alyonaminina/ropod.hardware

RoPod

RoPod is a compact, pod-like chamber for advanced microscopy of plant roots. Plants are grown and imaged in the same chamber - no need to move seedlings from the cozy growth medium onto a hostile microscopy glass and no need to worry about seedlings drying up during imaging! No stress!

RoPod's top and bottom is made out of microscopy-grade glass (coverslip with thickness 0.17 mm, #1.5). RoPods with watertight design allow drug treatments and stress-free long-term time-lapse imaging at low and high magnifications. RoPods with lanes for individual roots enable easy tracking of the same biological replicate during experiments.

RoPod is more than just a chamber, it is a toolkit that comprises of customizable chambers of various designs optimized for different applications, detailed protocol on how to use them and examples of semi-automated image analysis. Check out our semi-automated tool for root hair growth

Why use RoPod

• Plants are grown and imaged within the same chamber, thus sample mounting and imaging causes there little to no mechanical stress
  
  
• RoPods prevent samples from drying up during imaging, allowing for long-term time-lapse series
  
  
• Plant growth within RoPod requires very small volume of the medium, thus drug treatment will require small amount of compounds
  
  
• The watertight RoPods allow drug treatment in chambers positioned vertically and horizontally
  
  
• RoPods allows multiple biological replicates to be imaged simultaneously (up to 20 seedlings in RoPod v23)
  
  
• Plants are immobilized within the chamber, hence the same plant can be easily tracked in a RoPod in multiple imaging sessions
  
  
• RoPods with individual lanes prevent roots from crossing over each other during growth, thus making tracking of biological replicates easy
  
  
• RoPods are very cheap and reusable, printing one chamber with a lid takes ca 10g of a PETG or PLA filament and two 24 x 60mm microscopy coverglass (#1.5, i.e. 0.16-0.19 mm thick)
  
  
• The microscopy-grade glass is printed into the plastic and will never detach from the chamber in the middle of your experiment
  
  

How to grow Arabidopsis in RoPods

In this video you can find a short step-by-step demonstration of how to prep RoPod chambers for Arabidopsis thaliana growth.

• Step 1. Sterilize the RoPod. The quickest way to do it is using UV in a laminar flow hood. Place the open chamber and the lid (inner side up) under the UV light, 30 minutes of exposure is usually sufficient.

• Step 2. Cast the growth medium. Thaw 0.5x MS in a microwave, let it cool to 60 C and pipette 3-4 mL into the sterile chamber. Make sure the medium is evenly distributed in the chamber and there are no bubbles.

• Step 3. Remove a strip of the growth medium. Let the medium solidify and then use a tip or toothpick to remove ca 5 mm wide strip of the medium, this will give space for the aerial part of seedlings. For RoPod with arcs (5, v24 etc.) make sure arcs are not covered by the medium.

• Step 4. Transfer seeds. Use a sterile toothpick to place seeds on the bottom glass, by the edge of the medium. For RoPod 5, place the seeds “under” the arches, which are designed to guide young roots into the individual channels

• Step 5. Close the chamber. RoPods with watertight lids do not need to be sealed individually, other RoPods require it. Place the chamber into a square Petri plate and seal it.

• Step 6. Place the RoPod into a plant growth cabinet. Place the plate vertically under growth conditions. It is advisable to slightly tilt the chamber backwards to guide root growth along the bottom coverslip. Lids of some RoPod versions (e.g. v23.4, 23.4 etc) ensure such tilt
  
  

How to print RoPods

In this video you can find a short demo for printing the RoPod chambers.

For printing, you will need:

• access to an FDM 3D printer with a 0.4 mm nozzle
• clear PETG filament, ⌀ 1.75 mm. It is advisable to check the filament for toxicity. To our experience, clear PETG does not impact Arabidopsis growth.
• 24 x 60 mm microscopy coverslip #1.5, i.e. 0.16-0.19 mm thick ( VWR, 630-2108)
• superglue
• .3mf or .stl file for RoPods

Step 1. Generate the g-code:

• Files required for printing are provided in the tables below
• Prior to printing, the models can be tuned using the provided Autodesk .3fd files
• An example of recommended settings for printing each RoPod version is in the provided project file.
• Make sure that the chamber and the lid are positioned with the rims looking upwards and the glass insertion layer closest to the print bed
• Print settings-> Layer height = 0.1 mm (if possible we recommend using variable layer height). Variable layer height feature (available for Prusa printers) allows to use small layer height beneficial for efficient embedding of the glass into the print and a larger layer height for printing the rest of the structure. For an example see the provided Slic3r project files
• Print settings-> Fill density = 50%
• Print settings-> Fill pattern = 3D Honeycomb
• Print settings-> Layers and Perimeters-> Vertical shells-> Perimeters = 4
• Use of brim reduces warping and detachment of the first layers
• Slice the model and insert a pause at the layer right before the layer with the slot for the glass
• You can simultaneously print up to 17 RoPods on a Prusa MK2or MK3. However, we strongly recommend to start with just one

Step 2. Clean the coverslip glasses:

• Remove dust and fat from the glasses by wiping them clean with a paper tissue soaked in acetone​

Step 3. Start the print:

• Start the print and wait until it automatically pauses on the layer programmed into Step 1.
• The extruder will move to the back left of the print bed while waiting for the user to resume the print

Step 4. Place the glasses into the slots:

• The chamber and lid models have ca 0.18mm deep slots for the coverslips
• Add a small drop of super glue to two corners of a glass. This will secure the glass' position during printing
• Use forceps and patience to carefully place the glass into its slot. Make sure it does not stick upwards or sideways
• Press down on the the corners with glue and hold for a few seconds. sic! wear gloves! superglueing oneself to a 90C- hot print bed is not a good way to start your day

Step 5. Resume the print:

• Resume the print and make sure the the filament does not move the glass from its position and does not crack it
• When the chamber is ready make sure there is no filament sticking sideways. If needed, imperfections can be removed with a scalpel

RoPod.Hardware

Below you can find the most used versions of RoPods optimized for a number of different applicaitons. We provide a brief description, .3mf files for printing and Autodesk Fusion 360 .3fd file to be used if fine-tuning of a model is needed.

RoPod v26

• This large RoPod is designed for 64 x 48 x 0.17mm (#1.5) microscopy glass
• Various versions with separators for individual root growth are available
• Chamber with lid on measures 69.2 x 50.7 x 11.2 mm and is designed to fit into ZEISS Universal mounting frame K
• Unlike v25, these RoPods have a lid which fits on seamlessly
• We recommend using 7 ml of medium
• Glass is incorporated into the chamber and the lid during printing
• I recommend using a few drops of superglue not only in the corners, but also along the sides of the glass (due to the size of the glass)

Version	Preview	Files	Comments
v26 lid		Model files Individual model files: RoPod v26 lid.3mf Sliced file (ready to print): RoPod v26 lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v26 lid.f3d	v26 lid is suitable for all RoPods v26
v26.1		Model files Individual model files: RoPod v26.1.3mf Sliced file (ready to print): RoPod v26.1 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v26.1.f3d	---
v26.2		Model files Individual model files: RoPod v26.2.3mf Sliced file (ready to print): RoPod v26.2 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v26.2.f3d	21 channels
v26.3		Model files Individual model files: RoPod v26.3.3mf Sliced file (ready to print): RoPod v26.3 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v26.3.f3d	14 channels Suitable for imaging of roots even in the channels at the sides, as the chamber is redesigned to be more accessible for objectives with large magnification
v26.4		Model files Individual model files: RoPod v26.4.3mf Sliced file (ready to print): RoPod v26.4 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v26.4.f3d	4 compartments to grow 4 genotypes close to each other but avoid mix-ups Suitable for imaging of roots even in the channels at the sides, as the chamber is redesigned to be more accessible for objectives with large magnification

RoPod v25

• This large RoPod is designed for 64 x 48 x 0.17mm (#1.5) microscopy glass
• Various versions with separators for individual root growth are available
• Chamber measures 69.2 x 50.7 x 8.6 mm and is designed to fit into ZEISS Universal mounting frame K
• We recommend using 7 ml of medium
• Glass is incorporated into the chamber and the lid during printing I recommend using a few drops of superglue not only in the corners, but also along the sides of the glass (due to the size of the glass)

Version	Preview	Files	Comments
v25 lid		Model files Individual model files: RoPod v25 lid.3mf Sliced file (ready to print): RoPod v25 lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v25 lid.f3d	v25 lid is suitable for all RoPods v25
v25.1		Model files Individual model files: RoPod v25.1.3mf Sliced file (ready to print): RoPod v25.1 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v25.1.f3d	---
v25.2		Model files Individual model files: RoPod v25.2.3mf Sliced file (ready to print): RoPod v25.2 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v25.2.f3d	21 channels
v25.3		Model files Individual model files: RoPod v25.3.3mf Sliced file (ready to print): RoPod v25.3 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v25.3.f3d	15 channels
v25.4		Model files Individual model files: RoPod v25.4.3mf Sliced file (ready to print): RoPod v25.4 with lid adaptable layer height.3mf Autodesk Fusion 360 file: RoPod v25.4.f3d	14 (wider) channels Suitable for imaging of roots even in the channels at the sides, as the chamber is redesigned to be more accessible for objectives with large magnification

For general instructions see the information above ("How to print RoPods")

RoPod v24

• Optimal for imaging roots of 3-6 days old Arabidopsis seedlings
• A single-well chamber, can accommodate up to 11 seedlings
• Separators create lanes for individual root growth
• An arc on the top of each lane guides young root growth in the desired direction
• Outer measurements 30 x 76 x 11 mm, compatible with standard inverted microscope stages
• Chamber V = ca 6 ml
• Designed for microscopy glass 24 x 60 x 0.17mm (#1.5)
• Glass is incorporated into the chamber and the lid during printing

Version	Preview	Files	Comments
v24.3	Show more	Model files Individual model files: RoPod v24.3 chamber.3mf RoPod v24.3 lid.3mf Sliced file (ready to print): RoPod v24.3 with adjustable layer height.3mf Autodesk Fusion 360 file: RoPod v24.3.f3d Printing instructions 1. Printing one chamber with a lid takes ca 2 h and ca 10 g of filament 2. For general instructions see the information above ("How to print RoPods")	The bottom edge of the lid is angled to slightly tilt the chamber for the optimal root growth along the bottom glass

Previous versions of the RoPod v24

Version	Preview	Files	Comments
v24.2	---	---	---
v24.1	---	---	---

RoPod v23

• Optimal for imaging roots of 3-6 days old Arabidopsis seedlings
• A single-well chamber, can accommodate up to 20 seedlings
• Outer measurements 30 x 76 x 11 mm, compatible with standard inverted microscope stages
• Chamber V = ca 6 ml
• Designed for microscopy glass 24 x 60 x 0.17mm (#1.5)
• Glass is incorporated into the chamber and the lid during printing

Version	Preview	Files	Comments
v23.4	Show more	Model files Individual model files: RoPod v23.4 chamber.3mf RoPod v23.4 lid.3mf Sliced file (ready to print): RoPod v23.4 with adjustable layer height.3mf Autodesk Fusion 360 file: RoPod v23.4.f3d Printing instructions 1. Printing one chamber with a lid takes ca 2 h and ca 10 g of filament 2. For general instructions see the information above ("How to print RoPods")	The bottom edge of the lid is angled to slightly tilt the chamber for the optimal root growth along the bottom glass

Previous versions of the RoPod v23

Version	Preview	Files	Comments
v23.3	---	---	---
v23.2	---	---	---
v23.1	---	---	---

RoPod v22

• Optimal for imaging roots of 3-6 days old Arabidopsis seedlings
• A single-well chamber, can accommodate up to 20 seedlings
• Outer measurements 30 x 75 x 11 mm, compatible with standard inverted microscope stages
• A two-well chamber, can accomodate 5 seedlings/well. The chamber has been originally designed for plant cell culture and protoplasts imaging
• Well V = ca 3 ml
• The chamber bottom is made out of two 24 x 24mm coverslips, the lid contains one 24 x 60mm coverslip. All coverslips are 0.17mm thick (#1.5)

Version	Preview	Files	Comments
v22.1	Show more	Model files Individual model files: RoPod v22.1 chamber.3mf RoPod v22.1 lid.3mf Sliced file (ready to print): RoPod v22.1 with variable layer height.3mf Autodesk Fusion 360 file: RoPod v22.1.f3d Printing instructions 1. Printing one chamber + lid takes ca 2h and ca 14g of filament 2. For general instructions see the information above ("How to print RoPods")	RoPod v22 was originally designed for simultaneous imaging of two protoplast samples (with and without drug treatment). A version printed with clear filament was later also used for growing Arabidopsis seedlings

Previous versions of the RoPod v22

Version	Preview	Files	Comments

RoPod v5

• Optimal for imaging roots of 3-6 days old Arabidopsis seedlings
• A single-well chamber, can accommodate up to 15 seedlings
• An insert for an o-ring and clamps are added for proper liquid sealing
• A hole is inserted on top of the box for liquid medium injection
• No separators are added to allow the root hairs to freely develop on both sides of the root
• Outer measurements 29.2 x 76 x 14.6 mm
• Chamber V = ca 6 ml
• Designed for microscopy glass 24 x 60 x 0.17mm (#1.5)
• Glass is incorporated into the chamber and the lid during printing
• Suitable for liquid treatment on a vertical microscope

Version	Preview	Files	Comments
v5.2	Show more	Model files Individual model files: RoPod v5.2 chamber.3mf RoPod v5.2 lid.3mf Sliced file (ready to print): RoPod v5.2 with adjustable layer height.3mf Autodesk Fusion 360 file: RoPod v5.2.f3d Printing instructions 1. On the lid, force the seam of the print on the top of the chamber (on the same side as the injection hole) 2. Printing one chamber with a lid takes ca 2 h and ca 18 g of filament 3. For general instructions see the information above ("How to print RoPods")	The sealing property of this design makes this RoPod suitable for a liquid treatment on a vertical microscope during a time-lapse acquisition

Previous versions of the RoPod v5

Version	Preview	Files	Comments
v5.1		Model files Individual model files: RoPod v5.1 chamber.3mf RoPod v5.1 lid.3mf Sliced file (ready to print): RoPod v5.1 with adjustable layer height.3mf Autodesk Fusion 360 file: RoPod v5.1.f3d Printing instructions 1. For general instructions see the information above ("How to print RoPods")	Depending on the filament or printer used, the offset between the lid and the chamber might need to be adjusted