sealable glass jar</td>
			<td>McMaster-Carr</td>
			<td>3231T44</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ (Fiji)</td>
			<td></td>
			<td>imagej.net</td>
			<td></td>
		</tr>
		<tr>
			<td>ScanImage</td>
			<td>Vidrio Technologies, LLC</td>
			<td></td>
			<td>SRS imaging software</td>
		</tr>
		<tr>
			<td>Materials for live-cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass bottom sterile culture dishes (Dia.x H, 50 x 7 mm)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>70674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoSensor fluorescent dye DND-189</td>
			<td>ThermoFisher Scientific</td>
			<td>L7535 (Invitrogen)</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>MilliporeSigma</td>
			<td>364525</td>
			<td></td>
		</tr>
		<tr>
			<td>SKOV3 cell line</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We fabricated and assembled the flexible chamber system for time-lapse SRS imaging (Figure 1 and Figure 2), and then evaluated the performance of the system. The temperature inside the microscope environmental enclosure reached the expected 37 &#176;C within 1 h, which did not significantly affect the room temperature (Figure 3A). The temperature in the flexible chamber reached 37 &#176;C in 1.5 h, and it was stably maintained at 37...

Watch this Scientific Journal Video about A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy at JoVE.com

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

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