Name: design and building of a customizable, single-objective, light-sheet fluorescence microscope for the visualization of cytoskeleton networks
Uploaded: 2024-01-26T12:50:00.000+00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks at JoVE.com

This work was supported by the National Science Foundation (NSF) RUI Award (DMR-2203791) to J.S. We are grateful for the guidance provided by Dr. Bin Yang and Dr. Manish Kumar during the alignment process. We thank Dr. Jenny Ross and K. Alice Lindsay for the preparation instructions for the kinesin motors.

Customizable Fluorescence Microscope to Visualize Cytoskeleton Networks

<ol>
	<li>Girkin, J. M., Carvalho, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light-sheet+microscopy+revolution.">The light-sheet microscopy revolution.</a> Journal Optics. 20 (5), 053002 (2018).</li><li>You, R., McGorty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy+illuminating+soft+matter.">Light sheet fluorescence microscopy illuminating soft matter.</a> Frontiers in Physics. 9, 760834 (2021).</li><li>Fuchs, E., Jaffe, J. S., Long, R. A., Azam, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+laser+light+sheet+microscope+for+microbial+oceanography.">Thin laser light sheet microscope for microbial oceanography.</a> Optics Express. 10 (2), 145-154 (2002).</li><li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Dunsby, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+sectioned+imaging+by+oblique+plane+microscopy.">Optically sectioned imaging by oblique plane microscopy.</a> Optics Express. 16 (25), 20306-20316 (2008).</li><li>Bouchard, M. 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=Swept+confocally-aligned+planar+excitation+(SCAPE)+microscopy+for+high-speed+volumetric+imaging+of+behaving+organisms.">Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms.</a> Nature Photonics. 9 (2), 113-119 (2015).</li><li>Smith, C. W., Botcherby, E. J., Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+oblique-plane+images+in+sectioning+microscopy.">Resolution of oblique-plane images in sectioning microscopy.</a> Optics Express. 19 (3), 2662-2669 (2011).</li><li>Yang, 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=Epi-illumination+SPIM+for+volumetric+imaging+with+high+spatial-temporal+resolution.">Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.</a> Nature Methods. 16 (6), 501-504 (2019).</li><li>Wu, 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=Simultaneous+multiview+capture+and+fusion+improves+spatial+resolution+in+wide-field+and+light-sheet+microscopy.">Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.</a> Optica. 3 (8), 897-910 (2016).</li><li>Sahasrabudhe, A., Vittal, V., Ghose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peeping+in+on+the+cytoskeleton:+Light+microscopy+approaches+to+actin+and+microtubule+organization.">Peeping in on the cytoskeleton: Light microscopy approaches to actin and microtubule organization.</a> Current Science. 105 (11), 1562-1570 (2013).</li><li>Kumar, M., Kishore, S., Nasenbeny, J., McLean, D. L., Kozorovitskiy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+one-+and+two-photon+scanned+oblique+plane+illumination+(SOPi)+microscopy+for+rapid+volumetric+imaging.">Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging.</a> Optics Express. 26 (10), 13027-13041 (2018).</li><li>Kim, 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=Oblique-plane+single-molecule+localization+microscopy+for+tissues+and+small+intact+animals.">Oblique-plane single-molecule localization microscopy for tissues and small intact animals.</a> Nature Methods. 16 (9), 853-857 (2019).</li><li>Sapoznik, 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=A+versatile+oblique+plane+microscope+for+large-scale+and+high-resolution+imaging+of+subcellular+dynamics.">A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.</a> eLife. 9, e57681 (2020).</li><li>Bernardello, M., Marsal, M., Gualda, E. J., Loza-Alvarez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sheet+fluorescence+microscopy+for+the+in+vivo+study+of+microtubule+dynamics+in+the+zebrafish+embryo.">Light-sheet fluorescence microscopy for the in vivo study of microtubule dynamics in the zebrafish embryo.</a> Biomedical Optics Express. 12 (10), 6237-6254 (2021).</li><li>Shelden, E. A., Colburn, Z. T., Jones, J. C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+super+resolution+on+the+cytoskeleton.">Focusing super resolution on the cytoskeleton.</a> F1000Res. 5, (2016).</li><li>Wulstein, D. M., Regan, K. E., Garamella, J., McGorty, R. J., Robertson-Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology-dependent+anomalous+dynamics+of+ring+and+linear+DNA+are+sensitive+to+cytoskeleton+crosslinking.">Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.</a> Science Advances. 5 (12), (2019).</li><li>Sheung, J. Y., Garamella, J., Kahl, S. K., Lee, B. Y., McGorty, R. J., Robertson-Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor-driven+advection+competes+with+crowding+to+drive+spatiotemporally+heterogeneous+transport+in+cytoskeleton+composites.">Motor-driven advection competes with crowding to drive spatiotemporally heterogeneous transport in cytoskeleton composites.</a> Frontiers in Physics. 10, 1055441 (2022).</li><li>Zeiss Lightsheet 7. Light-Sheet Multiview Imaging of Living and Cleared Specimens. Zeiss Available from: <a target="_blank" href="https://www.zeiss.com/microscopy/en/products/light-microscopes/light-sheet-microscopes/lightsheet-7.html">https://www.zeiss.com/microscopy/en/products/light-microscopes/light-sheet-microscopes/lightsheet-7.html</a> (2023)</li><li>Zeiss Lattice Lightsheet 7. Long-Term Volumetric Imaging of Living Cells. Zeiss Available from: <a target="_blank" href="https://www.zeiss.com/microscopy/en/products/light-microscopes/light-sheet-microscopes/lattice-lightsheet-7.html">https://www.zeiss.com/microscopy/en/products/light-microscopes/light-sheet-microscopes/lattice-lightsheet-7.html</a> (2023)</li><li>Kumar, M., Kishore, S., McLean, D. L., Kozorovitskiy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossbill:+An+open+access+single+objective+light-sheet+microscopy+platform.">Crossbill: An open access single objective light-sheet microscopy platform.</a> bioRxiv. , (2021).</li><li>Olarte, O. E., Andilla, J., Gualda, E. J., Loza-Alvarez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sheet+microscopy:+A+tutorial.">Light-sheet microscopy: A tutorial.</a> Advances in Optics and Photonics. 10 (1), 111-179 (2018).</li><li>Pitrone, P. G., 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=OpenSPIM:+An+open-access+light-sheet+microscopy+platform.">OpenSPIM: An open-access light-sheet microscopy platform.</a> Nature Methods. 10 (7), 598-599 (2013).</li><li>Single Objective Light Sheet Microscope (SOLS) Guide. Sheung Lab Available from: <a target="_blank" href="https://sites.google.com/view/sheunglab/microscopy/single-objective-light-microscope-sols">https://sites.google.com/view/sheunglab/microscopy/single-objective-light-microscope-sols</a> (2023)</li><li>Lamb, J. R., Ward, E. N., Kaminski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+software+package+for+on-the-fly+deskewing+and+live+viewing+of+volumetric+lightsheet+microscopy+data.">Open-source software package for on-the-fly deskewing and live viewing of volumetric lightsheet microscopy data.</a> Biomedical Optics Express. 14 (2), 834-845 (2023).</li><li>. Harvard University. Mitchison Lab Available from: <a target="_blank" href="https://mitchison.hms.harvard.edu/home">https://mitchison.hms.harvard.edu/home</a> (2023)</li><li>Watkins, S. C., St. Croix, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+imaging+comes+of+age.">Light sheet imaging comes of age.</a> Journal of Cell Biology. 217 (5), 1567-1569 (2018).</li><li>Ndlec, F. J., Surrey, T., Maggs, A. C., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+microtubules+and+motors.">Self-organization of microtubules and motors.</a> Nature. 389 (6648), 305-308 (1997).</li><li>Sanchez, T., Chen, D. T. N., DeCamp, S. J., Heymann, M., Dogic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+motion+in+hierarchically+assembled+active+matter.">Spontaneous motion in hierarchically assembled active matter.</a> Nature. 491 (7424), 431-434 (2012).</li><li>Berezney, J., Goode, B. L., Fraden, S., Dogic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensile+to+contractile+transition+in+active+microtubule-actin+composites+generates+layered+asters+with+programmable+lifetimes.">Extensile to contractile transition in active microtubule-actin composites generates layered asters with programmable lifetimes.</a> Proceedings of the National Academy of Sciences of the United States of America. 119 (5), e2115895119 (2022).</li><li>Kim, 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=Isomorphic+coalescence+of+aster+cores+formed+in+vitro+from+microtubules+and+kinesin+motors.">Isomorphic coalescence of aster cores formed in vitro from microtubules and kinesin motors.</a> Physical Biology. 13 (5), 056002 (2016).</li><li>Millett-Sikking, A., 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=.">.</a> High NA single-objective lightsheet. , (2019).</li><li>Bourgenot, C., Saunter, C. D., Taylor, J. M., Girkin, J. M., Love, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+adaptive+optics+in+a+light+sheet+microscope.">3D adaptive optics in a light sheet microscope.</a> Optics Express. 20 (12), 13252-13261 (2012).</li><li>Bernardello, M., Gualda, E. J., Loza-Alvarez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+multimodal+platform+for+classical+and+high+throughput+light+sheet+microscopy.">Modular multimodal platform for classical and high throughput light sheet microscopy.</a> Scientific Reports. 12 (1), 1969 (2022).</li><li>Crombez, S., Leclerc, P., Ray, C., Ducros, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+hyperspectral+light-sheet+microscopy.">Computational hyperspectral light-sheet microscopy.</a> Optics Express. 30 (4), 4856-4866 (2022).</li></ol>

The authors have nothing to disclose. All research was conducted in the absence of commercial or financial relationships that could be interpreted as a conflict of interest.

Two important details regarding this protocol are the overall cost of the system and the expected build and alignment time. Although the exact cost is variable, we can comfortably estimate that the in toto cost of this SOLS or a similar DIY system would fall in the range of $85,000 USD. We note that this estimate considers the retail price of all the components, so this overall price may be greatly reduced by sourcing used components. In terms of the build time, it would be reasonable to expect a user with little optics experience to build and align this entire SOLS system within 1-2 months, provided that all of the components are available and ready. Despite the length and complexity of the protocol, we believe that the amount of detail in the written manuscript, paired with the video protocol, should make this protocol straightforward and fast to follow.
There are two critical steps in this protocol. First, the placement of the galvo determines the placement of many lenses as it is part of three separate 4f lens pairs. It is crucial that the galvo is both conjugated with the back focal planes of O1 and O2 and centered correctly to ensure tilt-invariant scanning. Second, the image quality is extremely sensitive to the alignment of O2 and O3 with respect to each other. Here, care must be taken to ensure that, first, the alignment angle of O3 to O2 matches the tilt of the excitation light sheet, thus providing maximally flat illumination across the similarly tilted FoV. Second, O3 must be placed at the correct axial distance to maintain a flat FoV with as large an area as possible. Third, O3 must be placed at the correct lateral distance from O2 to maximize the signal that passes through the O2-O3 interface.

In terms of the usable FoV, this system achieved a flat, reliable field with consistent illumination across an 80 &#181;m x 80 &#181;m area. This area is smaller than the maximum FoV provided by the camera, so the usable FoV is indicated by the yellow box in Figure 13. In terms of the resolving power, this system achieved a minimum resolvable distance of 432 nm along the x-axis and 421 nm along the y-axis, which was measured by finding the average sigma x and y of Gaussian fits to point spread functions (PSFs) in the good FoV and multiplying by two. We note that this system was not optimized in terms of its total NA, meaning there is room for significant improvement if users desire a resolving power higher than what this system achieved. There are a multitude of compatible objective options for this type of SOLS build, many of which would contribute to a higher system resolution but with the drawbacks of a higher cost, a smaller FoV, or more complicated alignment techniques at the relay interface8,11,13,20. Separately, should users desire a larger FoV, incorporateing a second galvo to allow for 2D scanning would achieve this goal but would require additional optics and control mechanics to be integrated into the design32. We have provided more detail regarding modifications to the system on our website page, alongside links to other helpful resources regarding the design process23.

Beyond improving the specific components for this particular design, it would be very feasible to add other high-resolution microscopy techniques or modalities to this build. One such improvement would be to incorporate multi-wavelength illumination, which would involve aligning additional excitation lasers to the original excitation path8. Furthermore, because this type of SOLS design leaves the sample accessible, adding additional functions to the microscope, including but limited to optical tweezing, microfluidics, and rheometry, is relatively straightforward2,33.

Compared to the myriad light-sheet guides that have been published, this protocol provides instructions at a level of understanding that a user without significant optics experience may find helpful. By making a user-friendly SOLS build with traditional sample slide mounting capabilities accessible to a larger audience, we hope to enable an even further expansion of the applications of SOLS-based research in all fields in which the instrument has or could be utilized. Even with the applications of SOLS instruments rapidly growing in number2,34,35, we believe that many benefits and utilizations of SOLS-type instruments still remain unexplored and express excitement at the possibilities for this type of instrument moving forward.

Light-sheet fluorescence microscopy (LSFM) represents a family of high-resolution fluorescence imaging techniques in which the excitation light is shaped into a sheet1,2, including selective plane illumination microscopy (SPIM), swept confocally-aligned planar excitation (SCAPE), and oblique-plane microscopy (OPM)3,4,5,6,7. Unlike other microscopy modalities such as epi-fluorescence, total internal reflection fluorescence microscopy (TIRFM), or confocal microscopy, phototoxicity is minimal in LSFM and samples can be imaged over longer timescales because only the plane of the sample being actively imaged is illuminated8,9,10. Therefore, LSFM techniques are extremely useful for imaging 3D samples over extended time periods, notably even those too thick for confocal microscopy techniques. Due to these reasons, since its original development in 2004, LSFM has become the imaging technique of choice for many physiologists, developmental biologists, and neuroscientists for the visualization of entire organisms such as live zebrafish and Drosophila embryos3,4,6,11. In these last two decades, the advantages of LSFM have been leveraged to visualize structure and dynamics at progressively smaller scales, including the tissue11,12, cellular, and subcellular scales, both in vivo and in vitro13,14,15,17.
Despite the reports of successful use cases in the literature, the high cost of commercial LSFM systems (~0.25 million USD as of the time of writing)18,19 prevents the widespread use of the technique. To make DIY builds a feasible alternative for researchers, multiple build guides have been published8,13,20,21, including the open-access effort OpenSPIM22. However, to date, researchers with minimal optics experience can only use earlier LSFM designs, which are incompatible with traditional slide-mounted samples (Figure 1A). Recent single-objective, light-sheet (SOLS) implementation use a single objective for both the excitation and detection (Figure 1C), thereby overcoming the limitation related to compatibility5,6,8,13,20. However, the cost for the versatility of the SOLS design is a substantial increase in the complexity of the build due to the requirement of two additional objectives to relay, de-tilt, and reimage the object plane onto the camera for imaging (Figure 1D). To facilitate access to the complex SOLS-style setups, this paper presents step-by-step guide on the design, build, alignment process, and use of a slide-compatible SOLS system, which would be useful to researchers with knowledge of only an entry-level optics course.
Although the protocol itself is succinct, readers must refer to other resources during the preparation steps to learn more about particular parts of the design or hardware considerations. However, if a reader intends to follow the specifications of this design, it may not be necessary to understand how to select particular optical components.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65411/65411fig01.jpg" /> 
Figure 1: Characteristics of different LSFM configurations. (A) The setup with two orthogonal objectives common in early LSFM designs. In this configuration, a capillary tube or a cylinder of gel is used to contain the sample, which is not compatible with traditional slide mounting techniques. (B) A schematic of an SOLS light-sheet design showing the following: (C) the single objective used for both the excitation and emission collection at the sample plane (O1); this allows a traditional slide to be mounted on top, and (D) the relay objective system in the SOLS emission path. O2 collects the emission light and demagnifies the image. O3 images the plane at the correct tilt angle onto the camera sensor. Abbreviations: LSFM = light-sheet fluorescence microscopy; SOLS = single-objective light-sheet; O1-O3 = objectives. <a href="https://www.jove.com/files/ftp_upload/65411/65411fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>1&quot; Plano-Concave Lens f = -50 mm</td><td>Thorlabs&amp;nbsp;</td><td>LC1715-A-ML</td><td>For alignment laser&lt;br/&gt; Estimated Cost: $49.5</td></tr><tr><td>1&quot; Achromatic Doublet f = 100 mm (x3)</td><td>Thorlabs</td><td>AC254-100-A-ML</td><td>L2, L4 and alignment laser&lt;br/&gt; Estimated Cost: $342.42</td></tr><tr><td>1&quot; Achromatic Doublet f = 125 mm</td><td>Thorlabs</td><td>AC254-125-A-ML</td><td>SL2&lt;br/&gt; Estimated Cost: $114.14</td></tr><tr><td>1&quot; Achromatic Doublet f = 150 mm</td><td>Thorlabs</td><td>AC254-150-A-ML</td><td>L3&lt;br/&gt; Estimated Cost: $114.14</td></tr><tr><td>1&quot; Achromatic Doublet f = 150 mm</td><td>Thorlabs</td><td>AC254-150-A-ML</td><td>TL2&lt;br/&gt; Estimated Cost: $114.14</td></tr><tr><td>1&quot; Achromatic Doublet f = 45 mm</td><td>Thorlabs</td><td>AC254-045-A-ML</td><td>L1&lt;br/&gt; Estimated Cost: $114.14</td></tr><tr><td>1&quot; Achromatic Doublet f = 75 mm</td><td>Thorlabs</td><td>AC254-075-A-ML</td><td>SL1&lt;br/&gt; Estimated Cost: $114.14</td></tr><tr><td>1&quot; Cylindrical Lens f = 100 mm</td><td>Thorlabs</td><td>LJ1567RM</td><td>CL3&lt;br/&gt; Estimated Cost: $117.62</td></tr><tr><td>1&quot; Cylindrical Lens f = 200 mm</td><td>Thorlabs</td><td>LJ1653RM</td><td>CL2&lt;br/&gt; Estimated Cost: $111.22</td></tr><tr><td>1&quot; Cylindrical Lens f = 50 mm</td><td>Thorlabs</td><td>LJ1695RM</td><td>CL1&lt;br/&gt; Estimated Cost: $117.62</td></tr><tr><td>1&quot; Mounted Pinhole, 30 &amp;micro;m Pinhole Diameter</td><td>Thorlabs</td><td>P30K</td><td>Estimated Cost: $77.08</td></tr><tr><td>1&quot; Silver Mirror (x3)</td><td>Thorlabs</td><td>PF10-03-P01</td><td>M1, M2, one for alignment&lt;br/&gt; Estimated Cost: $168.78</td></tr><tr><td>2&quot; Elliptical Mirror</td><td>Thorlabs</td><td>PFE20-P01</td><td>M3&lt;br/&gt; Estimated Cost: $179.98</td></tr><tr><td>2&quot; Post Holder (x11)</td><td>Thorlabs</td><td>PH2</td><td>For custom laser mount, ND wheel, safety screens&lt;br/&gt; Estimated Cost: $98.45</td></tr><tr><td>2&quot; Posts (x47)</td><td>Thorlabs</td><td>TR2</td><td>For custom laser mount and optical components&lt;br/&gt; Estimated Cost: $277.3</td></tr><tr><td>3&quot; Posts (x4)&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>TR3</td><td>For M3 supports and other mounts&lt;br/&gt; Estimated Cost: $24.6</td></tr><tr><td>3&quot; Post Holder (x4)</td><td>Thorlabs&amp;nbsp;</td><td>PH3</td><td>Estimated Cost: $38.48</td></tr><tr><td>30 to 60 mm Cage Adapter&amp;nbsp;</td><td>Thorlabs</td><td>LCP33</td><td>To mount O1&lt;br/&gt; Estimated Cost: $45.42</td></tr><tr><td>30mm Cage Filter Wheel</td><td>Thorlabs</td><td>CFW6</td><td>To mount ND filters&lt;br/&gt; Estimated Cost: $172.36</td></tr><tr><td>30mm Cage Plate (x6)</td><td>Thorlabs</td><td>CP33</td><td>To build alignment cage and alignment laser&lt;br/&gt; Estimated Cost: $114.54</td></tr><tr><td>30mm Right-Angle Kinematic Mirror Mount (x3)</td><td>Thorlabs</td><td>KCB1</td><td>To mount M1 and M2 and for alignment laser&lt;br/&gt; Estimated Cost: $463.95</td></tr><tr><td>4&quot; Post Holder (x30)</td><td>Thorlabs</td><td>PH4</td><td>Estimated Cost: $320.1</td></tr><tr><td>561 nm Laser and Power Supply&amp;nbsp;</td><td>Opto Engine LLC</td><td>MGL-FN-561-100mW</td><td>Excitation laser&lt;br/&gt; Estimated Cost: $6000</td></tr><tr><td>60mm Cage Plate (x2)</td><td>Thorlabs</td><td>LCP01</td><td>To mount TL1 and M3 mount&lt;br/&gt; Estimated Cost: $88.52</td></tr><tr><td>60mm Right-Angle Kinematic Mirror Mount</td><td>Thorlabs</td><td>KCB2</td><td>To mount M3&lt;br/&gt; Estimated Cost: $187.26</td></tr><tr><td>90&amp;deg; Flip Mount</td><td>Thorlabs&amp;nbsp;</td><td>TRF90</td><td>For alignment laser&lt;br/&gt; Estimated Cost: $95.5</td></tr><tr><td>Adapter with External C-Mount Threads and Internal SM1 Threads</td><td>Thorlabs&amp;nbsp;</td><td>SM1A9</td><td>To connect lens tube to camera&lt;br/&gt; Estimated Cost: $20.96</td></tr><tr><td>Adapter with External SM1 Threads and Internal C-Mount Threads</td><td>Thorlabs&amp;nbsp;</td><td>SM1A10</td><td>To connect tube lens to lens mount&lt;br/&gt; Estimated Cost: $21.82</td></tr><tr><td>Adapter with External SM1 Threads and Internal M25 Threads (x2)</td><td>Thorlabs</td><td>SM1A12</td><td>To mount O1 and O2&lt;br/&gt; Estimated Cost: $47.06</td></tr><tr><td>Adapter with External SM1 Threads and Internal M26 Threads</td><td>Thorlabs</td><td>SM1A27</td><td>To mount O3&lt;br/&gt; Estimated Cost: $22.38</td></tr><tr><td>Alignment Disk</td><td>Thorlabs&amp;nbsp;</td><td>SM1A7</td><td>Estimated Cost: $20.45</td></tr><tr><td>Alignment Laser</td><td>BISKEE</td><td></td><td>https://www.amazon.com/Tactical-Presentation-Teaching-Interactive-Adjustable/dp/B09B1VXPNM&lt;br/&gt; Estimated Cost: $16.98</td></tr><tr><td>Autoluorescent Plastic Slide, Red&amp;nbsp;</td><td>Chroma</td><td>92001</td><td>Estimated Cost: $20</td></tr><tr><td>Beam Shutter&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>SM1SH1</td><td>To block laser light&lt;br/&gt; Estimated Cost: $65.8</td></tr><tr><td>Cage Rotation Mount (x3)</td><td>Thorlabs</td><td>CRM1T</td><td>To mount CL1-3&lt;br/&gt; Estimated Cost: $282.15</td></tr><tr><td>Cage System Rods 1&quot; (x8)</td><td>Thorlabs&amp;nbsp;</td><td>ER1</td><td>To mount M3 and O1&lt;br/&gt; Estimated Cost: $44.8</td></tr><tr><td>Cage System Rods 3&quot; (x2)</td><td>Thorlabs&amp;nbsp;</td><td>ER3</td><td>To mount O3&lt;br/&gt; Estimated Cost: $14.28</td></tr><tr><td>Cage System Rods 4&quot; (x4)</td><td>Thorlabs&amp;nbsp;</td><td>ER4</td><td>To mount slit&lt;br/&gt; Estimated Cost: $30.76</td></tr><tr><td>Cage System Rods 8&quot; (x2)</td><td>Thorlabs&amp;nbsp;</td><td>ER8</td><td>For tube lens alignment&lt;br/&gt; Estimated Cost: $25.3</td></tr><tr><td>Cage System Rods 12&quot; (x8)</td><td>Thorlabs</td><td>ER12</td><td>For alignment cage&lt;br/&gt; Estimated Cost: $145.36</td></tr><tr><td>Camera</td><td>Andor</td><td>Zyla 4.2 sCMOS</td><td>Estimated Cost: ~$14,000</td></tr><tr><td>Clamping Fork (x35)</td><td>Thorlabs</td><td>CF125</td><td>To clamp down post mounts&lt;br/&gt; Estimated Cost: $338.8</td></tr><tr><td>Cover Glass, 22 x 22 mm&amp;nbsp;</td><td>Corning</td><td>2850-22</td><td>For slide samples&lt;br/&gt; Estimated Cost: $265</td></tr><tr><td>Dichroic&amp;nbsp;</td><td>AVR</td><td>DI01-R405/488/561/635-25x36</td><td>To split exciation/emission paths&lt;br/&gt; Estimated Cost: $965</td></tr><tr><td>Dovetail Translation Stage</td><td>Thorlabs</td><td>DT12</td><td>To translate pinhole&lt;br/&gt; Estimated Cost: $90.55</td></tr><tr><td>Emission Filter</td><td>Thorlabs</td><td>FELHO600</td><td>Estimated Cost: $140.99</td></tr><tr><td>Frosted Glass Alignment Disk (x2)</td><td>Thorlabs</td><td>DG10-1500-H1</td><td>For alignment cage and intermediate plane&lt;br/&gt; Estimated Cost: $75.14</td></tr><tr><td>Function Generator</td><td>Hewlett-Packard</td><td>HP 33120A 15 MHz</td><td>To control galvo&lt;br/&gt; Estimated Cost: $900</td></tr><tr><td>Galvanometer - 1D Large Beam Diameter System</td><td>Thorlabs</td><td>GVS011</td><td>Estimated Cost: $1715.78</td></tr><tr><td>Galvanometer Power Supply</td><td>Siglent</td><td>SPD3303C</td><td>Estimated Cost: $300</td></tr><tr><td>Gelrite</td><td>Research Products International</td><td>G35020-100.0</td><td>Gellan gum for 3D bead sample&lt;br/&gt; Estimated Cost: $68.25</td></tr><tr><td>FIJI Software</td><td>Open-source</td><td></td><td>Download from https://imagej.net/software/fiji/downloads&lt;br/&gt; Estimated Cost: Free</td></tr><tr><td>Hot Plate/ Stirrer</td><td>Corning</td><td>6795-220</td><td>For preparing sample solutions&lt;br/&gt; Estimated Cost: $550</td></tr><tr><td>K-Cube Brushed Motor Controller</td><td>Thorlabs</td><td>KDC101</td><td>Drives Z825B&lt;br/&gt; Estimated Cost: $757.51</td></tr><tr><td>Kinematic Mount</td><td>Thorlabs</td><td>KM100S</td><td>To mount dichroic&lt;br/&gt; Estimated Cost: $92.01</td></tr><tr><td>Kinesis Software</td><td>Thorlabs&amp;nbsp;</td><td></td><td>Download from https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=10285&lt;br/&gt; Estimated Cost: Free</td></tr><tr><td>Laser Light Blocker&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>LB1</td><td>For ND filter reflections&lt;br/&gt; Estimated Cost: $57.65</td></tr><tr><td>Laser Mount</td><td>custom made</td><td></td><td>3D printed&lt;br/&gt; Estimated Cost: N/A</td></tr><tr><td>Laser Safety Screen (x2)</td><td>Thorlabs&amp;nbsp;</td><td>TPS4</td><td>For blocking stray laser light&lt;br/&gt; Estimated Cost: $92.02</td></tr><tr><td>Laser Scanning Tube Lens</td><td>Thorlabs</td><td>TTL200MP</td><td>TL1&lt;br/&gt; Estimated Cost: $1491</td></tr><tr><td>Lens Mount (x10)&amp;nbsp;</td><td>Thorlabs</td><td>LMR1</td><td>To mount all lens and extra alignment mirror.&lt;br/&gt; Estimated Cost: $164.7</td></tr><tr><td>Magnetic Ruler</td><td>Thorlabs</td><td>BHM4</td><td>To check alignment&lt;br/&gt; Estimated Cost: $52.74</td></tr><tr><td>Micro-Manager Software&amp;nbsp;</td><td>Open-source</td><td></td><td>Download from https://micro-manager.org/Download_Micro-Manager_Latest_Release&lt;br/&gt; Estimated Cost: Free</td></tr><tr><td>Microscope Slides</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>12550400</td><td>For slide samples&lt;br/&gt; Estimated Cost: $123.9</td></tr><tr><td>Microscope Stage&amp;nbsp;</td><td>ASI</td><td>FTP-2000 with custom parts</td><td>To fine-translate samples&lt;br/&gt; Estimated Cost: ~$16,000</td></tr><tr><td>Mini Vortex Mixer&amp;nbsp;</td><td>VWR</td><td>10153-688</td><td>For sample preparation&lt;br/&gt; Estimated Cost: $152.64</td></tr><tr><td>Motorized Actuator</td><td>Thorlabs</td><td>Z825B</td><td>To fine-translate M1&lt;br/&gt; Estimated Cost: $729.07</td></tr><tr><td>Mounted Standard Iris (x2)</td><td>Thorlabs</td><td>ID20</td><td>At least 2 for alignment&lt;br/&gt; Estimated Cost: $118.02</td></tr><tr><td>ND Filter Set&amp;nbsp;</td><td>Thorlabs</td><td>NDK01&amp;nbsp;</td><td>To reduce excitation intensity&lt;br/&gt; Estimated Cost: $726.73</td></tr><tr><td>Objective Lens 1</td><td>Nikon&amp;nbsp;</td><td>Plan Apo 60X/ 1.20 WI</td><td>O1&lt;br/&gt; Estimated Cost: ~$15,000</td></tr><tr><td>Objective Lens 2</td><td>Nikon</td><td>TU Plan Fluor 100X/0.90&amp;nbsp;</td><td>O2&lt;br/&gt; Estimated Cost: ~$6,000</td></tr><tr><td>Objective Lens 3</td><td>Mitutoyo</td><td>Plan Apo HR 50X/0.75</td><td>O3&lt;br/&gt; Estimated Cost: ~$6,800</td></tr><tr><td>OPM Deskewing Software</td><td>Open-source</td><td></td><td>For image processing. Download from https://github.com/QI2lab/OPM&lt;br/&gt; Estimated Cost: Free</td></tr><tr><td>Photodiode Power Sensor</td><td>Thorlabs&amp;nbsp;</td><td>S121C</td><td>For measuring laser intensity&lt;br/&gt; Estimated Cost: $379.68</td></tr><tr><td>Positive Grid Distortion Target</td><td>Thorlabs</td><td>R1L3S3P</td><td>Brightfield alignment&lt;br/&gt; Estimated Cost: $267.87</td></tr><tr><td>Power Meter Digital Console</td><td>Thorlabs&amp;nbsp;</td><td>PM100D</td><td>For measuring laser intensity&lt;br/&gt; Estimated Cost: $1245.48</td></tr><tr><td>Rhodamine 6G</td><td>Thermo Scientific&amp;nbsp;</td><td>J62315.14</td><td>For fluorescent coated slide sample&lt;br/&gt; Estimated Cost: $27.7</td></tr><tr><td>Right-Angle Clamp for Posts</td><td>Thorlabs&amp;nbsp;</td><td>RA90</td><td>For M3 support and flip down mirror&lt;br/&gt; Estimated Cost: $32.46</td></tr><tr><td>RMS-Threaded Cage Plate (x2)&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>CP42</td><td>For alignment laser&lt;br/&gt; Estimated Cost: $70.56</td></tr><tr><td>Shear Plate 2.5-5.0 mm</td><td>Thorlabs</td><td>SI050P&amp;nbsp;</td><td>Estimated Cost: $182.85</td></tr><tr><td>Shear Plate 5.0-10.0 mm</td><td>Thorlabs</td><td>SI100P</td><td>Estimated Cost: $201.47</td></tr><tr><td>Shear Plate 10.0-25.4 mm</td><td>Thorlabs</td><td>SI254P</td><td>Estimated Cost: $236.42</td></tr><tr><td>Shear Plate Viewing Screen</td><td>Thorlabs</td><td>SIVS</td><td>Estimated Cost: $337.74</td></tr><tr><td>Shearing Interferometer with 1-3 mm Plate</td><td>Thorlabs</td><td>SI035</td><td>For checking collimation&lt;br/&gt; Estimated Cost: $465.85</td></tr><tr><td>Slip-On Post Collar (x35)</td><td>Thorlabs&amp;nbsp;</td><td>R2</td><td>To maintain post height&lt;br/&gt; Estimated Cost: $208.25</td></tr><tr><td>Slit</td><td>Thorlabs</td><td>VA100</td><td>Estimated Cost: $294.64</td></tr><tr><td>Slotted Lens Tube, 3&quot;</td><td>Thorlabs&amp;nbsp;</td><td>SM1L30C</td><td>For alignment laser&lt;br/&gt; Estimated Cost: $77.45</td></tr><tr><td>Square Mirror, 1 x 1&quot;</td><td></td><td></td><td>https://www.amazon.com/Small-Square-Mirror-Pieces-Mosaic/dp/B07FBNMDC1/ref=asc_df_B07FBNMDC1/?tag=hyprod-20&amp;amp;linkCode=df0&amp;amp;hva&lt;br/&gt; did=642191768069&amp;amp;hvpos=&amp;amp;hvne&lt;br/&gt; tw=g&amp;amp;hvrand=1336734911900437&lt;br/&gt; 4691&amp;amp;hvpone=&amp;amp;hvptwo=&amp;amp;hvqmt=&lt;br/&gt; &amp;amp;hvdev=c&amp;amp;hvdvcmdl=&amp;amp;hvlocint=&amp;amp;&lt;br/&gt; hvlocphy=9031212&amp;amp;hvtargid=pla-1&lt;br/&gt; 943952718742&amp;amp;gclid=Cj0KCQiA6L&lt;br/&gt; yfBhC3ARIsAG4gkF_AYBpn5EdGL&lt;br/&gt; q3mc-RU-nanT5vM4ac9r3-obbzqJoWKPkIPIJU6e1caAjWmEA&lt;br/&gt; Lw_wcB&amp;amp;th=1&lt;br/&gt; Estimated Cost: $14.76</td></tr><tr><td>Stackable Lens Tube 1/2&quot; (x3)</td><td>Thorlabs</td><td>SM1L05</td><td>To mount CL1-3&lt;br/&gt; Estimated Cost: $40.86</td></tr><tr><td>Stackable Lens Tube 1&quot;</td><td>Thorlabs</td><td>SM1L10</td><td>To mount O3&lt;br/&gt; Estimated Cost: $15.41</td></tr><tr><td>Stackable Lens Tube 2&quot; (x2)</td><td>Thorlabs</td><td>SM1L20</td><td>For camera path&lt;br/&gt; Estimated Cost: $35.7</td></tr><tr><td>Studded Pedestal Base Adapter (x37)</td><td>Thorlabs&amp;nbsp;</td><td>BE1</td><td>To attach post mounts to table&lt;br/&gt; Estimated Cost: $400.71</td></tr><tr><td>Translating Lens Mount (x3)</td><td>Thorlabs</td><td>LM1XY</td><td>To fine-translate pinhole, O2 and O3&lt;br/&gt; Estimated Cost: $441</td></tr><tr><td>Translation Stage with Standard Micrometer (x2)</td><td>Thorlabs</td><td>PT1/M</td><td>TS1-2&lt;br/&gt; Estimated Cost: $647.54</td></tr><tr><td>Travel Manual Translation Stage</td><td>Thorlabs</td><td>CT1A</td><td>O3 cage translation mount&lt;br/&gt; Estimated Cost: $497.3</td></tr><tr><td>Tube Lens</td><td>Nikon</td><td>MXA20696</td><td>TL3&lt;br/&gt; Estimated Cost: $359</td></tr><tr><td>White Mounted LED</td><td>Thorlabs&amp;nbsp;</td><td>MNWHL4</td><td>Brightfield light source&lt;br/&gt; Estimated Cost: $171.28</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>TOTAL ESTIMATED COST: $84,858.98</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>The authors note that many parts were bought used. Here, we have attempted to reflect the retail price of all items, so the total cost can be greatly reduced by buying particular items used, especially the more expensive ones.&amp;nbsp;</td></tr><tr><td>OPTIONAL COMPONENTS</td><td></td><td></td><td></td></tr><tr><td>Grasshopper3 USB3</td><td>FLIR</td><td>&amp;nbsp;GS3-U3-23S6C-C</td><td>For diagnostic checks during alignment. Acquisiton camera can be used instead, but requires realignment afterwards.&lt;br/&gt; Estimated Cost: $1089</td></tr></tbody></table>

design and building of a customizable, single-objective, light-sheet fluorescence microscope for the visualization of cytoskeleton networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

We performed volumetric scans of 1 &#181;m beads embedded in gellan gum. Figure 14 shows the maximum intensity projections of the deskewed volumetric scans along the x, y, and z directions.
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/65411/65411fig14.jpg" /> 
Figure 14: Volumetric imaging of 1 &#181;m fluorescent beads in gellan gum. Maximum intensity projections of deskewed volumetric scans are shown. Scale bars = 30 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65411/65411fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We have demonstrated the use of the single-objective, light-sheet microscope to characterize reconstituted cytoskeleton networks by performing volumetric scans of samples of microtubule asters. In brief, rhodamine-labeled, taxol-stabilized microtubules were polymerized from reconstituted dimers by GTP; then, following polymerization, streptavidin-based kinesin motor clusters were mixed into samples along with ATP for final concentrations of 6 &#181;M microtubules, 0.5 &#181;M kinesin dimers, and 10 mM ATP. Extensive protocols and guides for the preparation of taxol-stabilized microtubules and kinesin motor clusters can be found on the Mitchson Lab and Dogic Lab websites25,26. The samples were pipetted gently into microscope slides, sealed, and allowed to sit for 8 h before imaging to allow for motor activity to cease so that the samples reached a steady structural state that resembled asters.
Studies of reconstituted cytoskeleton systems most frequently employ confocal or epifluorescence microscopy to image labeled filaments. However, both of these techniques are limited in their capability to image dense 3D samples27. While much progress has been made in in vitro cytoskeleton-based active matter research by constraining samples to be quasi 2D28,29, cytoskeleton networks are inherently 3D, and many current endeavors lie in understanding the effects that can only arise in 3D samples29,30, thus creating a need for high-resolution 3D imaging.
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/65411/65411fig15.jpg" /> 
Figure 15: Facilitation of the 3D visualization of reconstituted cytoskeleton samples by single-objective light-sheet microscopy. (A) Images of fluorescent microtubule asters acquired on a Leica DMi8 laser-scanning confocal microscope. The images show different planes from a z-scan. Scale bar = 30 &#181;m. (B) Deconvolved deskewed images from a volumetric scan performed on the single-objective light-sheet setup of the same sample. Scale bar = 30 &#181;m. The deskewed image area here corresponds to the usable FoV (yellow box) demonstrated in Figure 13B. While the confocal excels at imaging single planes near the coverslip, the density of the fluorescent sample introduces complications when imaging at higher planes due to the additional signal from below the imaging plane. The light sheet circumvents this issue by only illuminating the imaging plane, thus allowing for uniformly sharp imaging at different planes in z. Abbreviations: SOLS = single-objective light-sheet; FoV = field of view. <a href="https://www.jove.com/files/ftp_upload/65411/65411fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In Figure 15, we demonstrate the volumetric imaging of a reconstituted microtubule network contracted into aster-like structures by kinesin motor clusters. As shown in previous research28,31, these 3D structures tend to grow dense toward the center, resulting in bright regions of fluorescence that are predominant in the signal. In imaging planes near the coverslip (low z level), confocal microscopy (Figure 15A) can resolve single filaments around the periphery of the aster, with additional background toward the center due to out-of-focus fluorescence signals from above. However, moving a few microns in z quickly reduces the quality of the images due to the out-of-focus dense sections of the aster being predominant in the signal in the imaging plane. The single-plane illumination of the light sheet (Figure 15B) eliminates the out-of-focus signals from the dense parts of the aster above and below the imaging plane, thus allowing for comparable image quality between the planes. The light sheet&#39;s ability to produce high-quality, reliable volumetric scan data opens up the possibility of visualizing and characterizing 3D phenomena in reconstituted cytoskeleton systems.

Watch this Scientific Journal Video about Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks at JoVE.com

Author Spotlight: Advancing Knowledge in Far-From-Equilibrium Materials Through Light-Sheet Microscopy

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...

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1.6K Views.  Scripps College. This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks. 

Video: Author Spotlight: Advancing Knowledge in Far-From-Equilibrium Materials Through Light-Sheet Microscopy

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.

A module for single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures. The sample is located within a rectangular capillary, and via a microfluidic system fluorescent dyes, pharmaceutical agents or drugs can be applied in small quantities.

A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and ...

Engineering

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Developmental Biology

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Biochemistry

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Bioengineering

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence signals were proven cumbersome until the advent of hyperspectral imaging, in which fluorescence sources can be separated from each other as well as from background signals and autofluorescence (given knowledge of their spectral signatures). However, traditional, emission-scanning hyperspectral imaging suffers from slow acquisition times and low signal-to-noise ratios due to the necessary filtering of both excitation and emission light. It has been previously shown that excitation-scanning hyperspectral imaging reduces the necessary acquisition time while simultaneously increasing the signal-to-noise ratio of acquired data. Using commercially available equipment, this protocol describes how to assemble, calibrate, and use an excitation-scanning hyperspectral imaging microscopy system for separation of signals from several fluorescence sources in a single sample. While highly applicable to microscopic imaging of cells and tissues, this technique may also be useful for any type of experiment utilizing fluorescence in which it is possible to vary excitation wavelengths, including but not limited to: chemical imaging, environmental applications, eye care, food science, forensic science, medical science, and mineralogy.

Spectral imaging has become a reliable solution for identification and separation of multiple fluorescence signals in a single sample and can readily distinguish signals of interest from background or autofluorescence. Excitation-scanning hyperspectral imaging improves on this technique by decreasing the necessary image acquisition time while simultaneously increasing the signal-to-noise ratio.

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence ...

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia.
Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells&#39; major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support.
This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at:&#160;https://github.com/badain/amebas.git

Current methods for analyzing the intracellular dynamics of polarized single cells are often manual and lack standardization. This manuscript introduces a novel image analysis pipeline for automating midline extraction of single polarized cells and quantifying spatiotemporal behavior from time lapses in a user-friendly online interface.

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Neuroscience

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Summary

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Chapters in this video

Single Plane Illumination Module and Micro-capillary Approach for a Wide-field Microscope

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development