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.remotexs.ntu.edu.sg/files/ftp_upload/65411/65411fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#34; Plano-Concave Lens f = -50 mm</td>
			<td>Thorlabs&#160;</td>
			<td>LC1715-A-ML</td>
			<td>For alignment laser 
			Estimated Cost: $49.5</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 100 mm (x3)</td>
			<td>Thorlabs</td>
			<td>AC254-100-A-ML</td>
			<td>L2, L4 and alignment laser 
			Estimated Cost: $342.42</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 125 mm</td>
			<td>Thorlabs</td>
			<td>AC254-125-A-ML</td>
			<td>SL2 
			Estimated Cost: $114.14</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 150 mm</td>
			<td>Thorlabs</td>
			<td>AC254-150-A-ML</td>
			<td>L3 
			Estimated Cost: $114.14</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 150 mm</td>
			<td>Thorlabs</td>
			<td>AC254-150-A-ML</td>
			<td>TL2 
			Estimated Cost: $114.14</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 45 mm</td>
			<td>Thorlabs</td>
			<td>AC254-045-A-ML</td>
			<td>L1 
			Estimated Cost: $114.14</td>
		</tr>
		<tr>
			<td>1&#34; Achromatic Doublet f = 75 mm</td>
			<td>Thorlabs</td>
			<td>AC254-075-A-ML</td>
			<td>SL1 
			Estimated Cost: $114.14</td>
		</tr>
		<tr>
			<td>1&#34; Cylindrical Lens f = 100 mm</td>
			<td>Thorlabs</td>
			<td>LJ1567RM</td>
			<td>CL3 
			Estimated Cost: $117.62</td>
		</tr>
		<tr>
			<td>1&#34; Cylindrical Lens f = 200 mm</td>
			<td>Thorlabs</td>
			<td>LJ1653RM</td>
			<td>CL2 
			Estimated Cost: $111.22</td>
		</tr>
		<tr>
			<td>1&#34; Cylindrical Lens f = 50 mm</td>
			<td>Thorlabs</td>
			<td>LJ1695RM</td>
			<td>CL1 
			Estimated Cost: $117.62</td>
		</tr>
		<tr>
			<td>1&#34; Mounted Pinhole, 30 &#181;m Pinhole Diameter</td>
			<td>Thorlabs</td>
			<td>P30K</td>
			<td>Estimated Cost: $77.08</td>
		</tr>
		<tr>
			<td>1&#34; Silver Mirror (x3)</td>
			<td>Thorlabs</td>
			<td>PF10-03-P01</td>
			<td>M1, M2, one for alignment 
			Estimated Cost: $168.78</td>
		</tr>
		<tr>
			<td>2&#34; Elliptical Mirror</td>
			<td>Thorlabs</td>
			<td>PFE20-P01</td>
			<td>M3 
			Estimated Cost: $179.98</td>
		</tr>
		<tr>
			<td>2&#34; Post Holder (x11)</td>
			<td>Thorlabs</td>
			<td>PH2</td>
			<td>For custom laser mount, ND wheel, safety screens 
			Estimated Cost: $98.45</td>
		</tr>
		<tr>
			<td>2&#34; Posts (x47)</td>
			<td>Thorlabs</td>
			<td>TR2</td>
			<td>For custom laser mount and optical components 
			Estimated Cost: $277.3</td>
		</tr>
		<tr>
			<td>3&#34; Posts (x4)&#160;</td>
			<td>Thorlabs&#160;</td>
			<td>TR3</td>
			<td>For M3 supports and other mounts 
			Estimated Cost: $24.6</td>
		</tr>
		<tr>
			<td>3&#34; Post Holder (x4)</td>
			<td>Thorlabs&#160;</td>
			<td>PH3</td>
			<td>Estimated Cost: $38.48</td>
		</tr>
		<tr>
			<td>30 to 60 mm Cage Adapter&#160;</td>
			<td>Thorlabs</td>
			<td>LCP33</td>
			<td>To mount O1 
			Estimated Cost: $45.42</td>
		</tr>
		<tr>
			<td>30mm Cage Filter Wheel</td>
			<td>Thorlabs</td>
			<td>CFW6</td>
			<td>To mount ND filters 
			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 
			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 
			Estimated Cost: $463.95</td>
		</tr>
		<tr>
			<td>4&#34; 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&#160;</td>
			<td>Opto Engine LLC</td>
			<td>MGL-FN-561-100mW</td>
			<td>Excitation laser 
			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 
			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 
			Estimated Cost: $187.26</td>
		</tr>
		<tr>
			<td>90&#176; Flip Mount</td>
			<td>Thorlabs&#160;</td>
			<td>TRF90</td>
			<td>For alignment laser 
			Estimated Cost: $95.5</td>
		</tr>
		<tr>
			<td>Adapter with External C-Mount Threads and Internal SM1 Threads</td>
			<td>Thorlabs&#160;</td>
			<td>SM1A9</td>
			<td>To connect lens tube to camera 
			Estimated Cost: $20.96</td>
		</tr>
		<tr>
			<td>Adapter with External SM1 Threads and Internal C-Mount Threads</td>
			<td>Thorlabs&#160;</td>
			<td>SM1A10</td>
			<td>To connect tube lens to lens mount 
			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 
			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 
			Estimated Cost: $22.38</td>
		</tr>
		<tr>
			<td>Alignment Disk</td>
			<td>Thorlabs&#160;</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 
			Estimated Cost: $16.98</td>
		</tr>
		<tr>
			<td>Autoluorescent Plastic Slide, Red&#160;</td>
			<td>Chroma</td>
			<td>92001</td>
			<td>Estimated Cost: $20</td>
		</tr>
		<tr>
			<td>Beam Shutter&#160;</td>
			<td>Thorlabs&#160;</td>
			<td>SM1SH1</td>
			<td>To block laser light 
			Estimated Cost: $65.8</td>
		</tr>
		<tr>
			<td>Cage Rotation Mount (x3)</td>
			<td>Thorlabs</td>
			<td>CRM1T</td>
			<td>To mount CL1-3 
			Estimated Cost: $282.15</td>
		</tr>
		<tr>
			<td>Cage System Rods 1&#34; (x8)</td>
			<td>Thorlabs&#160;</td>
			<td>ER1</td>
			<td>To mount M3 and O1 
			Estimated Cost: $44.8</td>
		</tr>
		<tr>
			<td>Cage System Rods 3&#34; (x2)</td>
			<td>Thorlabs&#160;</td>
			<td>ER3</td>
			<td>To mount O3 
			Estimated Cost: $14.28</td>
		</tr>
		<tr>
			<td>Cage System Rods 4&#34; (x4)</td>
			<td>Thorlabs&#160;</td>
			<td>ER4</td>
			<td>To mount slit 
			Estimated Cost: $30.76</td>
		</tr>
		<tr>
			<td>Cage System Rods 8&#34; (x2)</td>
			<td>Thorlabs&#160;</td>
			<td>ER8</td>
			<td>For tube lens alignment 
			Estimated Cost: $25.3</td>
		</tr>
		<tr>
			<td>Cage System Rods 12&#34; (x8)</td>
			<td>Thorlabs</td>
			<td>ER12</td>
			<td>For alignment cage 
			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 
			Estimated Cost: $338.8</td>
		</tr>
		<tr>
			<td>Cover Glass, 22 x 22 mm&#160;</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td>For slide samples 
			Estimated Cost: $265</td>
		</tr>
		<tr>
			<td>Dichroic&#160;</td>
			<td>AVR</td>
			<td>DI01-R405/488/561/635-25x36</td>
			<td>To split exciation/emission paths 
			Estimated Cost: $965</td>
		</tr>
		<tr>
			<td>Dovetail Translation Stage</td>
			<td>Thorlabs</td>
			<td>DT12</td>
			<td>To translate pinhole 
			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 
			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 
			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 
			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 
			Estimated Cost: Free</td>
		</tr>
		<tr>
			<td>Hot Plate/ Stirrer</td>
			<td>Corning</td>
			<td>6795-220</td>
			<td>For preparing sample solutions 
			Estimated Cost: $550</td>
		</tr>
		<tr>
			<td>K-Cube Brushed Motor Controller</td>
			<td>Thorlabs</td>
			<td>KDC101</td>
			<td>Drives Z825B 
			Estimated Cost: $757.51</td>
		</tr>
		<tr>
			<td>Kinematic Mount</td>
			<td>Thorlabs</td>
			<td>KM100S</td>
			<td>To mount dichroic 
			Estimated Cost: $92.01</td>
		</tr>
		<tr>
			<td>Kinesis Software</td>
			<td>Thorlabs&#160;</td>
			<td></td>
			<td>Download from https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=10285 
			Estimated Cost: Free</td>
		</tr>
		<tr>
			<td>Laser Light Blocker&#160;</td>
			<td>Thorlabs&#160;</td>
			<td>LB1</td>
			<td>For ND filter reflections 
			Estimated Cost: $57.65</td>
		</tr>
		<tr>
			<td>Laser Mount</td>
			<td>custom made</td>
			<td></td>
			<td>3D printed 
			Estimated Cost: N/A</td>
		</tr>
		<tr>
			<td>Laser Safety Screen (x2)</td>
			<td>Thorlabs&#160;</td>
			<td>TPS4</td>
			<td>For blocking stray laser light 
			Estimated Cost: $92.02</td>
		</tr>
		<tr>
			<td>Laser Scanning Tube Lens</td>
			<td>Thorlabs</td>
			<td>TTL200MP</td>
			<td>TL1 
			Estimated Cost: $1491</td>
		</tr>
		<tr>
			<td>Lens Mount (x10)&#160;</td>
			<td>Thorlabs</td>
			<td>LMR1</td>
			<td>To mount all lens and extra alignment mirror. 
			Estimated Cost: $164.7</td>
		</tr>
		<tr>
			<td>Magnetic Ruler</td>
			<td>Thorlabs</td>
			<td>BHM4</td>
			<td>To check alignment 
			Estimated Cost: $52.74</td>
		</tr>
		<tr>
			<td>Micro-Manager Software&#160;</td>
			<td>Open-source</td>
			<td></td>
			<td>Download from https://micro-manager.org/Download_Micro-Manager_Latest_Release 
			Estimated Cost: Free</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>12550400</td>
			<td>For slide samples 
			Estimated Cost: $123.9</td>
		</tr>
		<tr>
			<td>Microscope Stage&#160;</td>
			<td>ASI</td>
			<td>FTP-2000 with custom parts</td>
			<td>To fine-translate samples 
			Estimated Cost: ~$16,000</td>
		</tr>
		<tr>
			<td>Mini Vortex Mixer&#160;</td>
			<td>VWR</td>
			<td>10153-688</td>
			<td>For sample preparation 
			Estimated Cost: $152.64</td>
		</tr>
		<tr>
			<td>Motorized Actuator</td>
			<td>Thorlabs</td>
			<td>Z825B</td>
			<td>To fine-translate M1 
			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 
			Estimated Cost: $118.02</td>
		</tr>
		<tr>
			<td>ND Filter Set&#160;</td>
			<td>Thorlabs</td>
			<td>NDK01&#160;</td>
			<td>To reduce excitation intensity 
			Estimated Cost: $726.73</td>
		</tr>
		<tr>
			<td>Objective Lens 1</td>
			<td>Nikon&#160;</td>
			<td>Plan Apo 60X/ 1.20 WI</td>
			<td>O1 
			Estimated Cost: ~$15,000</td>
		</tr>
		<tr>
			<td>Objective Lens 2</td>
			<td>Nikon</td>
			<td>TU Plan Fluor 100X/0.90&#160;</td>
			<td>O2 
			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 
			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 
			Estimated Cost: Free</td>
		</tr>
		<tr>
			<td>Photodiode Power Sensor</td>
			<td>Thorlabs&#160;</td>
			<td>S121C</td>
			<td>For measuring laser intensity 
			Estimated Cost: $379.68</td>
		</tr>
		<tr>
			<td>Positive Grid Distortion Target</td>
			<td>Thorlabs</td>
			<td>R1L3S3P</td>
			<td>Brightfield alignment 
			Estimated Cost: $267.87</td>
		</tr>
		<tr>
			<td>Power Meter Digital Console</td>
			<td>Thorlabs&#160;</td>
			<td>PM100D</td>
			<td>For measuring laser intensity 
			Estimated Cost: $1245.48</td>
		</tr>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Thermo Scientific&#160;</td>
			<td>J62315.14</td>
			<td>For fluorescent coated slide sample 
			Estimated Cost: $27.7</td>
		</tr>
		<tr>
			<td>Right-Angle Clamp for Posts</td>
			<td>Thorlabs&#160;</td>
			<td>RA90</td>
			<td>For M3 support and flip down mirror 
			Estimated Cost: $32.46</td>
		</tr>
		<tr>
			<td>RMS-Threaded Cage Plate (x2)&#160;</td>
			<td>Thorlabs&#160;</td>
			<td>CP42</td>
			<td>For alignment laser 
			Estimated Cost: $70.56</td>
		</tr>
		<tr>
			<td>Shear Plate 2.5-5.0 mm</td>
			<td>Thorlabs</td>
			<td>SI050P&#160;</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 
			Estimated Cost: $465.85</td>
		</tr>
		<tr>
			<td>Slip-On Post Collar (x35)</td>
			<td>Thorlabs&#160;</td>
			<td>R2</td>
			<td>To maintain post height 
			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&#34;</td>
			<td>Thorlabs&#160;</td>
			<td>SM1L30C</td>
			<td>For alignment laser 
			Estimated Cost: $77.45</td>
		</tr>
		<tr>
			<td>Square Mirror, 1 x 1&#34;</td>
			<td></td>
			<td></td>
			<td>https://www.amazon.com/Small-Square-Mirror-Pieces-Mosaic/dp/B07FBNMDC1/ref=asc_df_B07FBNMDC1/?tag=hyprod-20&#38;linkCode=df0&#38;hva 
			did=642191768069&#38;hvpos=&#38;hvne 
			tw=g&#38;hvrand=1336734911900437 
			4691&#38;hvpone=&#38;hvptwo=&#38;hvqmt= 
			&#38;hvdev=c&#38;hvdvcmdl=&#38;hvlocint=&#38; 
			hvlocphy=9031212&#38;hvtargid=pla-1 
			943952718742&#38;gclid=Cj0KCQiA6L 
			yfBhC3ARIsAG4gkF_AYBpn5EdGL 
			q3mc-RU-nanT5vM4ac9r3-obbzqJoWKPkIPIJU6e1caAjWmEA 
			Lw_wcB&#38;th=1 
			Estimated Cost: $14.76</td>
		</tr>
		<tr>
			<td>Stackable Lens Tube 1/2&#34; (x3)</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
			<td>To mount CL1-3 
			Estimated Cost: $40.86</td>
		</tr>
		<tr>
			<td>Stackable Lens Tube 1&#34;</td>
			<td>Thorlabs</td>
			<td>SM1L10</td>
			<td>To mount O3 
			Estimated Cost: $15.41</td>
		</tr>
		<tr>
			<td>Stackable Lens Tube 2&#34; (x2)</td>
			<td>Thorlabs</td>
			<td>SM1L20</td>
			<td>For camera path 
			Estimated Cost: $35.7</td>
		</tr>
		<tr>
			<td>Studded Pedestal Base Adapter (x37)</td>
			<td>Thorlabs&#160;</td>
			<td>BE1</td>
			<td>To attach post mounts to table 
			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 
			Estimated Cost: $441</td>
		</tr>
		<tr>
			<td>Translation Stage with Standard Micrometer (x2)</td>
			<td>Thorlabs</td>
			<td>PT1/M</td>
			<td>TS1-2 
			Estimated Cost: $647.54</td>
		</tr>
		<tr>
			<td>Travel Manual Translation Stage</td>
			<td>Thorlabs</td>
			<td>CT1A</td>
			<td>O3 cage translation mount 
			Estimated Cost: $497.3</td>
		</tr>
		<tr>
			<td>Tube Lens</td>
			<td>Nikon</td>
			<td>MXA20696</td>
			<td>TL3 
			Estimated Cost: $359</td>
		</tr>
		<tr>
			<td>White Mounted LED</td>
			<td>Thorlabs&#160;</td>
			<td>MNWHL4</td>
			<td>Brightfield light source 
			Estimated Cost: $171.28</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>TOTAL ESTIMATED COST: $84,858.98</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</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.&#160;</td>
		</tr>
		<tr>
			<td>OPTIONAL COMPONENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Grasshopper3 USB3</td>
			<td>FLIR</td>
			<td>&#160;GS3-U3-23S6C-C</td>
			<td>For diagnostic checks during alignment. Acquisiton camera can be used instead, but requires realignment afterwards. 
			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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

design-building-customizable-single-objective-light-sheet

Nanyang Technological University

Without Section

Research

JoVE Journal

Biology

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

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.

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

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

Summary

Explore More Videos

Chapters in this video

Proper Care and Cleaning of the Microscope

Major Components of the Light Microscope

Analyzing and Building Nucleic Acid Structures with 3DNA

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

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

Design and Development of Aptamer–Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

A Customizable Chamber for Measuring Cell Migration