We thank the Institute for Clinical Hemostaseology and Transfusion Medicine for providing donor blood; Carmen H&#228;ssig and Cora Hoxha for excellent technical help. We thank Jens Rettig (Saarland University) for the modified pMAX vector, Roland Wedlich-S&#246;ldner (University of Muenster) for the original LifeAct-Ruby construct, and Christian Junker (Saarland University) for generating the LifeAct-mEGFP construct. This project was funded by Sonderforschungsbereich 1027 (project A2 to B.Q.) and 894 (project A1 to M.H.). The light-sheet microscope was funded by DFG (GZ: INST 256/4 19-1 FUGG).

Research carried out for this study with the human material (leukocyte reduction system chambers from human blood donors) is authorized by the local ethics committee (declaration from 16.4.2015 (84/15; Prof. Dr. Rettig-St&#252;rmer)) and follows the corresponding guidelines.
1. Preparation of Neutralized Collagen Solution (500 &#181;L)
<ol>
	<li>Transfer 400 &#181;L of chilled collagen stock solution (10.4 mg/mL) to a sterile 1.5 mL tube under the cell culture hood. Slowly add 50 &#181;L of chilled 10x PBS (pH 7.0 -&#160;7.3) to 400 &#181;L of chilled collagen stock solution. Mix the solution by gently tiling the tube. 
	Note: All steps in Part 1 should be done under a cell culture hood.</li>
	<li>Add 8&#160;&#181;L of 0.1 M NaOH into 500 &#181;L of the collagen solution from 1.1. to adjust the pH to 7.2-7.6. Use pH test strips (pH range: 6 - 10) to determine the pH value of the mixture. 
	Note: Volumes may vary for different batches of collagen. NaOH solution has to be mixed slowly to avoid air bubbles. The mixture should be kept on ice to avoid collagen gelation.</li>
	<li>Add 2&#160;&#181;L of sterile ddH2O to make the final volume to 500 &#181;L. Mix well and store this collagen solution (8.32 mg/mL) on ice or at 4 &#176;C until further use. 
	Note: Under this condition, neutralized collagen can be used for 24 h. Aliquots are not recommended to avoid air bubbles.</li>
</ol>
2. Sample Preparation for Light-sheet Fluorescence Microscopy Using Capillaries
<ol>
	<li>Fluorescently label the live cells of interest with the desired primary fluorescent dyes15 or fluorescent proteins11 as described previously.</li>
	<li>Transfer 1 &#215; 106 of cells into a sterile 1.5 mL tube under a cell culture hood. Centrifuge the tube at 200 x g for 8 min. Discard the supernatant and resuspend the pellet in 200 &#181;L of culture medium. 
	Note: The cell density of 5 &#215; 106 cells/mL is recommended for visualization of human immune cell migration, especially for cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.</li>
	<li>Add 85.9 &#181;L of neutralized collagen solution from 1.3. into the cell suspension from 2.2. and mix properly to reach a collagen concentration of 2.5 mg/mL. Leave the cell/collagen mix on ice in the hood. 
	Note: Suppose the desired concentration of collagen is N mg/mL, the volume of neutralized collagen solution (for 200 &#181;L cell suspension) = 200 &#215; N/(8.32 - N)</li>
	<li>Next, put the matching plunger into the capillary (inner diameter ~1 mm) until the plunger is 1 mm out of the capillary. Wet the plunger by dipping into culture medium&#160;(Figure 1A). 
	Note: This step could help to prevent air bubbles when the capillary is dipped into the cell/collagen mix. The capillary and the plunger do not have to be sterile.</li>
	<li>Dip the capillary into the cell/collagen mix from 2.3. Slowly pull the plunger back for 10 -&#160;20 mm (Figure 1B). Wipe the outer wall of the capillary with a paper towel moistened with 70% ethanol spray to remove the remaining collagen solution.</li>
	<li>Mount the capillary&#160;with modeling clay on the inner wall of a 5 mL tube and push the cell/collagen mix to the edge of the capillary&#160;(Figure 1C).</li>
	<li>Keep the capillary at 37 &#176;C with 5% CO2 for 1 h for collagen polymerization.</li>
	<li>Add the culture medium (around 1 - 2 mL) to a 5 mL tube. Carefully press the polymerized collagen rod out into the medium to around 3/4 of the collagen hanging in the medium (Figure 1D).</li>
	<li>Keep the capillary, like this, at 37 &#176;C with 5% CO2 for another 30 min to equilibrate the collagen rod with the medium. 
	Note: Afterwards, the collagen rod can be pulled back into the capillary and cultured further before further use.</li>
</ol>
3. Image Acquisition using Light-sheet microscopy
<ol>
	<li>Assembly the sample chamber according to the manufacturer&#8217;s instruction.</li>
	<li>Turn on the incubation and the microscope to heat the sample chamber to 37 &#176;C (for live cell imaging only).</li>
	<li>Place the capillary in the sample chamber and locate the sample to&#160;find the area of interest for image acquisition.</li>
	<li>Activate the corresponding laser(s). Set the following settings: laser power, exposure time, step-size of z-stack, start and end position of z-stack, and the time interval for live cell imaging. 
	Note: For example, the sample in Figure 2 was imaged every 40 s for 6 h at 37 &#176;C with a step-size of 1 &#181;m (total thickness: 538 &#181;m). The laser power was 1% with an exposure time of 30 ms. The pixel size at x-y direction is 0.23 &#181;m.</li>
	<li>Start the image acquisition.</li>
</ol>
4. Automated Tracking Analysis
<ol>
	<li>Open the file converter, click Add Files to choose the imaging file(s) to be converted into the software file format (*.ims). Click Browse and select a folder to save the converted file(s). Click Start All.</li>
	<li>Open the analysis software. Click Surpass. Go to File and click Open, then choose the imaging file to be analyzed. 
	Note: If the file size is big this step may take time. Files larger than 1 Terabyte (TB) are not recommended for a single experiment as the process such big files are highly computational capacity demanding.</li>
	<li>Click Add New Spots. Check the box Process Entire Image Finally. Click Next.</li>
	<li>Enter the coordinates for x and y (in pixel) to define the region of interest. Enter the number of frames (time and z-position) to analyze. Click Next.</li>
	<li>Select the target channel, which contains the objects to be tracked, in the Dropdown list of Source Channel. Enter Estimated xy Diameter (in &#181;m). Click Next. 
	Note: Estimated xy diameter is the average diameter in the x-y dimension of objects to be tracked.</li>
	<li>Click Quality. Set a threshold, in which most of the cells (objects) should be included. Click Next.&#160;</li>
	<li>Choose the desired algorithm (Autoregressive Motion is recommended). Enter the max distance (20 &#181;m is recommended) and the max gap size (3 or 2 is recommended). Click Process Entire Image finally. 
	Note: Max distance and Max gap size are two thresholds to break tracks. More specifically, in two continuous frames when the distance between the same object exceeds the Max distance, this object in the later frame will be considered as a new object. Sometimes during acquisition, the same object may disappear for a few frames and show up again. In this case, only when this object reappears within the Max gap size, it will be considered as the same object.</li>
	<li>Click Filter Type and choose the option to exclude undesired tracks. 
	Note: This step is optional.</li>
	<li>Click Next and then click Finish. 
	Note: This step can take hours up to days depending on computing performance.</li>
	<li>Click Edit Tracks and choose Correct Drift. Select the appropriate algorithm (Translational Drift is recommended). Select the desired Result Dataset Size (New Size Equal to Current Size is recommended). Click OK. 
	Note: This step is only required when the collagen drifted during image acquisition.</li>
	<li>Click Statistics and choose Configure List of Visible Statistics Values. Check the options of interest to be exported (e.g. coordinates, speed and so on). Click OK.</li>
	<li>Click Export All Statistics to File and enter the file name.</li>
</ol>
5. Fixation and Immunofluorescence Staining of Cells in Collagen Matrices
<ol>
	<li>Transfer 1,000 &#181;L of 4% paraformaldehyde (PFA, in PBS) into a 5 mL tube under a chemical hood. 
	Note: PFA should be balanced to room temperature.</li>
	<li>Dip the capillary with polymerized collagen from 2.9. into PFA solution (for ~ 5 mm) and mount the capillary on the inner wall of the 5 mL tube with modeling clay (as shown in Figure 1C).</li>
	<li>Press the plunger gently until half of the collagen rod is hanging in the PFA solution (Figure 1D). Keep the tube at room temperature for 20&#160;min.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Take out the capillary and discard PFA.</li>
	<li>Mount the capillary into a fresh tube and add 1 mL of PBS. Make sure that the capillary is immersed in the PBS.</li>
	<li>Press the plunger gently until half of the collagen rod is hanging in the solution. Mount the capillary on the inner wall with modeling clay.</li>
	<li>Keep the tube at room temperature for 5 min.</li>
	<li>Repeat 5.4. - 5.7 for another 2 times.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Discard PBS. Transfer 1 -&#160;2 mL of blocking/permeabilization buffer (PBS + 1-% BSA + 0.1% non-ionic surfactant) into the tube and repeat 5.6. 
	Note: The BSA (1%) can be replaced by&#160;5% serum of the animal the secondary Ab was raised in.</li>
	<li>Keep the tube at room temperature for 30 - 60 min.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Discard the permeabilization buffer. Transfer 200 - 500 &#181;L of the primary antibody in blocking/permeabilization buffer and expels the rod into the solution.</li>
	<li>Keep the tube at room temperature for 1 h.</li>
	<li>Wash the collagen rod 3 times with PBST (PBS + 0.1-% non-ionic surfactant) as described in 5.4. -&#160;5.8.</li>
	<li>Incubate the rod in&#160;secondary antibody in blocking/permeabilization buffer for 1 h at room temperature. Keep it from light.</li>
	<li>Wash the collagen rod 3 times with PBS as described in 5.4. -&#160;5.8.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Keep the samples in PBS until imaging.</li>
	<li>Scan the samples as described in 3.</li>
</ol>

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The authors declare no financial or commercial conflict of interest.

Most in vitro assays are carried out on a 2D surface, for example in cell-culture plates, Petri-dishes or on coverslips, whereas in vivo cells, especially immune cells, experience mostly a 3D microenvironment. Emerging evidence shows that migration patterns of immune cells differ between 2D and 3D scenarios17. Moreover, the expression profiles of tumor cells are also different in 2D- and 3D-cultured tissues18,19,20. Therefore, to best simulate the physiological conditions in vitro, it is of a great advantage to perform experiments in a 3D context, for instance in 3D collagen matrices. The newly developed approaches, in particular, the light-sheet fluorescence microscopy, enables reliable and reproducible experiments in 3D matrix systems under controlled environmental conditions.
In contrast to a conventional laser-scanning confocal microscopy which illuminates the whole probe, with light-sheet microscopy, only a thin sheet of laser light is generated to illuminate samples. The detection camera is perpendicular to the illuminating plane. Owing to this technique, only the section at the focal plane is exposed to the laser. Therefore, photo-bleaching and phototoxicity can be minimized. In addition, compared to point-scanning microscopy, the acquisition rate is drastically enhanced with light-sheet microscopy (100 - 1,000-fold faster) and the signal-to-noise ratio is also ameliorated. Combined with the incubation chamber of light-sheet microscopy setup used in this paper this microscope is a powerful option for image acquisition of large 3D volumes with minimized bleaching and photo-damage over extended periods of time (several days) under incubator conditions.
For sample preparation with the 3D collagen matrix, the most critical point is to avoid air bubbles. Given its high viscosity, when air bubbles are introduced to the collagen solution it is very difficult to remove it from the solution. The presence of air bubbles can induce heterogeneity of the polymerization of collagen matrix; in addition, it can block the path of light, which would diminish the quality of images. Therefore, when transfer or mix the collagen solution one must pipette very gently and do not expel the last drop in the pipette tip. Moreover, collagen starts to slowly polymerize at room temperature. Thus, to prevent polymerization before mixing the solution well, the collagen-containing tubes should be always kept on ice.
One big challenge we encounter in employing light-sheet microscopy is data handling. The data generated by light-sheet microscopy can easily reach the size of 500 gigabytes or even several terabytes, in particular for long-term experiments. To analyze these data takes several days, sometimes up to a week by a work-station with high calculation capacity. Since the size of analyzed images is not smaller than the original data the capacity of storage can quickly become a limiting factor for the application of light-sheet microscopy. Therefore, for tracking experiments or if complex image analysis is needed it is highly recommended to limit the file size per experiment to values that can be handled in a reasonable time frame by the available processing units.
Light-sheet fluorescence microscopy introduced in here has some features, which may affect the results if not handled carefully. The utilization of capillaries is particularly advantageous for a barrier-free illumination of the samples. Since the collagen rod is hanging in the medium, it may happen that the collagen rod drifts during image acquisition, especially for long-term measurements. This drift has to be corrected to avoid misinterpretation of the results. Of course, there are other light-sheet microscopy setups commercially available on the market, which do not require capillaries to mount the samples. Furthermore, with the light-sheet microscopy, besides the fact that compared to the conventional line scanning confocal microscopy phototoxicity has been substantially reduced, phototoxicity still remains a concern for long-term imaging21.
Apart from light-sheet microscopy, there are other approaches established for the 3D fluorescence imaging, among which the mostly applied are confocal microscopy, wide-field fluorescence microscopy, and multiphoton microscopy. In terms of penetration depth, confocal and wide-field fluorescence microscopy lie in the similar range, normally limited to 100 nm22. Owing to the longer wavelength light utilized for multi-photon microscopy, its penetration depth can be enhanced to the range of 0.5 - 1 mm23. In comparison, the size of multi-dimensional images generated by optical sectioning of light-sheet microscopy can be up to several millimeters24. In terms of lateral resolution, an elegantly performed experiment shows that light-sheet microscopy provides a better three-dimensional spatial resolution compared to confocal fluorescence microscopy in large samples25. Recently, light-sheet microscopy has been combined with two-photon technique, which further improves the penetration depth compared to normal one photon light-sheet microscopy and is ten times faster than point-scanning two-photon microscopy26.
Considering 3D matrix that provides the micro-architecture for cells, besides bovine collagen I used in this paper, there are many other options, such as extra-cellular matrix (ECM) derived from mouse sarcoma cells, agarose, synthetic hydrogels or other biocompatible polymeric materials. Mouse sarcoma ECM is a protein mixture including collagen IV, laminin as well as numerous growth factors. Compare to the synthetic materials, biological ECMs (collagen and mouse sarcoma ECM), on one hand, present a more physiologically relevant context; but on the other hand, the quality and composition biological ECMs may vary between batches and companies. In contrast, the reproducibility of synthetic materials is more guaranteed. Remarkably, fine-tuning of biochemical and mechanical properties, as well as desired modifications, is allowed for synthetic materials, but not for biological ECMs10,27.
In summary, in this paper we described a protocol to construct 3D collagen matrix for cell biology experiments using light-sheet fluorescence microscopy, and how to embed primary human CTL in the 3D collagen matrix to visualize and analyze CTL migration. We highlighted the key points in sample preparation, image acquisition, and the analysis. Our results show that formation of highly dynamic protrusions and fine actin structures can be visualized by light-sheet microscopy with a good spatiotemporal resolution. Moreover, the analysis protocol described here enables us to reliably track cells in an objective and automated manner. This method will be especially advantageous for human immunological research as mice and humans differ substantially in many immunological functions and reactivity to therapies28.

Most knowledge about migrating cells comes from 2D experiments1,2,3, which are normally conducted in a glass or plastic surface of a culture/imaging dish. However, a physiological scenario requires, in most cases, a 3D microenvironment, in which the extracellular matrix (ECM) plays a decisive role. ECM not only provides the 3D structure essential to maintain proper cell morphology but also offers survival signals or directional cues for an optimal functioning of many cells4,5 . Therefore, a 3D environment is required to better identify cellular functions and behavior in an environment better reflecting the physiological context.
In the human body, most cells especially immune cells, exert their functions under a 3D scenario. For example, activated T cells patrol tissues searching for target cells, na&#239;ve T cells migrate through lymph nodes in search for their cognate antigen-presenting cells during which the migration mode and machinery are adapted to the corresponding extracellular environment3,6,7. The 3D collagen gel has been widely used as a well-established and well-characterized 3D cell culture system8,9,10. Our previous work shows that primary human lymphocytes are highly mobile and migrate at an average speed of around 4.8 &#181;m/min in a 0.25 % collagen-based matrix11. Rearrangement of cytoskeleton plays a key role in the cell migration12. Accumulating evidence shows that lymphocytes do not apply only a single mode of migration yet can switch between certain migration behavior depending on the location, microenvironment, cytokines, chemotactic gradients, and extracellular signals which tune the migratory behavior in different ways 3.
To reliably analyze immune cell functions and behavior, for example, migration, protrusion formation or vesicular transportation, it is of great advantage to be able to acquire images in relatively large 3D volumes in a fast and reliable manner. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) offers a satisfactory solution13,14. During imaging acquisition, a thin static light sheet is generated to illuminate the sample. In this way, on the focus plane, a large area can be illuminated simultaneously without affecting the off-plane cells. This feature enables a high acquisition speed with a drastically reduced bleaching and photocytotoxicity. In this paper, we describe how to visualize primary human immune cells using light-sheet microscopy and how to analyze the migration in a 3D scenario.

<table><tbody><tr><td>Fibricol, bovine collagen solution</td><td>Advanced Biomatrix</td><td>&amp;nbsp;#5133-20ML</td><td>Collagen matrix</td></tr><tr><td>0.5 M NaOH Solution</td><td>Merck</td><td>1091381000</td><td>for neutralizing Fibricol solution</td></tr><tr><td>Ultra-Low melting agarose</td><td>Affymetrix</td><td>32821-10GM</td><td>Sample preparation in low c[Col]</td></tr><tr><td>Dynabeads Untouched Human CD8 T Cells Kit</td><td>Thermo Fisher</td><td>11348D</td><td>Isolation of primary human CD8&lt;sup&gt;+&lt;/sup&gt; T cells from PBMC</td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation</td><td>Thermo Fisher</td><td>11132D</td><td>Activation of CTL populations</td></tr><tr><td>Human recombinant interleukin-2</td><td>Thermo Fisher</td><td>PHC0023</td><td>Stimulation of cultured CTL</td></tr><tr><td>P3 Primary solution kit</td><td>Lonza</td><td>V4XP-30XX</td><td>Transfection</td></tr><tr><td>&amp;alpha;-PFN1 antibody, rabbit, IgG</td><td>Abcam</td><td>ab124904</td><td>IF</td></tr><tr><td>Alexa Fluor 633 Phalloidin</td><td>Thermo Fisher</td><td>A22284</td><td>IF</td></tr><tr><td>CellMask Orange Plasma membrane Stain</td><td>Thermo Fisher</td><td>C10045</td><td>Fluorescent cell label</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-250mL</td><td>IF</td></tr><tr><td>Triton X-100</td><td>Eurobio</td><td>018774</td><td>IF</td></tr><tr><td>DPBS Dulbecco&#039;s phopsphate buffered saline</td><td>Thermo Fisher</td><td>14190250</td><td>IF</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9418-100G</td><td>IF</td></tr><tr><td>Goat &amp;alpha; Rabbit 568, IgG, rabbit</td><td>Thermo Fisher</td><td>A-11011</td><td>IF</td></tr><tr><td>Lightsheet Z.1 (Light-sheet microscopy)</td><td>Zeiss</td><td>N.A.</td><td></td></tr><tr><td>Cell culture hood</td><td>Thermo Fisher</td><td>HeraSafe KS</td><td></td></tr><tr><td>Cell culture incubator HERACell 150i&amp;nbsp;</td><td>Thermo Fisher</td><td>N.A.</td><td></td></tr><tr><td>Centrifuge 5418 and 5452</td><td>Eppendorf</td><td>N.A.</td><td></td></tr><tr><td>Pippettes</td><td>Eppendorf</td><td>3123000039, 3123000020, 3123000063</td><td></td></tr><tr><td>Pippette tips</td><td>VWR</td><td>89079-444, 89079-436, 89079-452&amp;nbsp;</td><td></td></tr><tr><td>15 mL tubes</td><td>Sarstedt</td><td>&amp;nbsp;62.554.002</td><td></td></tr><tr><td>Capillaries 50 &amp;micro;L</td><td>VWR (Brand)</td><td>613-3373</td><td>Zeiss LSFM sample preparation</td></tr><tr><td>Plunger for capillaries</td><td>VWR (Brand)</td><td>BRND701934</td><td>&quot;Stamps with Teflon tip&quot; LSFM sample preparation</td></tr><tr><td>MColorPhast pH stips</td><td>Merck</td><td>1095430001</td><td>to test pH of neutralized Fibricol</td></tr><tr><td>BD Plastipak 1mL syringes</td><td>BD</td><td>Z230723&amp;nbsp;ALDRICH</td><td>Alternative sample preparation</td></tr><tr><td>Modeling clay (Hasbro Play-Doh A5417EU7)</td><td>Play-Doh</td><td>N.A.</td><td></td></tr><tr><td>Imaris file converter</td><td>Bitplane</td><td>available at http://www.bitplane.com&amp;nbsp;</td><td>Convert imaging files to Imaris file format</td></tr><tr><td>Imaris 8.1.2 (MeasurementPro, Track, Vantage)</td><td>Bitplane</td><td>available at http://www.bitplane.com&amp;nbsp;</td><td>Analysis of 3D and 4D imaging data</td></tr></tbody></table>

light-sheet microscopy for three-dimensional visualization of human immune cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Protrusion formation during T cell migration is a highly dynamic process, which is actin dependent. To visualize protrusion formation of primary human CTL, we transiently transfected a mEGFP fused protein to label the actin cytoskeleton in CTL as described before11. One day after transfection, the cells were embedded in the collagen matrix. Image stacks were acquired every 40 s with a step-size of 1 &#181;m at 37 &#176;C using light-sheet microscopy. As shown in Figure 2A and Supplementary Movie 1, during migration, human CTL form lemon-shaped major protrusions, fringed by fine spindle-like structures. In the experiment shown here, not only one single cell was imaged but a large volume (450 x 450 x 538 &#181;m3) (Figure 2B). Thus, the optical quality and resolution shown here were obtained with a low magnification objective (20X&#160;/ 1.0 DIC VIS IR). Therefore, the resolution could be further improved with higher NA objectives.
In the experiment shown in Figure 2 and Movie 1, CTLs were automatically tracked as described in the protocol Part 4. The trajectories of CTL are depicted in Figure 2B. Two parameters of migration were analyzed: velocity and persistence. Persistence is defined as the displacement divided by the total track length. The analysis shows that the mobility of CTL is very diverse in 3D: the velocities range from 0.01-0.19 &#181;m/s with an almost 20-fold difference, and the persistence ranges from 0 - 0.7 (Figure 2C). It was reported in mice that in vivo interstitial migration average velocity of T cells was 4 &#181;m/min16. Recently, we have determined the average velocity of CTL to be 4 - 5 &#181;m/min11 using the methods described in this paper. These results demonstrate that light-sheet microscopy is a powerful tool to visualize cell behavior, for example, cell migration and cell-cell interaction. In vivo, many factors and a non-homogeneous ECM of the complex composition including soluble factors that can affect migration will modify T cell behavior.
Application of a collagen matrix is not limited to live cells. Fixation and immunostaining can also be performed in the matrix. Figure 3 shows an example of fixed samples in 3D collagen gel, in which endogenous perforin1 (PFN1) and actin were stained. The sample was illuminated either from a single side (Single side illumination) or from both sides (Dual side illumination). We observe that cells at different z-positions are evenly stained, indicating that procedures described in our protocol achieve satisfactory penetration of antibodies into collagen gels. More importantly, after fixation CTL exhibited the same morphology as in live cell imaging (compare Figure 3 and Figure 2B), indicating that also the morphology is well maintained by this protocol. In addition, illumination from the single side or from both sides does not make a significant difference in the quality of images at the focal plane. Considering that less area is exposed to the laser with the mode of single side illumination compared to dual side illumination, single side illumination is more recommended.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57651/57651fig1.jpg" /> 
Figure 1: Illustration of handling capillaries during sample preparation. (A) Put the plunger into the capillary and how to wet the plunger with the culture medium. (B) Pull the cell/collagen mix into the capillary. (C) Mount capillaries using modeling clay. The plunger and the collagen rod are depicted in gray and red boxes, respectively. (D) Handle collagen rod for equilibration with the culture medium or for immunostaining. The plunger and the collagen rod are depicted in gray and red boxes, respectively. <a href="/files/ftp_upload/57651/57651fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57651/57651fig2v2.jpg" /> 
Figure 2: Tracking primary human CTL in 3D collagen and visualizing actin dynamics. (A) Maximum intensity projection of actin in primary human CTL. Actin was labeled in CTL as previously described11. Day one post transfection cells were embedded in 0.5 % collagen matrix. One representative cell is shown. Scale bars are 5 &#181;m. Fine actin structures at the protrusion edge are highlighted by the arrowhead. (B) Trajectories of CTL for 6 h. The trajectories are automatedly tracked by the software (Color code: blue = start, red = end of the track, 450 &#215; 450 &#215; 538 &#181;m3 volume). The scale bar is 50 &#181;m. (C) Quantification of velocity and persistence. One dot represents one cell. Results are shown as mean &#177; SEM from 4 donors. <a href="/files/ftp_upload/57651/57651fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57651/57651fig3v2.jpg" /> 
Figure 3: Immunostaining of endogenous profilin1 (PFN1) and actin in CD8+ T cells embedded in the 3D collagen matrix. Primary human CD8+ T cells were embedded in 0.25% collagen. After PFA fixation the sample was stained using anti-PFN1 (orange) and phalloidin (purple) and visualized by light-sheet microscopy at a step-size of 1 &#181;m for a total volume of 440 x 440 x 1,000 &#181;m3. The sample was illuminated either from a single side (Single side illumination) or from both sides (Dual side illumination). Maximum intensity projections (MIP) from 3D reconstruction are shown. The scale bars are 50 &#181;m. <a href="/files/ftp_upload/57651/57651fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/57651/57651mov1.jpg" /> 
Movie 1: Generation of protrusions during T cell migration. The cell shown in the movie was taken from the volume shown in Figure 2A. Cells were embedded in a collagen matrix (0.5%). Images were acquired every 40 s for 6 h at 37 &#176;C using light-sheet microscopy. <a href="/files/ftp_upload/57651/Movie_1.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells at JoVE.com

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

light-sheet-microscopy-for-three-dimensional-visualization-human

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.1K Views.  Saarland University. Hier presenteren we een protocol om te visualiseren van immune cellen die zijn ingesloten in een matrix van de driedimensionale (3D) collageen met behulp van licht vel microscopie. Dit protocol wordt ook ingegaan hoe bijhouden van cel migratie in 3D. Dit protocol kan worden gebruikt voor andere soorten schorsing cellen in de 3D-matrix.

Video: Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

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

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

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

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

Engineering

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Biochemistry

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

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 has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Bioengineering

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

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

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

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.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Medicine

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

Tissue clearing, combined with immunofluorescence microscopy, allows spatial visualization and quantification of immune-cell populations and virus proteins within intact tissues. Optical sectioning of cleared tissues with confocal and light sheet fluorescence microscopy can generate 3D models of complex tissue environments and reveal spatial heterogeneity exhibited during HIV infection.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 ...

Biology

Customizable Fluorescence Microscope to Visualize 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.

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.

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

Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells

Source: Rosenberg, J., et al. <a href="https://app.jove.com/v/59914">Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy.</a> J. Vis. Exp. (2020).
In this video, we describe the lattice light-sheet microscopy technique to visualize interactions between live T-cells and antigen-presenting cells. The method involves focusing an ultrathin lattice light sheet on an interacting cell pair to obtain a series of two-dimensional images, which are combined to obtain high-resolution four-dimensional images that reveal the spatiotemporal dynamics of the cell-cell interaction.

Source: Rosenberg, J., et al. Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy. J. Vis. Exp. (2020).
...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

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