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Tutorials

Drawing your micropatterns

The PRIMO solution can create with high flexibility any protein micropattern on all standard cell culture substrates. An image of the micropattern (no restriction on shape except for the minimal resolution of 1.2 μm) must first be loaded into Leonardo software, which will send it to the PRIMO module and project it in UV light through the microscope objective onto the substrate. Two file formats can be used with Leonardo software:

- .tif format: drawing in pixels (ratio is indicated in the Leonardo calibration parameters)

- .pdf format: drawing in millimeters (actual size of the pattern)

The tutorial video below presents how you can draw patterns and gradients for PRIMO with the Inkscape drawing program.

Preparing your substrates

PRIMO photopatterning solution is a so-called subtractive technology. The substrate must be coated with an antifouling polymer (we recommend PLL-PEG or PEG-SVA) that prevents adhesion of molecules and cells. The PRIMO system then degrades this non-adherent layer locally, through the combined action of UV and a photoactivatable reagent (PLPP).

- Passivation with PLL-PEG:

- Passivation with PEG-SVA:

Switching the PRIMO setup on

To avoid any communication problems between the computer and the various controlled external components of the setup, please turn on the patterning setup components in the following order:

1. Computer

2. Camera

3. All the microscope components

4. PRIMO

Starting the Leonardo software and Calibrating the system

Leonardo is operated through Micromanager software. To start it, first click on the Micromanager icon on the computer desktop. Then click on “Plugins” and select “Leonardo”. Check that the dedicated PRIMO filter cube is in the right position (optical path) in the turret filter.

Leonardo software automatically opens on the calibration step. Follow the onscreen instructions and complete the required fields.

Setting the tube lens position

Use the green knob on the side of the PRIMO module to fine tune the focus on the substrate for your experiment.

Photopatterning with Leonardo software

In order to allow you to conduct all the micropatterning experiments you can imagine, the Leonardo software enables the following functionalities:

- Replication of the same pattern (adjustment in rows and columns and setting the spacing)

- Realignment of successive patterns or on microstructures (preview, rotation)

- Management of grayscale images

The tutorial video below presents these functionalities and also the protocol for protein photopatterning.

Seeding cells on your protein micropatterns

Once the protein patterns have been made with PRIMO, the cells can be seeded and will adhere only to the protein patterns. The stability of cell patterns depends on the substrate used and cell type seeded. Under certain conditions, cells can adhere to protein patterns for up to 30 days.

FAQ

Photopatterning with Primo

What are the advantages of photopatterning with PRIMO compare to other micropatterning techniques?

The PRIMO solution gives researchers complete freedom and full control over their manipulations to optimize the experimental conditions easily. PRIMO allows any shape of protein pattern to be generated rapidly, to precisely align or overlay several proteins (up to 3 without restrictions depending on the proteins used and their interactions) and to control the local density of protein adsorbed to the substrate.

Where can PRIMO be applied?

Does the light-activated PLPP reagent always have to be used with PRIMO?

The PLPP accelerates the degradation reaction of the anti-fouling polymer during photopatterning (a few seconds instead of a few hours). However, microfabrication manipulations with SU8 resins or NOA do not require the use of PLPP, since these products are photosensitive in the PRIMO wavelength (375 nm).

How long does it take to create a protein pattern with the PRIMO solution?

The total time to create a pattern (excluding the design step) ranges from 10 to 15 minutes. The illumination time varies and must be optimized for the patterns projected and the objective used. After each UV illumination and degradation step for the anti-fouling polymer (anti-adhesive), about 5 minutes must be allowed for the proteins to adsorb (time varies with the concentration placed on the substrate).

Why is surface treatment important for photopatterning?

Photopatterning is a subtractive technology. It first aims to cover a substrate with an anti-fouling polymer that prevents molecules and cells from adhering. The PRIMO system is then used to degrade this anti-adhesive surface locally, under the combined action of UV and the photoactivatable reagent (PLPP).

What anti-fouling surface treatment can be carried out on the substrates?

We recommend using PLL-PEG. We regularly develop new protocols to enhance the stability of surface coatings.

Can the slide be coated in advance?

We recommend coating on the same day. However, our R&D team regularly develops new protocols for specific substrates making it possible to keep coated slides for later use.

What is the resolution of the device?

The resolution is 1.2 µm over the entire field of view of approximately 500x300 µm with a 20x objective.

What is the maximum size that can be patterned?

PRIMO allows all desired images to be patterned, without size limitation.

What substrates can be used for protein photo-patterning with PRIMO?

PRIMO can be used to pattern all standard cell culture substrates: PDMS, glass, plastic, NOA, SU8. New substrates are regularly tested and validated by our in-house R&D team. In-house data available on request.

What types of photoresist can be used for microfabrication with PRIMO?

All photoresist sensitive at 375 nm can be used with PRIMO. Our R&D team mainly works with SU8 photoresists.

Can PRIMO be used to pattern proteins on 3D structures?

PRIMO can be used to pattern all surfaces, whether flat, curved or microstructured. For example, it is possible to pattern proteins on microwell walls or PDMS micropillars. In-house data available on request.

What is the depth of field for the device?

By using a 20x objective, the depth of field for the laser passing through a transparent substrate is 200 µm to 300 µm.

Is it possible to pattern proteins on hydrogels using PRIMO?

We recommend using the transfer technique by patterning proteins with PRIMO onto a slide that will be laid on the hydrogel then removed. The UV illumination phase to photopattern proteins onto hydrogels modifies their physical properties and therefore the initially-determined experimental conditions.

Which proteins can be patterned with the PRIMO device?

Our team and users employ more than ten different proteins, including Fibrinogen-488, Fibrinogen‐647, Fibronectin, GFP, Neutravidin‐488, Neutravidin‐647, PLL‐PEG‐Biotin, Protein A‐647 and Streptavidin, as well as primary and secondary antibodies.

Does PRIMO allow experiments to be conducted under sterile conditions?

Yes, experiments conducted with PRIMO can be carried out under sterile conditions, even if the PRIMO device is not set up in a room assigned to this purpose. In such cases, we recommend performing all rinsing and incubation steps in a biosafety cabinet (BSC), then putting the sample in a closed culture plate that can then be placed on the motorized stage of the PRIMO photo-patterning device.

How long can the cell patterns be stable for?

The stability period of cell patterns depends on the substrate used and cell type cultured. Under certain conditions, the cells can adhere to the protein patterns for up to 2 weeks. Our R&D team regularly develops new protocols that are made available to our users.

The Primo module

What type of laser is built into the PRIMO unit?

The PRIMO unit contains a UV laser operating at 375 nm.

What is the power of the laser?

The power of the laser exiting the tube lens is between 6 mW and 8 mW. At objective outlet, the power is variable and depends on the objective. Using the Nikon S Plan Fluor ELWD 20x/ 0.45 reference objective, it is between 5 mW and 6 mW; the intensity is approximately 29 mW/mm².

What are the dimensions and weight of the PRIMO module?

The dimensions of the PRIMO unit are 46.2 x 32.5 x 7.2 cm (length x width x height). It weighs 15 kg and can be supported by a base supplied at installation.

The dedicated Leonardo software

What are the features of the Leonardo software?

What image file formats can be used?

The image file formats used to generate protein patterns must be in tif (pixels) or pdf (metric) format.

How are patterns drawn?

We recommend drawing patterns using the Inkscape® software (open source vector design software similar to Adobe Illustrator®), for which a user guide is available here. This software creates drawings in both pixels and millimeters and saves them in different formats recognized by the Leonardo software.

Photopatterning platform and reagents

What equipment is needed to use PRIMO?

  1. Full microscope configuration available on request.
  2. Optional depending on experimental conditions.

What consumables and reagents are needed for protein photopatterning with PRIMO?

  1. Optional
  2. Recommended to make the anti-fouling surface treatment on non-pretreated substrates

Webinar

PHOTOPATTERNING WITH PRIMO: watch the recording of the webinar organized by Alvéole to present PRIMO before it was launched, how its photopatterning technology works and the range of applications it covers in cell biology.