Name: Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks (Video) | JoVE Journal
Uploaded: 2024-01-26T12:50:00.000+00:00
Duration: 8 min 32 s
Description: Learn to build a customizable single-objective light-sheet fluorescence microscope for visualizing cytoskeleton networks in dense three-dimensional samples. This method minimizes photobleaching while enabling high-quality optical sectioning.

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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

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</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="Optical setup diagram; emission, excitation paths; microscopy; dichroic mirror; fluorescence study." class="xfigimg" src="/files/ftp_upload/65411/65411fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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

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 µm 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="Microscopy image of cellular structure in x-y, y-z, z-x planes for 3D visualization, 30 µm scale." class="xfigimg" src="/files/ftp_upload/65411/65411fig14.jpg" data-altupdatedat="1788304762000" data-alt="Figure 14"> 
Figure 14: Volumetric imaging of 1 µm fluorescent beads in gellan gum. Maximum intensity projections of deskewed volumetric scans are shown. Scale bars = 30 µm. <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 µM microtubules, 0.5 µM 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="Confocal vs SOLS microscopy, fiber structure at different depths z=0-15μm. Diagram, optical analysis." class="xfigimg" src="/files/ftp_upload/65411/65411fig15.jpg" data-altupdatedat="1788304762000" data-alt="Figure 15"> 
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 µm. (B) Deconvolved deskewed images from a volumetric scan performed on the single-objective light-sheet setup of the same sample. Scale bar = 30 µm. 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's ability to produce high-quality, reliable volumetric scan data opens up the possibility of visualizing and characterizing 3D phenomena in reconstituted cytoskeleton systems.

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

Research

JoVE Journal

Biology

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment.  Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

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.

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

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

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

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.

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

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.

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

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.

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

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.

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

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

A detailed instruction is described on how to build a highly inclined swept tile (HIST) microscope and its usage for single-molecule imaging.

A Guide to Build a Highly Inclined Swept Tile Microscope for Extended Field-of-view Single-molecule Imaging

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Learn to build a customizable single-objective light-sheet fluorescence microscope for visualizing cytoskeleton networks in dense three-dimensional samples. This method minimizes photobleaching while enabling high-quality optical sectioning.

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks (Video) | JoVE Journal

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

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