Name: light-induced in situ transmission electron microscopy for observation of the liquid-soft matter interaction
Uploaded: 2022-07-26T13:30:00
Description: Watch this Scientific Journal Video about Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction at JoVE.com

The research was supported by the Miniatura grant (2019/03/X/NZ3/02100, National Science Center, Poland).

1. Transmission electron microscope modification
<ol>
	<li>Modify the transmission electron microscope by connecting the optical fiber (see Table of Materials) to the microscope column at the top of the objective lens. Allow a few centimeters of space between the objective lens and the condenser lens, plus a free slot for accessories. 
	NOTE: A dedicated sample holder4 or a holder for cathodoluminescent observations in reverse configuration<sup class="xref"...

<ol>
	<li>Vadai, M., Angell, D. K., Hayee, F., Sytwu, K., Dionne, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+observation+of+plasmon-controlled+photocatalytic+dehydrogenation+of+individual+palladium+nanoparticles.">In-situ observation of plasmon-controlled photocatalytic dehydrogenation of individual palladium nanoparticles.</a> Nature Communications. 9 (1), 1-8 (2018).</li><li>Weng, B., 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=Visualizing+light-induced+dynamic+structural+transformations+of+Au+clusters-based+photocatalyst+via+in+situ+TEM.">Visualizing light-induced dynamic structural transformations of Au clusters-based photocatalyst via in situ TEM.</a> Nano Research. 12 (1), 1-5 (2021).</li><li>Cao, 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=In+situ+TEM+investigation+on+ultrafast+reversible+lithiation+and+delithiation+cycling+of+Sn@C+yolk-shell+nanoparticles+as+anodes+for+lithium+ion+batteries.">In situ TEM investigation on ultrafast reversible lithiation and delithiation cycling of Sn@C yolk-shell nanoparticles as anodes for lithium ion batteries.</a> Nano Energy. 40, 187-194 (2017).</li><li>Cavalca, F., 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=In+situ+transmission+electron+microscopy+of+light-induced+photocatalytic+reactions.">In situ transmission electron microscopy of light-induced photocatalytic reactions.</a> Nanotechnology. 23 (7), 075705 (2012).</li><li>Tung, C. -. W., 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=Light-induced+activation+of+adaptive+junction+for+efficient+solar-driven+oxygen+evolution:+In+situ+unraveling+the+interfacial+metal-silicon+junction.">Light-induced activation of adaptive junction for efficient solar-driven oxygen evolution: In situ unraveling the interfacial metal-silicon junction.</a> Advanced Energy Materials. 9 (31), 1901308 (2019).</li><li>&#379;ak, A. M., Kaczmarczyk, O., Piksa, M., Matczyszyn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+in+situ+transmission+electron+microscopy:+Novel+approach+for+antimicrobial+photodynamic+therapy+imaging.">Light-induced in situ transmission electron microscopy: Novel approach for antimicrobial photodynamic therapy imaging.</a> Photodiagnosis and Photodynamic Therapy. 35, 102463 (2021).</li><li>Shindo, D., 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=Development+of+a+multifunctional+TEM+specimen+holder+equipped+with+a+piezodriving+probe+and+a+laser+irradiation+port.">Development of a multifunctional TEM specimen holder equipped with a piezodriving probe and a laser irradiation port.</a> Journal of Electron Microscopy. 58 (4), 245-249 (2009).</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=Photosensing+performance+of+branched+CdS/ZnO+heterostructures+as+revealed+by+in+situ+TEM+and+photodetector+tests.">Photosensing performance of branched CdS/ZnO heterostructures as revealed by in situ TEM and photodetector tests.</a> Nanoscale. 6 (14), 8084-8090 (2014).</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=Statistically+analyzed+photoresponse+of+elastically+bent+CdS+nanowires+probed+by+light-compatible+in+situ+high-resolution+TEM.">Statistically analyzed photoresponse of elastically bent CdS nanowires probed by light-compatible in situ high-resolution TEM.</a> Nano Letters. 16 (10), 6008-6013 (2016).</li><li>Yoshida, K., Yamasaki, J., Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+high-resolution+transmission+electron+microscopy+observation+of+photodecomposition+process+of+poly-hydrocarbons+on+catalytic+TiO2+films.">In situ high-resolution transmission electron microscopy observation of photodecomposition process of poly-hydrocarbons on catalytic TiO2 films.</a> Applied Physics Letters. 84 (14), 2542-2544 (2004).</li><li>Yoshida, K., Nozaki, T., Hirayama, T., Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+high-resolution+transmission+electron+microscopy+of+photocatalytic+reactions+by+excited+electrons+in+ionic+liquid.">In situ high-resolution transmission electron microscopy of photocatalytic reactions by excited electrons in ionic liquid.</a> Journal of Electron Microscopy. 56 (5), 177-180 (2007).</li><li>Textor, M., De Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+preparing+graphene+liquid+cells+for+transmission+electron+microscopy.">Strategies for preparing graphene liquid cells for transmission electron microscopy.</a> Nano Letters. 18 (6), 3313-3321 (2018).</li><li>Ring, E. A., De Jonge, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+system+for+transmission+electron+microscopy.">Microfluidic system for transmission electron microscopy.</a> Microscopy and Microanalysis. 16 (5), 622-629 (2010).</li><li>Inayoshi, Y., Minoda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+carbon+sandwich+environmental+cell+for+wet+specimens.">A carbon sandwich environmental cell for wet specimens.</a> Journal of Electron Microscopy. 62 (6), 623-628 (2013).</li><li>Koo, K., Dae, K. S., Hahn, Y. K., Yuk, J. M. <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+electron+microscopy+using+graphene+veils.">Live cell electron microscopy using graphene veils.</a> Nano Letters. 20 (6), 4708-4713 (2020).</li><li>De Jonge, N., Peckys, D. B. <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+electron+microscopy+is+probably+impossible.">Live cell electron microscopy is probably impossible.</a> ACS Nano. 10 (10), 9061-9063 (2016).</li><li>Kennedy, E., Nelson, E. M., Damiano, J., Timp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+electron-beam-irradiated+bacteria+in+reply+to+&amp;#34;live+cell+electron+microscopy+is+probably+impossible.&amp;#34.">Gene expression in electron-beam-irradiated bacteria in reply to &amp;#34;live cell electron microscopy is probably impossible.&amp;#34.</a> ACS Nano. 11 (1), 3-7 (2017).</li><li>Alves, 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=An+insight+on+bacterial+cellular+targets+of+photodynamic+inactivation.">An insight on bacterial cellular targets of photodynamic inactivation.</a> Future Medicinal Chemistry. 6 (2), 141-164 (2014).</li><li>Cieplik, F., 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=Antimicrobial+photodynamic+therapy-What+we+know+and+what+we+don't.">Antimicrobial photodynamic therapy-What we know and what we don't.</a> Critical Reviews in Microbiology. 44 (5), 571-589 (2018).</li><li>Huang, 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=Type+I+and+Type+II+mechanisms+of+antimicrobial+photodynamic+therapy:+An+in+vitro+study+on+gram-negative+and+gram-positive+bacteria.">Type I and Type II mechanisms of antimicrobial photodynamic therapy: An in vitro study on gram-negative and gram-positive bacteria.</a> Lasers in Surgery and Medicine. 44 (6), 490-499 (2012).</li><li>Caminos, D. A., Spesia, M. B., Pons, P., Durantini, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Escherichia+coli+photodynamic+inactivation+by+an+amphiphilic+tricationic+porphyrin+and+5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl)+porphyrin.">Mechanisms of Escherichia coli photodynamic inactivation by an amphiphilic tricationic porphyrin and 5,10,15,20-tetra(4-N,N,N-trimethylammoniumphenyl) porphyrin.</a> Photochemical and Photobiological Sciences. 7 (9), 1071-1078 (2008).</li><li>Chu, J. C. H., Chin, M. L., Wong, C. T. T., Hui, M., Lo, P. C., Ng, D. K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-pot+synthesis+of+a+cyclic+antimicrobial+peptide-conjugated+phthalocyanine+for+synergistic+chemo-photodynamic+killing+of+multidrug-resistant+bacteria.">One-pot synthesis of a cyclic antimicrobial peptide-conjugated phthalocyanine for synergistic chemo-photodynamic killing of multidrug-resistant bacteria.</a> Advanced Therapeutics. 4 (3), 1-10 (2021).</li><li>Rogers, S., Honma, K., Mang, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+fluorescence+imaging+to+evaluate+the+effect+of+antimicrobial+photodynamic+therapy+depth+on+P.+gingivalis+and+T.+denticola+biofilms.">Confocal fluorescence imaging to evaluate the effect of antimicrobial photodynamic therapy depth on P. gingivalis and T. denticola biofilms.</a> Photodiagnosis and Photodynamic Therapy. 23, 18-24 (2018).</li><li>Maldonado-Carmona, N., 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=Porphyrin-loaded+lignin+nanoparticles+against+bacteria:+A+photodynamic+antimicrobial+chemotherapy+application.">Porphyrin-loaded lignin nanoparticles against bacteria: A photodynamic antimicrobial chemotherapy application.</a> Frontiers in Microbiology. 11, 606185 (2020).</li><li>&#379;ak, A. M., Kaczmarczyk, O., Piksa, M., Grz&#281;da, J., Matczyszyn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-optic+sample+illuminator+design+for+the+observation+of+light+induced+phenomena+with+transmission+electron+microscopy+in+situ:+Antimicrobial+photodynamic+therapy.">Fiber-optic sample illuminator design for the observation of light induced phenomena with transmission electron microscopy in situ: Antimicrobial photodynamic therapy.</a> Ultramicroscopy. 230, 113388 (2021).</li><li>Dinh, V. 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=Insight+into+the+adsorption+mechanisms+of+methylene+blue+and+chromium(III)+from+aqueous+solution+onto+pomelo+fruit+peel.">Insight into the adsorption mechanisms of methylene blue and chromium(III) from aqueous solution onto pomelo fruit peel.</a> RSC Advances. 9 (44), 25847-25860 (2019).</li><li>&#379;ak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+controlling+the+electron+dose+to+improve+low-dose+imaging+of+sensitive+samples.">Guide to controlling the electron dose to improve low-dose imaging of sensitive samples.</a> Micron. 145, 103058 (2021).</li><li>Drulyte, I., 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=Approaches+to+altering+particle+distributions+in+cryo-electron+microscopy+sample+preparation.">Approaches to altering particle distributions in cryo-electron microscopy sample preparation.</a> Acta Crystallographica Section D. 74 (6), 560-571 (2018).</li><li>Scarff, C. 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The installation and commissioning of the illuminator require basic service knowledge and can damage the microscope. The simplest way to introduce the light to the microscope is to connect the optical fiber from the top of the objective lens, where there is usually room for the TEM deflection coils and additional detectors. More free space can also be expected from older top-entry devices, where the same location houses the sample vacuum lock and the sample installation mechanism. This configuration is widely described i...

Light-induced phenomena in high resolution are interesting in many fields such as nanoengineering1,2,3, catalysis4,5, and biophotonics6. Some original designs that allow such experiments can be found in the literature,including modifications of the sample holders1,4,7,8,9&#160;and the optical fiber attached to the microscope10,11.
The combination of light illumination, a liquid environment, and transmission electron microscopy (TEM) gives a great opportunity for detailed, dynamical studies of photo-induced processes. However, the high-vacuum condition inside the microscope is rather unfavorable for many liquids, especially water solutions. Liquid encapsulation, which protects it from the environment, can be achieved using a few techniques based mainly on graphene12, silicon nitride13, or carbon14 substrates. In addition to research in materials science2, so-called liquid cells offer possibilities for conducting unconventional microscopic observations on biological specimens near their native conditions15. Such observations are extremely demanding, especially for living microorganisms such as bacterial cells. The electron beam as ionizing radiation causes irreversible damage to the hydrated specimens, so the electron dose must be specified16. This is necessary to minimize unfavorable effects, control the damage, and avoid confusing artifacts. The optimal maximum electron dose that allows observations of living cells is still a questionable topic16, but the dose of 30 e&#8722;/nm2 appears to be the threshold value, at least for bacteria17.
Some of the subjects of interest for such microscopic studies are processes during antimicrobial photodynamic therapy (APDT)18. In short, the therapy proceeds as follows. The bacterial cells are surrounded by the photosensitive liquid called photosensitizer. When light illumination is given at a specific wavelength, the cytotoxic reactive oxygen species (ROS) are generated from energy or charge transfer from the excited photosensitizer molecules to the oxygen naturally present in the solution. Pathogens exposed to ROS are quickly inactivated with very high efficiency, with no side effects19. The response to the therapy varies for distinct microbes &#8211; for example, the impact of the same photosensitizer may be quite different for Gram-positive and Gram-negative bacteria20. In general, it has been established that the main target of ROS is the outer structures of cells, where damage results in functional disorders of the cell membrane and, consequently, lead to the death of bacteria21,22. However, damage to nucleic acids and proteinscan also be considered a cause of inactivation18, so it is still unknown which cell structures are the main targets during this process19. A deeper understanding of the damaging processes could help to improve this definitive therapy. Compared to the light microscopy methods used in APDT research23, TEM techniques give more possibilities to look at the APDT mechanism with higher resolution and magnification24. TEM has already been successfully used for cell observation during ongoing therapy, which allowed us to study Gram-positive bacteriadamage and describe the changesoccurring within the cell wall in detail6,25.
The current protocol presents a suitable experimental setup for high-resolution imaging of light-induced bacteria inactivation using TEM, which requires a proper light illumination system, the encapsulation of cells with a liquid, and strict electron dose control. The bacteria used for the observation was Staphylococcus aureus,&#160;and a methylene blue solution was used as a photosensitizer. The special light illumination setup comprises a tunable semiconductor laser connected directly to the microscope column using the light fiber. This design provides uniform irradiation throughout the sample because of the almost-parallel placement of the optical fiber to the microscope axis. Monochromatic light of high intensity generated by the laser can then be used to study various photochemical effects. The light used in the experiment had a wavelength equal to 660 nm because, in the visible region, methylene blue has absorption peaks at 613 nm and 664 nm26. The protocol for liquid encapsulation is based on carbon substrates, which makes the procedure quick and uncomplicated. Finally, a method for low-dose in situ TEM observation of cells in liquid is presented. The difficulties regarding sample preparation, the electron dose effects on the sensitive specimen, and reasonable image interpretation are discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbon film on 200 mesh copper grid</td>
			<td>Agar Scientific</td>
			<td>AGS160</td>
			<td>The standard TEM grids for observations and liquid cell preparation</td>
		</tr>
		<tr>
			<td>Crossover Tweezers</td>
			<td>Dumont</td>
			<td>N5</td>
			<td>The tweezers are neecesarry for liquid cell preparation</td>
		</tr>
		<tr>
			<td>Photodiode Power Sensor</td>
			<td>ThorLabs</td>
			<td>S130C</td>
			<td>The sensor used for light intensity measurement</td>
		</tr>
		<tr>
			<td>Polyimide-Coated Multimode Fiber</td>
			<td>Thorlabs</td>
			<td>FG400UEP</td>
			<td>Must be built into the microscope using the on-site built adapter, according to the 10.1016/j.ultramic.2021.113388</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Hitachi</td>
			<td>H-800</td>
			<td>Can be replaced with any side-entry microscope, available for modification</td>
		</tr>
		<tr>
			<td>Tuneable Diode Laser</td>
			<td>CNI</td>
			<td>MRL-III-660D</td>
			<td>The light wavelength must be chosen basing on photosensitizer&#39;s absorption spectrum</td>
		</tr>
	</tbody>
</table>

light-induced in situ transmission electron microscopy for observation of the liquid-soft matter interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass optical fiber inserted into the electron column above the objective lens polepiece, and a laser, an adjustable light source, was used to fabricate the device. After the illuminator has been calibrated using an external measuring system, it allows one to adjust the intensity of the lighting to the needs of the observed process. This lighting system was utilized to image antimicrobial photodynamic therapy phenomena, which are currently the subject of intense research. The sample was prepared by spotting a suspension of bacteria on a carbon, graphene, or silicon nitride substrate, blotting the excess solution, spotting the photosensitizer solution, blotting the excess liquid again, and then assembling the liquid cell with a second substrate or graphene film. The process of the imaging experiment itself includes choosing the right place for observation with the use of low magnification and a minimum dose of electrons, and then cyclical activation of the light source to capture subsequent images at specified intervals with the minimum amount of electrons necessary. The electron dose of each exposure and the time and intensity of lighting used need to be carefully recorded due to the complexity of the observed phenomena as, at the same time, the process is both light- and electron-driven. After the actual experiment is performed, additional control observations must be made, in which the same doses of electrons are used but without additional light influence and smaller doses of electrons are used for higher doses of light. This makes it possible to distinguish light-induced microstructural effects from those caused by electrons in both the fields of life and materials science.

The experimental setup was designed to observe the light-induced processes occurring in a liquid with high resolution. The competitive analysis of the images allowed us to distinguish the damage caused by the electron beam from changes related to the photochemical reaction. The effect of the electron beam on the sensitive specimen (in this case, the cells encapsulated with liquid) was visible all over the irradiated area as the electrons penetrated through it uniformly. Of course, the effect depends on the electron dose,...

Watch this Scientific Journal Video about Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction at JoVE.com

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The present protocol describes transmission electron microscope (TEM) modifications with a light illumination system, the fabrication of liquid cells, and in situ TEM observations of light-induced interactions between bacterial cells and a photosensitizer. The sample preparation methods, electron beam damage, and imaging are also discussed.

Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction

The current protocol describes the modifications of the transmission electron microscope (TEM) setup for in situ light-induced observations. A glass ...

The protocol presents how the combination of liquid cell TEM and light illumination can be used to observe the structural changes in the bacteria encapsulated with photosensitizer upon light illumination. The main advantage of this technique is its simpleness. The protocol does not require difficult sample preparation and provides sufficient encapsulation of bacteria with photosensitizer solution.To begin, modify the transmission electron microscope by connecting the optical fiber to the microscope column at the top of the objective lens. Allow a few centimeters of space between the objective lens and the condenser lens, plus a free slot for accessories. Place two untreated TEM grids covered with amorphous carbon between two crossed tweezers, facing the carbon coated side to the top.Hold the grids as close to the edge as possible. Put four microliters of bacteria solution on one of the grids. The optical density of the sample must be in the range of five to 10.Wait for 60 seconds, and carefully blot the liquid using filter paper. Try to spread the liquid throughout the entire grid surface during the process. Put four microliters of photosensitizer on the same grid.After five seconds, blot out the liquid. Leave a very thin layer of liquid on the substrate. Quickly place the dry grid on top of the one covered with liquid.Gently move the upper grid to the edge of the second one, and squeeze the substrates at the edges using tweezers. Carefully repeat the squeeze. Leave the liquid cell between the tweezers for one minute.Insert the sample into the TEM holder right after finishing the preparation. After inserting the sample into the microscope, start the observations in low magnification mode to find a suitable observation area. During the observations keep the electron dose as low as possible.Expand the exposure time. Find the cells surrounded by liquid at the appropriate magnification with extended exposure time, and capture the first image immediately. Keep the electron dose constant, and collect images every 30 seconds to observe the changes.Carefully examine the images and calculate the total electron dose that corresponds to the first visible changes in the cells. Before starting the experiment, set the laser light intensity by adjusting the current value. Insert the sample into the microscope and find the observation area.Capture the first image of the cell before starting illumination of the sample, and use the beam blanker to turn off the electron beam to avoid the effects of electron irradiation. Turn on the laser. Start with a short illumination time, as the laser light has relatively high intensity.After that time, turn off the light source. Turn off the beam blanker, and capture the image immediately. If possible, use an automatic algorithm to expose the sample only for the time needed for taking the image.Carefully measure the electron irradiation time to calculate the electron dose used for imaging. A notable degradation of the outer layer of the cell caused by the reactive oxygen species generated by lighter radiation of the photosensitizer after one minute is observed. Further light illumination made the changes more visible.Proper observation spots with enough liquid can be easily found at low magnification, as these areas appear darker and blurred. The liquid covered bacteria are less visible than the dry ones, but can still be distinguished. It's useful to look at the liquid boundary during the imaging, and its movement can be easily detected, which implies that the chosen area might be unsuitable and unstable.Another issue during observations is the evaporation of the water and precipitation of the solution which can occur in random areas of the sample, including the areas surrounding the bacteria. Encapsulation of bacteria can be also performed using graphene and silicon nitrate membranes. The sample preparation is more difficult, but the stability of the liquid cell will be better.The presented method can be used for observations of light-induced reactions on liquid. For example, the influence of light on metal materials, light-induced catalysts and photochemical reactions.

Research

JoVE Journal

Engineering

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