Supplementary Manual 1. Instructions for Building the Upright and Inverted Macroscopes

Supplementary Manual 1. Instructions for building the upright and inverted macroscopes.

We strongly recommend that users build and use the upright fluorescence macroscope made from PVC tubing. This macroscope is completely water proof, can be easily assembled in a few hours using simple tools (a PVC cutter, a drill, and wire snips), and provides better images that the inverted macroscope. By contrast, the inverted LED fluorescent macroscope requires knowledge of woodworking and special tools including saws, routers, and glasscutters. Moreover, wood will break down over time when exposed to water and is hard to clean. We describe both here for illustrative purposes.

Building an inverted LED Fluorescent Macroscope using wood

1. Cut wood to size using a circular saw or chop saw where appropriate:

Wood comes in standard sizes when purchased commercially, however, sizing can be misleading. For example, a 12” x1” board is really 11 ¼” x ¾”. We provide the commercial sizing below and true sizes in parenthesis. For consistency, we will describe wood pieces as real sizes in the remainder of this manual.

19 1/2” x 12” x 1” pine board (true size is 19 ½” x 11 ¼” x ¾”) for back 21 ½” x 12” x 1” pine board for two sides (true size is 21 ½” x 11 ¼” x ¾”). 19 ½” x 2” x 1” pine board x 2 (true size is 19 ½” x 1 ½” x ¾”) for support bars on the front of macroscope. 19 ½” x 20” plywood board 5/8” thick for top (sizing correct) z19 ¼” x 19 ¾” plywood board 5/8” thick for inside bottom piece (sizing correct) 19 ¼ x 19 ¾” plywood board ¼” thick for bottom (sizing correct). 14” x 5/8” hard wood dowel for supports of macroscope shelf (sizing correct) 20” x 5/8” hard wood dowel for supports of macroscope shelf (sizing correct) 19 ¼” x 2”x 1” pine board (true size is 19 1/4” x 1 ½” x ¾”). Two pieces are required to make up the bottom supports of macroscope. 18” x 3”x 1” pine board (true size is 18” x 2 ½” x ¾”). Two pieces are required to make up the front and back of the camera housing. 3 ½” x 2” x 1” pine board (true size is 3 ½” x 1 ½” x ¾”). Two pieces are required to make up the sides of the camera housing.

2. Cut the hole for the light switch box:

(A) Mark a 3 ¾” wide by 6” long box on one of the sidepieces (21 ½” x 11 1/4” x ¾” pine board). The box should be 2 ¼” from the bottom and 1 ½” from the front (see Supplementary Figure 1A). (B) Remove the 3 ¾” wide by 6” piece of wood using the ¼” router bit. CRITICAL STEP The router should be run on the inside of the lines to remove the correct amount of wood and the cut should be completed using several passes removing only about ¼” of wood per pass. A guide will aid in making straight cuts. Alternatively, a jigsaw can be used to make this cut.

1 3. Make the inside bottom piece of the macroscope:

(A) Take the pre-cut 19 ¼” x 19 ¾” plywood board 5/8” thick and mark lines 4 ½” from each of sides extending to within 1 ½” of the ends. Also mark a line at 9 ¾” from one of the sides and extending to within 1 ½” of the ends (see Supplementary Figure 1C). Lines should run parallel to the 19 ¾” edge of the plywood. (B) Using the ¼” router bit, follow the lines drawn on the plywood. A guide should be used to ensure that the router cuts are straight. The router cut should bisect the line and should be completed using several passes with the router.

4. Make the wooden top of the macroscope:

(A) Take the pre-cut 19 1/2” x 20” plywood board 5/8” thick and mark a rectangle 15 ½” wide by 5” long. The 15 ½” line should run parallel to the 19 ½ edge, while the 5” lines run parallel to the 20” side. This rectangle should be positioned 2” from the sides and 7” and 8” from the front and back edges of the plywood (see Supplementary Figure 2E). (B) Using the ¼” router bit, follow the lines drawn on the plywood. A guide should be used to ensure that the router cuts are straight. The router cut should be on the inside of the line exposing a perfect 15 1/2” x 5” rectangle cut out.

5. Assemble the sides of the macroscope:

(A) The side of the macroscope is made of a piece of 19 1/2” x 11 1/4” x ¾” pine board and should be positioned inside of the two sidepieces (Supplementary Figure 1C). The sidepiece with the cut out for the electric switch box should be on the right side positioned closest to the front (Supplementary Figure 1B,E). The sidepieces are 21 ½” x 11 1/4” x ¾” pine board. Three #6 wood 1 ¼” screws should be used on each side connection to attach the side to the back piece. (B) Having made a three-sided box, add support bars to the front of the macroscope. These are the 19 ½” x 1 1/2” x ¾” pine board pieces and are positioned inside of the two sidepieces. These pieces should be connected so that the top and bottom edges are flush with the box (see Supplementary Figure 1B). Two screws per side are enough to secure the pieces together. (C) Next, attach the base of the macroscope. Turn the macroscope box over so that the bottom is facing up. Measure 1” from the bottom and mark a line on the front and back of the macroscope. This line demarks were the 19 ¼” x 1 1/2” x ¾” pine board bottom supports will be attached. Line up the supports so that the wider face is exposed and so that the top is on the line (Supplementary Figure 1D, denoted by arrows). Only 1/4” of macroscope box structure can be viewed from below. Screw bottom supports into place using four #6 wood 1 ¼” screws from the outside of the macroscope (Supplementary Figure 2D, red arrows). Turn the macroscope right side up. (D) With the macroscope right side up, place the grooved inside bottom piece into the macroscope box so that the grooves are parallel to the right and left sides. 1 ¼” #6 wood screws should be used to attach the bottom of the macroscope to the 19 ¼” x 1 1/2” x ¾” pine board bottom supports (Supplementary Figure 2D, green arrows).

2 (E) A 1” hole should drilled near the electric switch box opening to receive the wires (see Supplementary Figure 1 A-E).

6. Install the light fixtures

(A) The lights and bases should be screwed together as per the manufacturer’s instructions. (B) The wires for each light are passed through the grooves in the bottom of the macroscope (Supplementary Figure 1E,F). (C) The lights are fastened in place by one 1 1/2” 8-32 knob screw, a 8-32 wing nut, and a #10 zinc patted washer (Supplementary Figure 2C). Specifically, the washer is placed on the screw and then threaded through the grove so that the head and washer is exposed from the bottom of the macroscope. The screw is then inserted in the front hole of the light and held in place with a wing nut, allowing the position of the lights to be moved by sliding the lights along the grove and securing in place by tightening the wing nut (Supplementary Figure 2C). (D) Repeat this procedure for all six lights.

7. Wire the macroscope for power: CAUTION Some knowledge of basic electrical wiring is required.

(A)A detailed outline concerning proper wiring techniques is shown in Supplementary Figure 3A-D. Briefly, all connections should be made by twisting the exposed wires together (1/2” exposed wire per connection, Supplementary Figure 3A). Wire nuts, also called wire connectors (Buchanan #73208, Home Depot, Supplementary Figure 3B), are then screwed into place. Use black electrical tape to wrap the connections at least 3 times to ensure that connectors cannot be disengaged and expose live wires (Supplementary Figure 3C-D). (B) Turn the macroscope over so that the bottom is facing upwards. The first connections to be made are the black wires from opposing light sources (Supplementary Figure 3E). Specifically, a single wire should be cut leaving enough play to allow the lights to be moved easily (20-24”). The wire should be exposed by removing ½” of the wire plastic coating from each end. Black/Positive wires are connected by twisting them together. Do not secure with wire nuts. (C) Cut a 24” length of 16 Gauge Indoor Light-duty cord (15’, #0070296, Home depot). Wire on all four ends should be exposed. One wire should be attached to the black wire connection closest to where the electric switch box will be positioned. Repeat for all three circuits and then attach wire nuts and secure with electrical tape (Supplementary Figure 3E, denoted by red arrows). An electrical diagram is shown in Supplementary Figure 3F to aid in wiring the macroscope. (D) The white wires can now be attached in series with a 12” length of wire (Supplementary Figure 3F). Twist the wires together but do not add the wire nuts. (E) A 6-foot length of 16 Gauge Indoor Light-duty cord that includes the plug should now be cut. The ends of both wires should be exposed. One end should be attached to the set of white connections that is closest to the switch box. This now completes the neutral circuit. Six wire nuts should be fastened to all six white/negative connections and secured with electrical tape.

3 (F) The remaining exposed wires (three total that are associated with the black connections) should be connected together with the remaining exposed wire from the cord that includes the plug. There will be FOUR wires twisted together in this instance. If there is not enough play in the wires, the wires can be separated by pulling. This connection should be secured with a wire nut and black electrical tape. (G)At this point, no exposed wires should be visible with exception of the three long cords each having two exposed wires. (H)The three wires should be thread through the 1” hole in the base of the macroscope (Supplementary Figure 1F, blue arrow). At this point, the macroscope should be turned so that it is upright. (I) Next all three cords should be thread through the same port of the electric switch box (Carlon Three Gang, 44 Cu. In., Blue, Nonmetallic Switch and Outlet Box (Model # B344AB, Home depot, Supplementary Figure 4B). CRITICAL STEP The plastic wings with associated nails on the electric switch box should be removed prior to this step using a coping saw. The tab at the top of the switch box should NOT be cut and will be the primary attachment point for the electric switch box to the macroscope. (J) The electric switch box should now be fastened to the macroscope by both hot glue on the top tab (Supplementary Figure 2A) and the bottom edge. The tab should be secured from the inside of the macroscope. A small nail can also be used to further secure the electric switch box. (K)The end of each cord protruding into the electric switch box should be fastened to a toggle switch as shown in Supplementary Figure 5A. Next, the toggle switches are attached to the electric switch box by two screws. Make sure all switches are oriented in the same direction (Supplementary Figure 5B). Finally, a plastic plate cover is positioned over the toggle switches (Supplementary Figure 5C). (L) The macroscope is now correctly wired. Screw the six LED fluorescent light bulbs into the fixtures so that the same LED lights are across from one another. Once the bulbs have been inserted, plug in the macroscope. Turn on and off each switch to test. Once you have ensured correct wiring the ¼” plywood bottom (19 ¼ x 19 ¾”) can be attached by flipping over the macroscope and securing with hot glue and/or nails.

8. Retrofit the cameras:

(A)Remove the flexible arms from the Logitech QuickCam with wire cutters. (B) Cut a 2 ¼” x 1 ½” piece of ¼” plastic. A ¼” hole should be drilled completely through the middle of this plastic piece (Supplementary Figure 6A). Next, a ½” drill bit should be used to create a recess for the bolt head (Supplementary Figure 6B). The plastic piece should be affixed to a scrap piece of lumber by clamps to ensure that the piece does not rotate and to allow the drill to pass into all the way through the plastic piece. (C) Next, a 1 ½” 8-32 knob screw (Model # 28992, Home depot) is inserted into the hole of the plastic piece. The head of the bolt should be completely recessed. Secure the bolt to the plastic with superglue (Supplementary Figure 6B). (D)The plastic piece can now be attached to the bottom of the camera using super glue (Supplementary Figure 6C). (E) Cut a 1” PCV pipe to create a ¼” ring. The internal diameter of the 1” PVC pipe is approximately 25mm and is the correct size to receive the optical filter. This PVC ring is

4 attached to the camera by hot glue so it can be removed if necessary (Supplementary Figure 6D).

9. Assemble and install the camera housing:

(A)Draw a line 1” from the long edge of one of the 18” x 2 1/4” x 3/4” board pieces. The line should extend to 1 1/2” from the sides. Using a ¼” router, cut a groove completely through the wood piece. This will provide the attachment site for the cameras (Supplementary Figure 6E). (B) Make a box by gluing the four boards together (two pieces are 18” x 2 1/4” x 3/4” and two are 3 ½ x 2 1/4” x 3/4”, Supplementary Figure 6G). (C) The bolt for each camera can now be inserted into the groove of the camera housing. A washer and wing nut are used to secure the camera to the housing (Supplementary Figure 6E). Position the camera by loosening the wing nut and sliding it along the grove and by rotating the camera directly. A second camera can be used for multispectral imaging (Supplementary Figure 6F,G). (D)Position the completed camera within the macroscope between the light fixtures (Supplementary Figure 6H). Ensure that the wing nuts are positioned at the front of the macroscope to aid in fine adjustments when in use.

10. Assemble the top of the inverted macroscope:

(A) Use a pencil to mark 2 1/2” below the top of the macroscope on the left and right side (Supplementary Figure 2C). With this line in place, hot glue can be used to attach the 5/8” hard wood dowel macroscope shelf supports (Supplementary Figure 2B). The line should be even with the top of each support (see Supplementary Figure 2C). Affix a 20” square dowel to the left side and the 14” piece to the right (Supplementary Figure 2B). The 14” piece does not extend the full length of the box. (B) The wood top of the macroscope can now be positioned (Supplementary Figure 2D). The imaging field can be adjusted by moving the wood top front to back. (C) Make a pexiglass cover by cutting a 1/8” sheet Plexiglass with a glasscutter. The glasscutter should be used with a straight edge and scratched 10 times into the full-length of the Plexiglass. The plexiglass is cut by exerting pressure on the plastic from above while aligning with the edge of a desk, causing the plastic to snap apart. A plexiglass cover of 19 ½” x 20” should be cut. CAUTION Do not remove the protective cover until the plexiglass has been cut to ensure scratches are not created during the cutting process. (D) The plexiglass cover can now be positioned over the wood top.

5 Building an upright LED Fluorescent Macroscope

The upright fluorescent macroscope allows the investigator much more functionality at reduced cost and ease of building; however, it must be used in a dedicated dark room space. Since the wiring is contained within the body of the PCV shell, the upright macroscope (Supplementary Figures 7 and 8) is completely waterproof and can be easily cleaned. Imaging occurs from above and therefore necessarily has different uses than the inverted macroscope. We strongly encourage users to build the upright LED fluorescent macroscope.

11. Cut ¾” PVC pipe to the appropriate sizes using either a PVC cutter (recommended) or a chop saw: 2 x 1 ½” The quantity needed is denoted on the left, and length on the right. 8 x 2” 1 x 2 ¾” 8 x 3” 1 x 4” 3 x 5 ¾” 1 x 7” 2 x 11 ¼” 1x 14 ¾” 3x18”

12. Thread light fixture wires into the ½” threaded opening of a ¾” x ¾” x ½” threaded T shape PVC connector (Supplementary Figure 10A,B). Then screw each of the light fixtures into place. The lights may not screw entirely into this T connection.

13. Create 6 corner joints by connecting a ½” threaded ¾” slip straight PVC connector with a slip ¾” PVC adaptor, as shown in Supplementary Figure 9B-D.

14. Cut five 1/4” holes through the center of one of the 18” PVC pipes (Supplementary Figure 12A,B). Holes should be centered in the middle of the pipe and lie 7”, 8”, and 9” from the end of the pipe accordingly. Drill all the way through both sides of the pipe as shown in Supplementary Figure 12.

15. Dry fit the macroscope together as per the diagrams depicted in Supplementary Figure 7 and 8.

16. Wire the macroscope for power: CAUTION: Some knowledge of basic electrical wiring is required.

(A)A detailed outline concerning proper wiring techniques is shown in Supplementary Figure 3A-D. Briefly, all connections should be made by twisting the exposed wires together (1/2” exposed wire per connection, Supplementary Figure 3A). Next, wire nuts, also called wire connectors (Buchanan #73208, Home depot, Supplementary Figure 3B), are screwed into place. Finally, wrap the connection with black electrical tape to ensure that connectors cannot be disengaged and expose live wires

6 (Supplementary Figure 3C). Each connection should be wrapped at least three times with electrical tape (Supplementary Figure 3D). (B) The first connections to be made are the black wires from opposing light sources (Supplementary Figure 11). All wiring is internal and thus, wires should be threaded through the PVC tubing leaving enough wire for connection with the switch box. A single wire should be cut leaving (36-48”) and ½” of the plastic removed to expose the wire. Wires can be twisted together. Attach wire nuts and secure with black electrical tape as depicted in Supplementary Figure 3. (C) The white/negative wires can now be attached in series with 12”-48” lengths of wire. Wire should be threaded through the PVC tubing and excess wire removed when new connections are made. (D)The three wires should now be threaded through the switch box from the back of the macroscope. The white wire (negative) will NOT make connections to switches in the box. Connect one black wire (+) to one switch and the second to the other. Make sure to screw the connection down tightly. (E) The remaining switch ports can now be wired together in series through use of a 6” wire. Tighten one of the ports with the screw. The remaining connection should not be tightened as it will receive a second wire connection in step h. (F) Drill a ¼” hole in the ¾” PVC end cap (Supplementary Figure 10D). (G)Cut an 8-foot length of 16 Gauge Indoor Light-duty cord that includes the plug. The ends of both wires should be exposed. Thread the wire through the drilled hole in the end cap and then through the body of the macroscope. The wire should connect into the switch box from the front of the macroscope. (H)Attach the wire ends of the 8’ length of 16 Gauge Light-duty cord to complete the circuit. Specifically, one end should be attached to the negative wire and secured with wire nuts and electrical tape. The second wire should be twisted with the 6” wire from step e and the two connected to the switch by securing to tightening the screw. (I) Secure the faceplate of the switch box with screws and install the waterproof cover (Supplementary Figure 7F and 10C). (J) The macroscope is now correctly wired and can be tested by screwing in four LED fluorescent light bulbs. The machine has been wired to turn on lights that are catty- corner from one another (Supplementary Figure 11). Once the bulbs have been inserted, plug in the macroscope, and turn on and off each of the switches to test. CRITICAL STEP Do not use PVC glue to make the connections. This allows modifications to connections as needed and allows for potential upgrades (i.e addition of another set of lights in the future). The macroscope is waterproof without glue.

17. Retrofit the cameras and attach them to the macroscope:

(A) Follow steps outlined above in Section 8a-d. (B) Cut a 1” PCV pipe to create a 1/2” ring and a ¾” PVC pipe to create a 1/8” ring. The 1” PVC internal diameter is approximately 25mm or ¾”. Insert the 1/8” ring made from ¾” PVC into the other ring. A filter can now be inserted into the housing and attached to the camera by hot glue so it could be removed in the future if necessary. CAUTION It is not possible to change filters that are attached to cameras as outlined above. The inverted macroscope used in this protocol has four cameras affixed with different filters that can

7 be easily interchanged as needed. Cameras with attached filters should be removed from the macroscope when not in use and stored in a dry, dust-free place for long-term storage. (C) The bolt for each camera can now be inserted into the holes in the PVC tubing Supplementary Figure 12C). A wing nut is used to secure the camera to the housing (Supplementary Figure 12D). The camera angle can be moved by rotating the camera directly. A second camera can be added for multispectral imaging. (D) Plug the cameras into the USB port of a computer. The upright LED fluorescence macroscope is now functional and ready for use.