Name: conducting multiple imaging modes with one fluorescence microscope
Uploaded: 2018-10-28T13:00:00
Description: Watch this Scientific Journal Video about Conducting Multiple Imaging Modes with One Fluorescence Microscope at JoVE.com

J.F. acknowledges support from the Searle Scholars Program and the NIH Director&#39;s New Innovator Award. The authors acknowledge useful suggestions from Paul Selvin&#39;s lab (University of Illinois, Urbana-Champaign) for positioning the 3-D lens.

1. Microscope Design and Assembly
<ol>
	<li>Excitation path 
	NOTE: The excitation path includes lasers, differential interference contrast (DIC) components, the microscope body, and its illumination arm.
	<ol>
		<li>Prepare a vibration-isolated optical table. For example, a structural damping table of 48 x 96 x 12&#8217;&#8217; gives enough space for all the components. 
		NOTE: Build the set-up in a room with temperature control (e.g., 21.4 &#177; 0.55 &#176;C). Temperature stabili...

<ol>
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C., Jia, S., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabright+photoactivatable+fluorophores+created+by+reductive+caging.">Ultrabright photoactivatable fluorophores created by reductive caging.</a> Nature Methods. 9 (12), 1181-1184 (2012).</li><li>Ha, T., 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=Probing+the+interaction+between+two+single+molecules:+fluorescence+resonance+energy+transfer+between+a+single+donor+and+a+single+acceptor.">Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (13), 6264-6268 (1996).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nature Methods. 5 (6), 507-516 (2008).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Hua, 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=An+improved+surface+passivation+method+for+single-molecule+studies.">An improved surface passivation method for single-molecule studies.</a> Nature Methods. 11 (12), 1233-1236 (2014).</li><li>Fei, J., 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=RNA+biochemistry.+Determination+of+in+vivo+target+search+kinetics+of+regulatory+noncoding+RNA.">RNA biochemistry. Determination of in vivo target search kinetics of regulatory noncoding RNA.</a> Science. 347 (6228), 1371-1374 (2015).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nature Methods. 5 (10), 877-879 (2008).</li><li>Stracy, M., Kapanidis, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+and+super-resolution+imaging+of+transcription+in+living+bacteria.">Single-molecule and super-resolution imaging of transcription in living bacteria.</a> Methods. 120, 103-114 (2017).</li><li>Wang, S., Moffitt, J. R., Dempsey, G. T., Xie, X. S., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+development+of+photoactivatable+fluorescent+proteins+for+single-molecule-based+superresolution+imaging.">Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (23), 8452-8457 (2014).</li><li>Sekar, R. B., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+(FRET)+microscopy+imaging+of+live+cell+protein+localizations.">Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.</a> The Journal of Cell Biology. 160 (5), 629-633 (2003).</li><li>Shi, 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=Super-resolution+microscopy+reveals+that+disruption+of+ciliary+transition-zone+architecture+causes+Joubert+syndrome.">Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome.</a> Nature Cell Biology. 19 (10), 1178-1188 (2017).</li><li>Youn, Y., Ishitsuka, Y., Jin, C., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+nanoimprint+lithography+for+drift+correction+in+super-resolution+fluorescence+microscopy.">Thermal nanoimprint lithography for drift correction in super-resolution fluorescence microscopy.</a> Optics Express. 26 (2), 1670-1680 (2018).</li></ol>

This hybrid microscope system eliminates the need to purchase multiple microscopes. The total cost for all parts, including the optical table, table installation labor, software, and workstation, is about $230,000. Custom-machined parts, including the mag lens and 3-D lens, cost around $700 (the cost depends on the actual charges at different institutes). Typical commercially available integrated systems for single-molecule detection-based SR microscopy cost more than $300,000 ~ 400,000 and are not readily available for ...

Fluorescence microscopes are important tools for the modern biological science research and fluorescent imaging is routinely performed in many biology laboratories. By tagging biomolecules of interest with fluorophores, we can directly visualize them under the microscope and record the time-dependent changes in localization, conformation, interaction, and assembly state in vivo or in vitro. Conventional fluorescence microscopes have a diffraction-limited spatial resolution, which is ~200 - 300 nm in the lateral direction and ~500 - 700 nm in the axial direction1,2, and are, therefore, limited to imaging at the 100s of nanometers-to-micron scale. In order to reveal finer details in the molecular assembly or organization, various SR microscopies that can break the diffraction limit have been developed. Strategies used to achieve SR include non-linear optical effects, such as stimulated emission depletion (STED) microscopy3,4 and structured illumination microscopy (SIM)5,6,7, stochastic detection of single molecules, such as stochastic optical reconstruction microscopy (STORM)8 and photoactivated localization microscopy (PALM)9, and a combination of both, such as MINFLUX10. Among these SR microscopies, single-molecule detection-based SR microscopes can be relatively easily modified from a single-molecule microscope set-up. With repetitive activation and imaging of photoactivatable fluorescent proteins (FPs) or photo-switchable dyes tagged on biomolecules of interest, spatial resolution can reach 10 - 20 nm11. To gain information on molecular interactions and conformational dynamics in real-time, angstrom-to-nanometer resolution is required. smFRET12,13 is one approach to achieve this resolution. Generally, depending on the biological questions of interest, imaging methods with different spatial resolutions are needed.
Typically, for each type of imaging, specific excitation and/or emission optical configuration is needed. For instance, one of the most commonly used illumination methods for single-molecule detection is through total internal reflection (TIR), in which a specific excitation angle needs to be achieved either through a prism or through the objective lens. For smFRET detection, emissions from both donor and acceptor dyes need to be spatially separated and directed to different parts of the electron-multiplying, charge-coupled device (EMCCD), which can be achieved with a set of mirrors and dichroic beam splitters placed in the emission path. For three-dimensional (3-D) SR imaging, an optical component, such as a cylindrical lens14, is needed to cause an astigmatism effect in the emission path. Therefore, homebuilt or commercially available integrated microscopes are, usually, functionally specialized for each type of imaging method and are not flexible to switch between different imaging methods on the same set-up. Here we present a cost-effective, hybrid system that provides adjustable and reproducible switches between three different imaging methods: conventional epi-fluorescent imaging with diffraction-limited resolution, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging (Figure 1A). Specifically, the set-up presented here contains fiber-coupled input lasers for multi-color excitation and a commercial illumination arm in the excitation path, which allows programmed control of the excitation angle, to switch between epi-mode and TIR mode. In the emission path, a removable homebuilt cylindrical lens cassette is placed within the microscope body for 3-D SR imaging, and a commercial beam splitter is placed before an EMCCD camera that can be selectively enabled to detect multiple emission channels simultaneously.

<table>
	<tbody>
		<tr>
			<td>Nikon Ti-E microscope stand</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td>100X NA 1.49 CFI HP TIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy imaging software</td>
			<td>Nikon</td>
			<td>NIS-Elements Advanced Research/HC</td>
			<td>HC includes &#34;JOBS&#34; module, the programmed acquisition module being used for SR imaging.</td>
		</tr>
		<tr>
			<td>The illumination arm</td>
			<td>Nikon</td>
			<td>Ti-TIRF-EM Motorized Illuminator Unit M</td>
			<td>This arm has a slot for a magnification lens</td>
		</tr>
		<tr>
			<td>Analyze block</td>
			<td>Nikon</td>
			<td>Ti-A</td>
			<td>This is installed in the filter turret.</td>
		</tr>
		<tr>
			<td>Z-drift correction system</td>
			<td>Nikon</td>
			<td>PFS</td>
			<td>This system is composed by the stepmotor on the objective nosepiece, IR LED, and a detector.</td>
		</tr>
		<tr>
			<td>Optical table top</td>
			<td>TMC</td>
			<td>783-655-02R</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical table bases</td>
			<td>TMC</td>
			<td>14-426-35</td>
			<td></td>
		</tr>
		<tr>
			<td>647 nm laser</td>
			<td>Cobolt</td>
			<td>90346 (0647-06-01-0120-100)</td>
			<td>Modulated Laser Diode 647nm 120mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)</td>
		</tr>
		<tr>
			<td>561 nm laser</td>
			<td>Coherent</td>
			<td>1280721</td>
			<td>OBIS 561nm LS 150mW Laser System</td>
		</tr>
		<tr>
			<td>488 nm laser</td>
			<td>Cobolt</td>
			<td>90308 (0488-06-01-0060-100)</td>
			<td>Modulated Laser Diode 488nm 60mW incl. laser head, CDRH control box, USB cable and PSU (Power Supply Unit)</td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Crystalaser</td>
			<td>DL405-025-O</td>
			<td>405 (+/-5)nm, 25mW, Circular , M2 &#60;1.3, Low Noise, CW, TTL up to 20MHz. 2 BNC connectors for TTL &#38; Analog adjust</td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>Cobolt</td>
			<td>11658 (HS-03)</td>
			<td>Two units, Heat sink without fan HS-03, Heat sink for 647 nm and 488 nm lasers</td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>Coherent</td>
			<td>1193289</td>
			<td>Obis heat sink with fan, 165 x 50 x 50 mm for the 561 nm laser</td>
		</tr>
		<tr>
			<td>CAB-USB-miniUSB</td>
			<td>Cobolt</td>
			<td>10908</td>
			<td>Two units, communication cable for 647 nm and 488 nm lasers</td>
		</tr>
		<tr>
			<td>aluminum for height adjustment</td>
			<td>McMaster-Carr</td>
			<td>9146T35</td>
			<td>Multipurpose 6061 Aluminum, Rectangular Bar, 4MM X 40MM, 1&#39; Long for raising 561 nm laser</td>
		</tr>
		<tr>
			<td>aluminum for height adjustment</td>
			<td>McMaster-Carr</td>
			<td>8975K248</td>
			<td>Multipurpose 6061 Aluminum, 1-1/4&#34; Thick X 3&#34; Width X 1&#39; Length for raising 405 nm laser</td>
		</tr>
		<tr>
			<td>BNC cable</td>
			<td>L-com</td>
			<td>CC58C-6</td>
			<td>RG58C Coaxial Cable, BNC Male / Male, 6.0 ft</td>
		</tr>
		<tr>
			<td>BNC adapter</td>
			<td>L-com</td>
			<td>BA1087</td>
			<td>Coaxial Adapter, BNC Bulkhead, Grounded</td>
		</tr>
		<tr>
			<td>SMA to BNC Adapter</td>
			<td>HOD</td>
			<td>SMA-870</td>
			<td>Cobolt MLD lasers have SMA interface, so this adapter is used for BNC connection.</td>
		</tr>
		<tr>
			<td>SMB to BNC Adapter</td>
			<td>Fairview Microwave</td>
			<td>FMC1638316-12</td>
			<td>SMB Plug to BNC Female Bulkhead Cable RG316 Coax in 12 Inch for Coherent Obis lasers</td>
		</tr>
		<tr>
			<td>Data Acquisition Card</td>
			<td>National Instruments</td>
			<td>PCI-6723</td>
			<td>13-Bit, 32 Channels, 800 kS/s Analog Output Device for controlling lasers, DIC LED, and etc</td>
		</tr>
		<tr>
			<td>Barrier Filter Wheel controller</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-B</td>
			<td>Optical Filter Changer</td>
		</tr>
		<tr>
			<td>Emission Splitter</td>
			<td>Cairn</td>
			<td>OptoSplit III</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>T640LPXR-UF2</td>
			<td>Dichroic beamsplitter separating red emission from green emission in OptoSplit III</td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>T565LPXR-UF2</td>
			<td>Dichroic beamsplitter separating green &#38; red emission from blue emission in OptoSplit III</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET700/75M</td>
			<td>Two units, Emission filter for red emission (like Alexa Fluor 647) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET595/50M</td>
			<td>Two units, Emission filter for yellow/green emission (like Cy3B) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Chroma</td>
			<td>ET525/50M</td>
			<td>Two units, Emission filter for blue emission(like Alexa Fluor 488/GFP) in OptoSplit III as well as in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Emission filter</td>
			<td>Semrock</td>
			<td>FF02-447/60-25</td>
			<td>Emission filter for violet emission (like DAPI/Alexa Fluor 405), installed in the Barrier filter wheel</td>
		</tr>
		<tr>
			<td>Dichroic beamsplitter</td>
			<td>Chroma</td>
			<td>zt405/488/561/647/752rpc-UF3</td>
			<td>Multiband dichroic beam splitter for 647, 561, 488, and 405 nm laser excitations inside of the microscope body</td>
		</tr>
		<tr>
			<td>DAPI Filter set</td>
			<td>Chroma</td>
			<td>49000</td>
			<td>installed in the microscope body</td>
		</tr>
		<tr>
			<td>Nikon laser/TIRF filtercube</td>
			<td>Chroma</td>
			<td>91032</td>
			<td></td>
		</tr>
		<tr>
			<td>590 long pass filter</td>
			<td>Chroma</td>
			<td>T590LPXR-UF1</td>
			<td>for combining 647 nm laser and 561 nm laser</td>
		</tr>
		<tr>
			<td>525 long pass filter</td>
			<td>Chroma</td>
			<td>T525LPXR-UF1</td>
			<td>for combining already combined 647 nm and 561nm lasers with 488 nm laser</td>
		</tr>
		<tr>
			<td>470 long pass filter</td>
			<td>Chroma</td>
			<td>T470LPXR-UF1</td>
			<td>for combining already combined 647 nm, 561 nm and 488 nm lasers with 405 nm laser</td>
		</tr>
		<tr>
			<td>Laser clean-up filter (647)</td>
			<td>Chroma</td>
			<td>zet640/20x</td>
			<td>for cleaning up other wavelengths from the 647 nm laser</td>
		</tr>
		<tr>
			<td>Laser clean up filter (488)</td>
			<td>Semrock</td>
			<td>LL01-488-25</td>
			<td>for cleaning up other wavelengths from the 488 nm laser</td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>Excelitas</td>
			<td>X-Cite120LED</td>
			<td>used only for DAPI imaging</td>
		</tr>
		<tr>
			<td>Mirror mount</td>
			<td>Newport</td>
			<td>SU100-F3K</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical posts</td>
			<td>Newport</td>
			<td>PS-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping fork</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Meter</td>
			<td>Newport</td>
			<td>PMKIT</td>
			<td>For measuring laser power</td>
		</tr>
		<tr>
			<td>Dichroic beamcombiner mount</td>
			<td>Edmund Optics</td>
			<td>58-872</td>
			<td>C-Mount Kinematic Mount, for holding dichroic beamcombiners in the laser excitation assembly</td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs</td>
			<td>CMRR</td>
			<td>used for dichroic beamcombiner mounts</td>
		</tr>
		<tr>
			<td>Fiber Adapter Plate</td>
			<td>Thorlabs</td>
			<td>SM1FC</td>
			<td>FC/PC Fiber Adapter Plate with External SM1 (1.035&#34;-40) Thread</td>
		</tr>
		<tr>
			<td>Z-axis translational mount</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>Z-Axis Translation Mount, 30 mm Cage Compatible</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens</td>
			<td>Thorlabs</td>
			<td>AC050-008-A-ML</td>
			<td>&#216;5 mm, Mounted Achromatic Doublets, AR Coated: 400 - 700 nm</td>
		</tr>
		<tr>
			<td>Cage Plate</td>
			<td>Thorlabs</td>
			<td>CP1TM09</td>
			<td>30 mm Cage Plate with M9 x 0.5 Internal Threads, 8-32 Tap</td>
		</tr>
		<tr>
			<td>Cage Assembly Rod</td>
			<td>Thorlabs</td>
			<td>ER4</td>
			<td>Cage Assembly Rod, 4&#34; Long, &#216;6 mm</td>
		</tr>
		<tr>
			<td>Cage Mounting Bracket</td>
			<td>Thorlabs</td>
			<td>CP02B</td>
			<td>30 mm Cage Mounting Bracket</td>
		</tr>
		<tr>
			<td>Single mode optical fiber</td>
			<td>Thorlabs</td>
			<td>P5-405BPM-FC-2</td>
			<td>Patch Cable, PM, FC/PC to FC/APC, 405 nm, Panda, 2 m</td>
		</tr>
		<tr>
			<td>Multi mode optical fiber</td>
			<td>Thorlabs</td>
			<td>M42L01</td>
			<td>&#216;50 &#181;m, 0.22 NA, FC/PC-FC/PC Fiber Patch Cable, 1 m</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens (mag lens)</td>
			<td>Thorlabs</td>
			<td>ACN127-025-A</td>
			<td>ACN127-025-A - f=-25.0 mm, &#216;1/2&#34; Achromatic Doublet, ARC: 400-700 nm , a concave lens in the &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Achromatic Doublet lens (mag lens)</td>
			<td>Thorlabs</td>
			<td>AC127-050-A</td>
			<td>f=50.0 mm, &#216;1/2&#34; Achromatic Doublet, ARC: 400-700 nm, a convex lens in the &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Retaining ring</td>
			<td>Thorlabs</td>
			<td>SM05PRR</td>
			<td>SM05 Plastic Retaining Ring for &#216;1/2&#34; Lens Tubes and Mounts, for &#34;mag lens&#34;</td>
		</tr>
		<tr>
			<td>Nylon-tipped screw</td>
			<td>Thorlabs</td>
			<td>SS3MN6</td>
			<td>M3 x 0.5 Nylon-Tipped Setscrew, 6 mm Long, for holding &#34;3D lens&#34;</td>
		</tr>
		<tr>
			<td>3D lens</td>
			<td>CVI Laser Optics</td>
			<td>RCX-25.4-50.8-5000.0-C-415-700</td>
			<td>f=10 m, rectangular cylindrical lens</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon Ultra 888</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm multichannel beads</td>
			<td>Thermo</td>
			<td>T7279, TetraSpeck microspheres</td>
			<td></td>
		</tr>
		<tr>
			<td>red dye</td>
			<td>Thermo</td>
			<td>Alexa Fluor 647</td>
			<td></td>
		</tr>
		<tr>
			<td>yellow-green dye</td>
			<td>GE Healthcare</td>
			<td>Cy3</td>
			<td></td>
		</tr>
		<tr>
			<td>green dye</td>
			<td>GE Healthcare</td>
			<td>Cy3B</td>
			<td></td>
		</tr>
		<tr>
			<td>blue dye</td>
			<td>Thermo</td>
			<td>Alexa Fluor 488</td>
			<td></td>
		</tr>
	</tbody>
</table>

conducting multiple imaging modes with one fluorescence microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

This microscope allows flexible and reproducible switching between different imaging methods. Here we show sample images collected with each imaging module.
Figure 5D demonstrates the PSF of the blinking-on molecule during the SR acquisition. Thousands of such images are reconstructed to generate the final SR image (Figure 5E). Figure 5E sh...

Watch this Scientific Journal Video about Conducting Multiple Imaging Modes with One Fluorescence Microscope at JoVE.com

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

This method can help answer key questions in the optical microscopy field, such as how to take super-resolution images as well as FRET images using the same microscope. The main advantage of this technique is that we could combine three different imaging modules into one microscope, significantly reducing the overall cost. Visual demonstration of this method is critical as assembly of the excitation path is difficult to learn.They require proper choice of parts and a sensitive optical alignments. To begin, install a data acquisition card through a PCI interface and use it to connect the lasers to the computer. Control the lasers'on and off behaviors by transistor-transistor logic output and their power adjustment by the analog output of this card.Next, prepare a vibration-isolated optical table with mirrors and beam splitters as shown here and described in the accompanying text protocol. Combine the laser beams into a single-mode optical fiber by first mounting a fiber adaptor plate in a z-axis translation mount. Next, mount an achromatic doublet lens in a cage plate.Use an extension rod to connect the adapter and lens to form a cage. Then, use one-inch thick optical posts to mount the cage on the optical table. Align the 647-nanometer laser by adjusting the components to gain maximum laser power output through the fiber.Once the alignment of the first laser is done, temporarily install a pair of irises and align the rest of the lasers one by one. Check the alignment efficiency of each laser with a power meter. Be sure to leave one iris in front of the adapter plate to reduce the reflections of the lasers.Next, design and install the magnification lens as described in the accompanying text protocol. In order to create the astigmatism effect necessary for extracting the z-coordinates of every single molecule, place a 3D lens with a 10-meter focal length into the cassette and insert it into the emission beam path. To minimize the vibrations during sequential multicolor epifluorescence imaging, use emission filters placed in a barrier filter wheel connected next to the microscope.For simultaneous multicolor detection during single-molecule FRET experiments, place another filter set in an emission splitter. To set up for diffraction-limited imaging using epi-excitation, first adjust the excitation laser's incidental angle to epi mode in the illumination arm. Next, disengage the 3D lens, and insert the bypass cube into the emission splitter.Then, insert the mag lens for broadened illumination. Once set up, take multi-channel, z-stack, and/or time lapsed images of your sample based on your desired results. To set up multicolor single-molecule detection of surface-immobilized molecules, first move the filter wheel to an empty position.This will allow the lasers to pass through. Then, adjust the excitation lasers'incidental angle to the turf angle, and disengage both the mag and 3D lenses. Next, engage the three-channel mode in the emission splitter by first replacing the bypass cube with a calibration cube that allows all of the light to pass through all channels.Then, turn on the camera under DIC and adjust the aperture of the emission splitter until three, fully separated channels appear on the screen. Turn the vertical-horizontal adjustment control knobs on the emission splitter, and roughly align the three channels. Next, turn off the camera and replace the calibration cube with the triple cube.Place a sample of 100-nanometer multi-channel beads. Upon excitation at 488 nanometers, the 100-nanometer multi-channel beads emit different wavelengths of light, enabling three-channel alignment. Then, turn on the camera and the 488-nanometer laser, zoom in on one of the bright beads, and finally align the three channels by turning the adjustment control knobs again.With the sample now in place, using a weak laser, navigate to an area with a reasonable spot density and adjust the laser power and exposure time to achieve acceptable signal-to-noise and photobleaching levels. Then use the imaging software to take time-lapse images. For super-resolution imaging, begin by inserting the 3D lens and remove the mag lens.Then determine the optimal excitation laser's incidental angle to be the turf angle. In order to find the proper objective height for SR imaging, use DIC imaging to find the middle plane of the cells. Identify the plane by the height at which the cells become transparent.Once the desired focal plane is determined, begin super-resolution imaging. While imaging, change the 405-nanometer laser's power to maintain a reasonable density of blinking-on spots. Begin imaging without violet laser power.Count the number of blinking-on spots in a certain period and increase the violet laser power so that the number of blinking-on spots is kept above a user-defined counting threshold in the field of view. Analyze the data by detecting the centroids of each spot in the imaging frames and extract z-values for each spot from X and Y widths. Build a reconstructed image and visualize objects in 3D.This microscope setup allows for flexible and reproducible switching between different imaging methods, including conventional epifluorescent imaging, single-molecule detection-based super-resolution imaging, and multicolor single-molecule detection. In order to reveal finer details in the molecular assembly, super-resolution microscopy combines thousands of images such as this one. These images are then reconstructed to generate a final super-resolution image.Super-resolution techniques allow for high spatial resolution, providing details that can't be seen with other techniques. This is highlighted in the two images shown here. The super-resolution image shows the same bacterial regulatory RNase as the epifluorescence image but allows for single molecule detection.SmFRET is another method capable of angstrom to nanometer resolution. Here, folded RNA molecules were labeled with a green donor dye and a red acceptor dye. Fluorescence intensity trajectories can be extracted from individual single molecules, generating FRET efficiency as a function of time.Don't forget that working with lasers can be hazardous, and precautions such as wearing eye protection or lowering laser powers should always be taken where performing this procedure.

Research

JoVE Journal

Bioengineering

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...