An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

Summary

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Abstract

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing.

Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral Förster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2^D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

Introduction

RASopathies are genetic syndromes that impair normal development and affect various organs and tissues. These conditions are often caused by germline gain-of-function (GoF) mutations in the key genes and players involved in RAS/MAK signaling, resulting in a hyperactivation (increased phosphorylation) of the extracellular signal-regulated kinase (ERK). ERK regulates some fundamental processes important during development-tissue growth-by translocating to the nucleus^1,2. Somatic mutations in genes involved in the RAS-MAPK pathway are the most common events leading to cancer³. Thus, not surprisingly, cancer predisposition is also observed in RASopathies. Noonan syndrome (NS), characterized by developmental delay, short stature, cognitive deficits with variable severity, and cardiomyopathy, is the most common form of RASopathy². In most cases, the disease is caused by GoF mutations in PTPN11, the first RASopathy gene to be discovered in early 2000⁴ encoding for the protein tyrosine phosphatase SHP2, which acts as a positive regulator of the pathway.

Since then, thanks to the exponential use of exome sequencing approaches in undiagnosed patients, potentially pathogenic variants affecting factors involved in the RAS-MAPK, and likely linked to various forms of RASopathies, continue to be discovered and await functional characterization for efficient patients' stratification². To achieve this goal, experimental protocols that guarantee fast and informative functional validation at the organismal level are required. Employing classical and standardized mammalian models to test variants with unknown significance would be costly, extremely time-consuming, and require invasive methods in non-transparent large animals. Such a strategy is clearly not compatible with the requirement for fast testing, given the societal burden represented by poor or undiagnosed RASopathy patients, currently without management or treatment. Protocols for quantitative assessment of key phenotypic traits and molecular correlates in entire organisms would also serve to accelerate the possible clinical translation of drugs possibly available to RASopathy patients by repurposing/repositioning.

Zebrafish is an ideal vertebrate model to study diseases that affect early development. As a start, zebrafish share a high level of genetic homology with humans. The high fecundity of adult fish results in a large production of embryos that are small and develop fast. Embryos are transparent at early stages, such that major developmental processes-epiboly, gastrulation, axes, and body plan formation-can be visualized effortlessly using standard microscopy. In addition, the availability of transgenic lines that can be used to track specific cellular behavior and dynamic molecular events in space and time during development, in conjunction with advanced techniques to generate genetic models, is unbeatable. Furthermore, phenotypic readouts can be assessed at multiple levels in zebrafish (from organismal to cellular defects), and dedicated assays are already established for several diseases, including RASopathies⁵. Moreover, relatively simple bath-immersion methods for drug administration during the early stages, at least for water-soluble compounds, permit high-throughput drug screening in vivo in a 96-well format.

From a molecular point of view, studies using standard approaches, such as immunohistochemistry and immunoblot, robustly demonstrate the correlation between ERK activation and RASopathy-associated developmental defects in fish embryos^6,7. The recently developed EKAR-type FRET biosensor in zebrafish (Tg[ef1a:ERK biosensor-nes], Teen) provides a reliable in vivo tool to register ERK activation during embryogenesis in a spatiotemporally resolved manner. Hence, it could be valuable for better assessment of dynamic ERK alterations and pharmacological modulations in RASopathy fish models.

In the Teen sensor, a specific ERK substrate in the reporter is phosphorylated upon ERK activation, triggering a conformational change that brings in close vicinity the fluorescent CFP donor (D) and the fluorescent Ypet (improved YFP) acceptor (A). If the D emission spectrum overlaps considerably with the absorption spectrum of the A, FRET can occur (energy absorption from D to A). This is proportional to the distance between D and A and, therefore, in Teen, to the ERK activation status. Different imaging protocols can be set up using both standard and advanced imaging modules of standard or confocal microscopes in both live and fixed samples. Upon D excitation, the acquisition of multispectral scans along a defined spectrum of emission (λ) from CFP to YFP followed by spectral "unmixing" algorithms is among the most reliable methods to register and quantify FRET data⁸. It can be applied also to live zebrafish specimens to record in vivo tissue dynamics.

Following previous reports^6,9 and our recent application⁷, here, we detail the step-by-step workflow using Teen fish to assess ERK activation in cells at the margin of the animal pole of NS models at the beginning of gastrulation and correlate it with characteristic body axes defects visible only later in development. We show how to obtain and examine quantitative FRET data from live NS gastrulae before and after treatment with an available MEKi and how to cross-validate the results via standard immunohistochemistry against phosphorylated (active) ERK or perform correlative morphometric analysis of embryo elongation defects.

The workflow could be applied to boost the functional test of emerging variants and disease genes putatively associated with RASopathies and to get insights into the correlation of ERK activation dynamics spatially and temporally during vertebrate development and the morphological defects in embryos. We show that this protocol can also be used to test the efficacy of candidate drugs acting to modulate ERK activation.

Protocol

All experimental procedures involving animals' housing and breeding were conducted according to ARRIVE guidelines for the use of zebrafish in animal research and authorized by the Italian Ministry of Health (Direzione Generale della Sanità Animale e dei Farmaci veterinari - DGSAF). All the DNA/RNA reactions and imaging sessions may be scaled up or down as desired, depending on the final material required or the number of genes and variants tested.

1. Generation and drug treatment of transient zebrafish RASopathy models

NOTE: To monitor the expression of RASopathy-associated variants, specific constructs harboring the desired coding sequence (cds) of the protein of interest in frame with the cds of small non-fluorescent tags (such as myc or similar) can be used. This way, expression levels of the mutant protein can be assessed by standard western blot against the tag. If antibodies against the specific protein of interest are available, tags can be avoided. Immunofluorescence can also be used to assess protein expression within embryo tissue following standard protocols. This type of a control experiment can be useful to correlate mutant protein expression with induced ERK activation levels. The use of fluorescent tags is not advisable in combination of FRET imaging, given the possible fluorescence emission cross-talks during microscopy.

1. Plasmid linearization (Days 1-2) (Figure 1A)
   NOTE: When generating a transient disease model in zebrafish (RAsopathy here) caused by GoF mutation affecting a specific gene, a rapid approach is to prepare the mRNA encoding the wild-type (WT) and the mutant protein of interest, which should then be injected in zebrafish embryos following the steps below.The plasmid used as template to transcribe the mRNA should contain the desired full-length CDS (coding sequence) for the gene of interest cloned downstream a T7, Sp6, or T3 polymerase promoter and upstream of polyadenylation (polyA) signal. If not available, the first step is to produce it by cloning the zebrafish or human desired CDS in a suitable vector¹⁰ and validate by SANGER sequencing. For the cloning, consider extra time (on average 1 week including cloning, screening colonies, and isolating and expanding the correct clones).
   1. Linearize the plasmid with suitable restriction enzymes cutting only once downstream the polyA signal (here KpnI). To achieve efficient plasmid linearization, mix 3 γ (µg/µL) of plasmid DNA, 2 µL of restriction enzyme (20,000 Unit/mL), and 10 µL of 10x reaction buffer in a final volume of 100 µL in nuclease-free water. Mix the components thoroughly by pipetting a few times and incubate the reaction mix at 37 °C for 2-4 h.
   2. Check the outcome of the linearization by electrophoresis on a 1.5% agarose gel.
      1. Dilute 10x stock solution of TBE (48.5 g of Tris, 11.4 mL of glacial acetic acid, 20 mL of 0.5 M EDTA [pH 8.0]) with nuclease-free water.
      2. Prepare the agarose gel by dissolving in the microwave 1.5 g of agarose in a final volume of 100 mL of 1x TBE solution. Then, add 3.5 µL of dye gel stain.
      3. Cast the gel by pouring the agarose solution into chambers of suitable size, depending on the number of conditions/plasmids. Set the combs and wait approximately 1 h until the gel is solidified; then, remove the combs and position the polymerized gel in the appropriate gel tray for the electrophoresis.
      4. Mix a few µL of the digested plasmid ("cut") and 1.5 µL of 6x loading buffer (containing a dye to monitor the electrophoresis) in a final volume of 10 µL of nuclease-free water. Prepare in the same manner also a separate control sample with the undigested plasmid ("uncut"). Load the samples in the agarose lanes, and in the first lane, load a 1 Kb DNA ladder. Perform the DNA electrophoresis at 100 V for 30 min.
      5. Visualize and document the DNA bands resulting from the run on a standard UV transilluminator. Check the efficiency of plasmid linearization by inspecting the pattern of DNA bands comparing "cut" and "uncut."
         NOTE: Sufficiently linearized plasmids should run faster as one sharp band. Make sure that the DNA cleavage is complete because initiation of the transcription reaction is a key limiting step that can be hindered by the presence of circularized plasmid forms, resulting in poor mRNA yield.
   3. Proceed to purify the linearized plasmid using commercially available spin column-based DNA purification kits and store the purified DNA preparation at -20 °C until required.
      NOTE: Instead of linearization, it is possible to use a PCR product as the template for mRNA transcription, provided it includes the polymerase promoter sequence upstream and the polyA signal sequence downstream the stop codon. PCR products should also be purified and inspected on an agarose gel before proceeding.
2. In vitro mRNA transcription, purification, and quality control (Days 2-3) (Figure 1A)
   NOTE: clean carefully the working area and the pipettes with RNAse decontamination solutions. Use nuclease-free materials and reagents; wear gloves. This will minimize RNase contamination and allow a better yield of full-length mRNA.
   Transcribe capped and polyadenylated mRNAs encoding WT and mutant proteins from linearized plasmids using standard kits for in vitro mRNA transcription and following manufacturer's instructions.
   1. Centrifuge the purified linearized plasmid for a few seconds at 17,949 × g to ensure that debris is collected at the bottom of the tube and not transferred into the transcription reaction.
   2. Thaw all the components at room temperature (RT) except the mixture of NTP and CAP analogs, which are kept on ice. Keep the polymerase at -20 °C until use.
   3. Centrifuge briefly all the reagents at 17,949 × g before use to prevent reagent loss or accidental contamination and briefly vortex the 10x reaction buffer and the ready-to-use NTP/CAP analog mixture until they are completely in solution. Keep the 10x Reaction Buffer at RT.
   4. Prepare the transcription reaction at RT by adding 600-800 ng of the linearized and purified plasmid to a solution containing 10 µL of 2x NTP/CAP analog mixture, 2 µL of 10x reaction buffer, 2 µL of SP6, T7, or T3 enzyme (depending on the specific promoter upstream the CDS in the plasmid) in a final volume of 20 µL made up with nuclease-free water. Mix thoroughly by pipetting up and down a few times.
   5. Incubate the transcription reaction at 37 °C in a thermocycler. Make sure to also set the temperature of the lid to 37 °C and run the reaction for 2 h.
   6. To remove residual plasmid that was not transcribed during the reaction, add 1 µL of standard DNAse solution (2 Units/µL). Mix the reaction thoroughly by pipetting a few times and incubate at 37 °C for an additional 30 min.
   7. Before proceeding, check the quality and integrity of in vitro synthesized mRNA by TBE/formamide agarose electrophoresis. Dissolve 1.0 g of agarose in 50 mL of 1x TBE solution, add 5.5 mL of 37% formamide and 3.5 µL of dye gel stain, mix, and cast the gel as explained above.
      CAUTION: When handling formamide, wear Personal Protective Equipment (PPE) and use a chemical hood.
   8. Mix 1 µL of the transcribed mRNA with 2.5 µL of 2x Formamide Dye Loading Buffer in nuclease-free water in a total volume of 5 µL. Mix the components thoroughly by pipetting a few times and load the samples in the gel lanes. Run the gel at 100 mV for 10-15 min in prechilled 1x TBE buffer. Run also a 1 kb DNA and/or RNA Ladder.
   9. Optional: Denature the ladder and the mRNA at 70 °C for 10 min.
   10. Visualize and document the resulting mRNA bands on a standard UV transilluminator.
       NOTE: Long runs and high temperatures increase the likelihood of RNA degradation.
   11. If the integrity and size of the synthesized capped RNA are optimal, proceed with polyadenylation of the C-terminal by adding to the reaction: 36 µL of nuclease-free water, 20 µL of 5x denaturing loading buffer, 10 µL of 25 mM MnCl₂, 10 µL of 10 mM ATP, and 4 µL of polyadenylation enzyme (such as purified E. coli Poly(A) Polymerase). Mix the reaction thoroughly by pipetting a few times and incubate the 100 µL of the final reaction at 37 °C for an additional 1 h.
       NOTE: It is not advisable to check the RNA quality via gel electrophoresis after polyA reaction because the RNA will appear smeared due to the polyA tails.
   12. Precipitate and recover the synthesized capped and polyadenylated mRNA using appropriate salts such as LiCl. Briefly, mix equal volumes of nuclease-free water and standard 2.5 M LiCl solution (30 µL) and incubate for >30 min at -20 °C.
       NOTE: Efficient precipitation can be obtained after 2 h, but overnight incubation typically increases the final yield of mRNA.
   13. To pellet the purified mRNA, centrifuge the reaction for 30 min at 4 °C at 17,949 × g and remove the supernatant. Now, wash the pellet with ~1 mL of 70% ethanol (EtOH), and centrifuge for 15 min at 4 °C at 17,949 × g to remove contaminants and residual nucleotides from the reaction.
   14. Air dry the pellet and then re-suspend it in 20 µL of nuclease-free water. Dissolve the RNA well by gently and repeatedly pipetting at RT and incubate at 10 min.
       NOTE: Check the pellet every 5 min to ensure EtOH evaporation. When resuspending, given the stickiness of the RNA, pipette it until it is properly dissolved in water.
   15. Assess the concentration and purity of the prepared mRNA by measuring the absorbance at 260 nm and 280 nm (260/280 ratio) in a spectrophotometer. Measure different scalar dilutions of the RNA preparation to ensure correct concentration estimation.
       NOTE: Normally, the concentration of the synthesized RNA ranges from 1 to 2 µg/mL, but the final amount can differ depending on the CDS length and amount of starting material.
   16. While assessing the quality, keep the mRNA stock in ice, then aliquot it into 5 µL aliquots and store at -80 °C until required for injection.
3. Preparation of standard solutions and materials for embryo injection and drug treatment (Days 2-3, Figure 1B) 
   1. Pull microinjection needles for zebrafish embryos using sterile capillaries (standard capillary dimensions: 1.0 OD x 0.58 ID x 100 L mm). Use a commercially available puller apparatus and apply the desired settings, in terms of output voltage, pulling mode (one step or two steps), and pulling force to obtain variable tip length, diameter, and needle shape. For commonly used needle features for zebrafish, refer to Abdelrahman and colleagues¹¹.
      NOTE: Optimal settings might vary across laboratories because environmental conditions (such as humidity or room temperature) can influence the needle-pulling results. Settings should be regularly re-tested and calibrated. Pulled needles can be stored at RT in clean containers for several months.
   2. Prepare the 30x "Danieau" stock solution at pH 7.6 by dissolving 101.7 g of NaCl, 1.56 g of KCl, 2.96 g of MgSO₄ x 7H₂O, 4.25 g of Ca(NO₃)₂, and 35.75 g of HEPES in 1 L of double-distilled (DD) water for preparing the microinjection solution. Prepare also a stock solution of Phenol red at 5% in DD water for use as visible tracer of the injection mixture.
   3. Prepare E3 medium solution for embryo growth by diluting 4 mL of NaCl (5 M), 680 µL of KCl (1 M), 1.32 mL of CaCl₂ x 2H₂O (1 M), and 1.32 mL of MgSO₄ (1 M) in a final volume of 4 L of reverse-osmosis water.
   4. Prepare stock solutions of the desired drugs (in this example: the MEK inhibitor [MEKi] PD032590) as stock solutions according to the product datasheet and solubility (10 mM of the MEKi of choice by dissolving 5 mg in 1.036 mL of DMSO). Mix thoroughly and generate small aliquots to store at -80 °C until use.
      NOTE: DMSO is a common solvent used to dissolve both polar and nonpolar compounds and shows solubility in a wide range of organic solvents as well as water.
   5. Prepare a live brine shrimp suspension by allowing Artemia salina cysts to hatch in hatching solution (2 g/L of Artemia salina cysts in 30 g/L of ocean salt previously dissolved in reverse-osmosis water) at 28-30 °C for at least 18 h, providing constant aeration.
      NOTE: Culturing conditions and hatching time might be influenced by facility environmental conditions and cysts lots and should be re-tested regularly and re-set if necessary.
4. Preparing breeding pairs for Tg[ef1α:ERK biosensor-nes] (Teen) fish (Day 3)
   1. The day of mating, feed the adult pairs regularly according to the approved animal facility protocol.
      1. Clean the culture containing hatched brine shrimps by filtering out the non-hatched or empty cysts with two strainers of different filter sizes (filter step I: 180 µm, filter step II: 112 µm).
      2. Collect and wash the filtered brine shrimps in 200 mL of reverse-osmosis water to feed 10-15 adult fish with approximately about 5-10 mL of the brine shrimp solution.
         NOTE: Check the quality of the hatched brine shrimp solution looking at their vitality, motility, size, and color and be sure to remove unhatched or empty cysts, which are indigestible for fish and can cause harm to the fish gastrointestinal tract. For breeding pairs, it is preferable to provide the last meal of the day at least 3 h before isolating pairs in the breeding tank to allow food digestion and keep high water quality levels.
   2. Select fish pairs who have not been paired for mating in (at least) the previous 2 weeks, to minimize distress and allow gamete development. Acclimate the selected adult Teen fish pairs (normally with this composition: 1 ♂: 2 ♀) in appropriate breeding tanks. Separate females from males by using a tank divider until the next morning. Ensure a standard light/dark cycle of 14/10 h.
      NOTE: Group crosses can also be arranged. However, in this case, spawning can occur at different times and selecting embryos at the same stage from mixed clutches becomes more difficult. The transition from dark to light is critical for successful mating.
5. Embryo collection and microinjection of WT and mutant mRNAs (Day 4)
   1. As soon as the light is switched on in the facility, remove the tank divider separating the males and females and, if necessary, change approximately 20% of the tank water with fresh water from the recirculating aquaculture system to keep the water quality high without diluting the hormones released by the fish excessively.
   2. Leave the fish undisturbed and check regularly for egg laying (usually in the first 30 min to 1 h of daylight). The eggs (2-3 mm) are laid and fall to the bottom of the tank separated by a grid such that adults cannot eat them. Isolate the adults who have successfully bred and strain the tank water containing the eggs through a standard strainer (such as a tea strainer) to retain the eggs.
      NOTE: It is possible to put the same fish back in the breeding tank for another mating round or return the fish to the original housing tanks, recording the mating pair, date, and performance.
   3. Wash the collected embryos with fresh E3 medium and remove unfertilized, degenerated eggs, feces, and other kinds of debris from the pairs by using a Pasteur pipet.
      NOTE: Collect ~n = 100 fertilized Teen eggs in 90 mm diameter Petri dish to avoid embryo overpopulation.
   4. Arrange and align fertilized Teen eggs in a custom microinjection plate obtained by molding 2% agarose in E3 medium to generate appropriate lanes that must contain and restrict the eggs during microinjection.
      NOTE: To this aim, custom-made or commercial molds can be used.
   5. Prepare fresh microinjection mix to generate mutant embryos as follows: 30-60 pg of capped and polyadenylated mRNA (here shp2) dissolved in 0.3x Danieau solution (diluted from stock solution) for a final volume of 20 µL. Add 0.2 µL (0.05%) of Phenol Red stock solution as microinjection tracer.
   6. Backload the needle with 2 µL of injection material using a microloader pipette. Inject the solution into one-cell stage of Teen zebrafish embryos using a commercially available pressure microinjection device, manually adjusting the pressure and time settings to calibrate each injection based on the needle and embryo quality. Refer to standard zebrafish microinjection protocols available in the literature^11,12.
   7. Raise microinjected Teen embryos under controlled husbandry conditions (temperature: 28 °C, humidity: 70%, light/dark cycle: 14/10 h) for optimal development and clean out any eggs looking cloudy or degenerated in the next 3 hours after deposition.
   8. At ~4 h post fertilization (hpf), screen the embryos for fluorescence (reporter expression) using a standard fluorescence stereomicroscope with appropriate lamp and filter settings (465-500 nm). Use Teen-positive fish for FRET imaging and negative siblings for immunohistochemical (IHC) assessments (see below).
      NOTE: Given the known variability in the expression of Tol2-based transgenes¹³, embryos can show variable levels of Teen fluorescence. It is recommended to inspect not-injected siblings to control for inter-individual variability in fluorescence within a batch and, considering the low dynamic range of FRET imaging, discard embryos with extremely low levels of basal fluorescence.
6. Treatment of zebrafish embryos with the MEK inhibitor (MEKi) (Day 4)
   1. Prepare an intermediate solution of MEKi PD0325901 (1 mM) by diluting 1 µL of 10 mM stock solution with 9 µL with E3 medium. Use the intermediate solution to obtain final low dose solutions. Prepare a low-dose (0.25 µM) of the MEKi PD0325901 in a total volume of 3 mL by mixing 0.75 µL of the 1 mM intermediate solution with 2.25 µL of E3 medium. Dilute 0.75 µL of 10% DMSO with 2.25 mL of E3 medium to obtain the control (Ctrl) solution.
   2. Place embryo pools of equal numbers in different wells of a 6-well plate and start treatment by bath immersion: for each well, exchange the E3 medium with E3 medium containing vehicle control (0.0025% DMSO) or diluted drugs at the desired concentration (0.25 µM, 0.0025% DMSO) as mentioned before. Use 3 mL of drug solution per well.
      NOTE: To eliminate any effects caused by varying concentrations of the carrier (DMSO), it is important to ensure that both control and treatment solutions have the same final DMSO concentration.
   3. Keep the treated embryos at 28 °C before collecting them until the desired developmental stage for followup assays (here: morphometric analysis and IHC validation).

2. Live multispectral FRET imaging of RASopathy zebrafish models at gastrula stage and data analysis

1. Mounting gastrulae for live imaging (Day 4) (Figure 2A)
   1. Prepare mounting medium for alive embryos by dissolving 1.5 g of low-melting agarose (LMA) in 1x PBS to make 1.5% LMA/E3.
      NOTE: Choose the agarose concentration from 0.8 to 1.5% depending on the developmental stage and specific need to restrain the fish. Distribute freshly made LMA solution in aliquots of the desired format and store LMA in a polymerized state at RT (stable for a few months). This minimizes bacterial and fungal contamination but check the quality of the LMA aliquot regularly before use.
   2. Before sample mounting, melt the 1.5% LMA aliquot in a thermomixer or sterile water bath at 50 °C. Once dissolved, decrease the temperature of the thermomixer to approximately 30 °C.
   3. Position a single injected Teen⁺ embryo (here a gastrula expressing Shp^D61G) at the center of a glass bottom dish of 35 mm diameter for live imaging and orient it using a thin hair. Remove the excess E3 medium and immobilize the embryo by putting a drop of LMA on top and leaving it to polymerize at RT.
      NOTE: In the beginning of the polymerization process, the fish can be oriented to the desired position. It is strictly recommended to use the LMA at a temperature not higher than 35 °C to avoid tissue damage.
2. Setting microscopy parameters and performing multispectral FRET imaging of gastrulae (Day 4) (Figure 2B)
   NOTE: The protocol applies to the use of any confocal microscopy platforms equipped with all the necessary hardware and software to perform FRET imaging. Here we used confocal microscope equipped with an argon-ion laser with the wavelength lines of 458-476-488-496-514 nm, a programmable acousto-optical beam splitter (AOBS) that separates excitation and emission light, two spectral Hybrid (HyD) detectors, and a stage incubator to maintain stable conditions of temperature (at 28 °C) and humidity during live imaging of samples.
   The live multi-spectral FRET imaging protocol as described can be applied to embryos at different developmental stages beyond early gastrulae, as shown by Fasano et al.⁷. A larger z-stack should be considered for later developmental stages (e.g., 24 hpf) as well as appropriate z- and t- intervals' settings to permit multi-spectral detection steps during spectral FRET image acquisition.
   1. Turn on the incubator controller at least 1 h before starting the acquisition and set the temperature to 28 °C to keep zebrafish embryos in a healthy state. Once the incubator temperature has stabilized, place the imaging dish with the embryo on the sample holder and use a 10x/dry objective (numerical aperture of 0.4) to quickly visualize the sample.
   2. In the Laser Configuration of the referenced software, switch on the argon-ion laser and use the slider to adjust the laser power to 50%. In Hardware Settings, choose 8-bit depth resolution at which the images will be acquired.
   3. Select the spectral detection XYλZ scanning mode in the acquisition panel and set the following acquisition parameters: format image: 512 x 512 px; scanning speed of 400 Hz; optical zoom of 0.75.
   4. Activate the 458 nm laser line of the argon-ion laser and set its intensity value, normally <10% (here 8.5%).
   5. Select a HyD detector and adjust the sensitivity of the detector (gain) by entering the desired value (here 500).
      NOTE: For live, weakly fluorescent samples and to accumulate less background noise, it is advisable to use hybrid detectors rather than Photomultiplier Tubes (PMTs).
      ​In the referenced software, displaying the emission spectrum of a dye requires activating a detector.
   6. To begin, activate the first detector (HyD) and select the cyan fluorescence protein (CFP) to display its emission curve. Open the dropdown menu of the detector bar of the software to open the selection list for the CFP emission curve. Now, activate a second detector to display the emission curve of the second dye yellow fluorescence protein (YFP). To display also the Ypet emission curve, activate a second detector, then select the YFP emission curve that will appear in the spectrum image, after turning off the second detector.
      NOTE: Once this step is complete, the second detector should be deactivated, as only one detector is utilized in spectral acquisition mode.
   7. To start live acquisitions, position the detection cursor in the range of the most intense signal emission (in this case, that of YFP) to visualize the sample, decide and set the start and end positions of the sample thickness in the z-stack LAS X window.
   8. For λ-scan range properties, set the following parameters: begin (460 nm) and end (570 nm) of the detection range; Detection Band Width: 5 nm; λ-scan Stepsize: 5 nm.Start z-stack acquisition.
      NOTE: The parameters indicated allow users to obtain a relatively fast imaging with acceptable resolution, but different settings can be used. In the Fluorifier Disc Settings, the automatically inserted NOTCH filter can be deselected to avoid single intensity loss.
   9. To assess real-time signal changes upon MEKi exposure, mount a single mutant embryo (here Shp2^D61G) in a glass dish and image before and after drug treatment. For these live experiments, directly perform drug treatment by bath immersion during imaging session with a maximum of 2 mL of the solution containing the dissolved drug.
3. Image postprocessing and multispectral dye separation to obtain ratiometric FRET images (Day 5) (Figure 2C)
   1. To separate the donor (CFP) and acceptor (Ypet) emission, be sure to eliminate the contribution of the fluorescence emission of both molecules in the FRET channel, called spectral bleedthrough (SBT).
      1. Refer to publicly available dye/fluorescent proteins databases to display and download the .cvs file relative to a standard CFP emission spectrum (excitation and emission spectra).
      2. In the Data panel of the .cvs file, choose the Text to columns option and split the data into columns using a comma as the delimiter. The resulting file will include additional information (e.g., CFP excitation, CFP 2P) that should be removed. Retain only the column data relative to wavelength and emission.
      3. In the CFP emission column, remove all intensity data from 501 nm onwards.
      4. Save the adapted CFP emission spectrum file from 460 to 500 nm in .xls format (called "eCFP modified" in Figure 2C).
      5. Repeat the same procedure to download and process the YFP protein emission spectrum and retain only the column data relative to wavelength and emission. Remove all emission values up to 524 nm. Save the adapted YFP emission spectrum file from 525 to 650 nm in.xls format (called "Ypet from 525 nm" in Figure 2C).
      6. To allow exclusion of spectral bleed-through (SBT) in the dye database available in the Configuration window of the software, insert and save two adapted reference emission spectra for CFP and YFP.
   2. Select the resulting spectral image file from the imaging session, open the Process window, and select Spectral Dye Separation in the Dye Separation tool. Configure the settings for the dye separation as follows: in the dropdown lists on the left side of the dialog, select the new CFP emission spectrum (eCFP modified) in the first position and the new YFP emission spectrum (Ypet from 525 nm) from the spectrum database.
   3. Under Rescale, select Per Channel to scale the channels individually then, in the λ-scan of the images, choose the one with the greatest signal intensity (corresponding to step 14 of the spectral scan at the level of the FRET channel emission peak, indicated by white arrow in the panel showing detection steps on the right of Figure 2B). Then, move along the Z scan of the sample, and choose the optical section that highlights the area of interest on margin zone.
   4. Use the ROI selection mode on the margin zone of the animal pole to define the area with the best spectrum. Click on ROICrosshair indicated at the top in the display window to call up the crosshair and adjust the size of the reference ROI by entering the value of 40 voxels in the Measurements Area. Define precisely the area of interest by the ROICrosshair. In the image diagram, choose data normalization, then click Apply to perform the dye separation with the configured settings.
   5. Open this newly generated file obtained with the two channels separated and produce a two-dimensional projection image from the three-dimensional image series (maximum intensity projection) for data visualization.
   6. In the Process window, select Crop to separate channels into two separate files, the CH1 and CH2 (or FRET) channels.
   7. In the Process window, select Combine Images, select the CH2 file and insert it in the first option, and then select the CH1 file and insert it in the second option. Set a rescale with factor 5 and choose the Ratio operation. Then, click on Apply to generate the new file containing the ratiometric image (YFP/CFP). Save the file for followup data analysis.
4. Quantitative analysis of FRET signals and image rendering (Days 5-6) (Figure 2D)
   1. For quantitative measurements of FRET signals from FRET/CFP ratiometric images, perform the experiment on N>2 embryos (replicates), depending on an a priori statistical assessment based on the expected effect. Employ an image processing package such as the open-source software Fiji. Import the image files obtained from spectral imaging and dye separation into an image analysis package such as the open source Fiji.
   2. Before starting with the ROI selection on the gastrulae images, set the parameters as readout measurements by using the Analyze function | Set Measurements | select the parameters of interest such as Area, Integrated density, and Mean grey value.
   3. Select the region of interest (in this specific study: the margin of gastrula) using the polygon selection tool from the toolbar. Save the ROI x,y specifications by clicking on Analyze | Tools | ROI Manager.
   4. To perform measurements in a selected ROI, click on Analyze | Measure. Repeat these steps for each ROI and image/condition.
      NOTE: If needed, save the image as .TIFF format. It is possible to save all ROI measurements deriving from multiple images as one ROI file. Rename each measure in the ROI manager list with the correct name relative to each sample/image.
   5. For each condition (mutant and mutant + treatment here), extract all the values of FRET/CFP ratio for each selected ROI obtained and organize them in a worksheet by organizing, for example, experimental groups in columns and each raw value in rows.
   6. Use any suitable software to assess statistical differences of FRET signals among different experimental conditions and for graph generation and data visualization.
      NOTE: Open-source and licensed software are available for data analysis and graphing solutions.
   7. Organize a specific project for the statistical assessment considering all raw measurements and the number of replicates for each experimental condition.For the statistical analysis of a single biological replicate, create a column table having one grouping variable, with each group defined by a column. For the statistical analysis of more than one biological replicate, create grouped tables having at least two grouping variables, one defined by columns (e.g., experimental conditions) and the other defined by rows (e.g., mean values of replicates).
   8. After appropriately arranging the experimental groups in the worksheet, assess data distribution (Normality test, e.g., D'Agostino-Pearson, Anderson-Darling) and based on the result, choose the most appropriate statistical test.
      1. Perform a One-Way ANOVA for parametric data (following Gaussian distribution) and Kruskal-Wallis test for non-parametric data. If more factors are present (genetic and drug concentrations), consider a Two-Way ANOVA. Compare the mean FRET value for each condition against each other (multiple comparisons) and correct for multiple comparisons using a post hoc test (e.g., Tukey's test and Dunn's for parametric and non-parametric data, respectively).

3. IHC validation of the FRET results and correlative morphometric analysis of gastrulation defects

1. Preparation of solutions and embryo fixation and mounting for IHC against tERK and pERK (Day 7) (Figure 3A)
   1. Prepare working solutions required for IHC.
      1. Prepare a stock solution of 10x PBS by dissolving 80 g of NaCl, 2 g of KCl, 14.4 g of Na₂HPO₄, and 2.4 g of KH₂PO₄ in 1 L of DD water. Autoclave the solution to keep it sterile.
      2. Prepare a 0.8% PBS-Triton (PBSTr) solution by dissolving 8 mL of 10% Triton X-100 in a final volume of 100 mL of 1x PBS.
      3. Prepare 20%, 50%, and 80% glycerol solutions by diluting 3 mL, 7.5 mL, and 12 mL of 100% glycerol, respectively, in a final volume of 15 mL of 1x PBS.
   2. Prepare 4% paraformaldehyde (PFA)/0.25% PBSTr (fixative solution) by mixing 2.5 mL of 16% PFA in 250 µL of 10% Triton X-100 previously dissolved in a final volume of 10 mL of 1x PBS. Use this solution to fix embryos at 6 hpf overnight at 4 °C. Use 2 mL tubes and a maximum of five embryos per tube to allow efficient fixation and washes.
      NOTE: It is recommended to prepare fresh fixative for each fixation round. CAUTION: When handling formaldehyde at 16%, wear Personal Protective Equipment (PPE) and under a chemical hood.
   3. Wash the embryos in the 2 mL tubes by filling the tubes up with 1.5 mL of 0.8% PBSTr for a few times. Transfer the embryos to new 2 mL tubes and store the embryos in 1x PBS until use.
      NOTE: If not familiar with the handling of early zebrafish embryos, work under a stereomicroscope to avoid embryo loss during pipetting. Use "low binding" plasticware to reduce possible stickiness of the embryos to the tube wall.
2. Tissue permeabilization and IHC against pERK and tERK (Days 7-9)
   NOTE: IHC outcomes depend upon multiple variables that can differ across labs and specific optimization of the protocols might be necessary.
   1. Wash the fixed zebrafish embryos in 1 mL of 0.8% PBSTr for 3 x 10 min at RT.
   2. Permeabilize the samples with 2 µL of Proteinase K diluted in 0.8% PBSTr (1:1,000) for 2 min at RT.
   3. Stop the permeabilization of the tissue by washing the samples in 1 mL of 0.8% PBSTr for 3 x 10 min, and postfix the samples in 500 µL of 4% PFA/1x PBS for 20 min at RT. Then, wash the samples in 1 mL of 0.8% PBSTr for 3 x 10 min.
      NOTE: The method and the time used for tissue permeabilization are critical steps that can be influenced by the antigen type, the quality of tissue preservation, and environmental factors. Optimize the protocol before applying it on precious samples.
   4. Incubate the samples with 1x blocking buffer (BB) containing 5% normal goat serum (NGS), 1% Bovine Serum Albumin (BSA), 1% DMSO, prepared as follows: 75 µL of 100% NGS, 150 µL of 10% BSA, and 15 µL of 100% DMSO in a final volume of 1.5 mL of 0.8% PBSTr. Use 200 µL of the solution per tube and incubate the embryos for 20-30 min at RT in the solution.
   5. Remove the 1x BB from the previous step and incubate the samples in a solution containing a mix of the desired primary antibodies (here: diluting 1 µL of primary mouse monoclonal antibody p44/42 MAPK, total ERK, tERK, + 1 µL of rabbit polyclonal phospho-p44/42 MAPK, phosphorylated ERK, pERK) in 250 µL of freshly prepared 1x BB solution. Use 200 µL of the solution per tube and incubate the embryos in the solution overnight at 20 °C.
   6. To eliminate nonspecific binding, wash the samples with 1 mL of 0.8% PBSTr several times (first wash every 10 min and then every 30 min).
   7. Incubate the samples in freshly prepared 1x BB for 20 min at RT. Use 200 µL per tube.
   8. Remove the 1x BB from the previous step and incubate the samples with a mix of the secondary antibodies (1 µL of fluorescently 488 conjugated-goat anti-mouse, + 1 µL of fluorescently conjugated 633 goat anti-rabbit in 600 µL of 1x BB solution). Leave the embryos gently shaking overnight at 20 °C.
      NOTE: We performed IHC on negative (not transgenic) Teen fish from the same batch used for FRET imaging. Alternatively, Teen⁺ fish can also be used, but in this case, secondary antibodies conjugated to fluorophores that do not overlap with the emission spectra of CFP or YFP should be employed instead.
   9. Eliminate nonspecific binding as described above.
   10. Wash the samples in a gradient of glycerol/1x PBS solutions (20%, 50%, 80%) for 15 min per each solution at RT. Store the samples in 90% glycerol/1x PBS at 4 °C.
       NOTE: To minimize possible fluorescence decay, store the samples in the dark.
3. Confocal microscopy of immunostained tissue and quantitative analysis (Days 10-11) (Figure 3B-E)
   1. Mount the embryos as described before.
   2. Set the confocal microscopy acquisition parameters as follows: white laser at 80%, fluorescence emission range at 507 - 551 nm for fluorescently conjugated goat anti-mouse (in this case with the emission spectrum of 488 nm, used for tERK), 644 - 740 nm for fluorescently conjugated goat anti-rabbit (in this case with the emission spectrum of 633 nm, used for pERK). Select sequential mode of acquisition, a format scanning of 512 x 512 px, and a speed of 400 Hz. Define start and end of the scan with a z-step size thickness of 4 - 5 µm and standard digital zoom of 1. Use water immersion 25x objective (with a 0.05 numerical aperture) to acquire the whole gastrula at 6 hpf
      CAUTION: This protocol includes the application of lasers that can be harmful; therefore, it requires personnel training according to the specific national requirements.
   3. After confocal image acquisition, inspect the raw image file obtained using an image processing package such as the open-source software Fiji.
      1. In Fiji, use the command Split channels from the menu Image and submenu Color to obtain a single channel image (total ERK: channel = 0, C = 0; pERK: channel =1, C = 1) and generate a maximum intensity z-projection image for both channels by clicking on Image | Stacks | Z Project.
      2. Repeat the procedure for ROI intensity measurements as described before and extract the data to a worksheet.
      3. To infer p-ERK signal intensity changes in the desired ROI among experimental groups, calculate the p-ERK/t-ERK ratio by dividing the raw integrated density values of p-ERK staining by raw integrated density of the t-ERK signal. Proceed to the assessment of statistical significance as in step 2.4.
         NOTE: Imaging parameters' settings can be modified based on the specific needs and considering the time duration for each sample, the desired quality of images, and the number of planes to be acquired.
4. Axes measurements in 11 hpf embryos (Day 4 for embryo fixation, Day 12 for data acquisition and analysis) (Figure 4A-D)
   1. At the end of gastrulation (11 hpf), fix the embryos with 4% PFA in 1x PBS for 20 min at RT, wash several times in 1x PBS, and store at 4 °C until image acquisition.
   2. Orient the embryos laterally using a Pasteur pipette in a single well of a 12-well plate containing fresh 1x PBS.
   3. To evaluate the presence of an oval shape (Y/X embryo axes ratio > 1), a characteristic Rasopathy phenotype in zebrafish, and morphological rescue of embryo axes ratio as a result of the treatment efficacy, acquire images of whole embryos using a stereo microscope with a 3.4x magnification objective (0.63x scanning objective x 8.60 zoom factor) by simply capturing images in a brightfield mode.
   4. Import the image file into an image analysis package such as the open source Fiji.
      1. Measure the embryos' axes length (x, minor axis; y, major axis) by selecting the straight tool from the toolbar and clicking on Analyze | Measure. After performing selected measurements, add them to the ROI manager list and save the ROI file as explained for FRET imaging analysis above.
         NOTE: Repeat these steps for each embryo image.
   5. Proceed to export the data in a worksheet and calculate the axes ratio by dividing the length of the major axis (y) by the length of the minor axis (x). Calculate the mean, standard deviation of mean, or standard error of mean of different replicates.
   6. Proceed to statistical data analysis as in step 2.4.

תוצאות

This protocol shows a simple workflow to quickly generate transient RASopathy models in zebrafish embryos and assess ERK fluctuations in early mutants with a standard live FRET imaging method applied to a recently established ERK zebrafish sensor^6,9. As recently shown^6,7 within the same experimental workflow, FRET results can be cross-validated by standard IHC against phosphorylated and total ERK on ...

Discussion

Despite decades of research and myriads of mutations leading to highly heterogeneous forms of RASopathies now mapped, genetic variants with unknown significance continue to emerge from sequencing efforts on undiagnosed patients. Indeed, in many cases, diagnosis based solely upon clinical features can be challenging and functional genomic approaches to validate sequencing results remain crucial. Moreover, despite some available anticancer molecules (i.e., MEK inhibitors) being proposed to treat a subset of RASopathie...

Materials

Name	Company	Catalog Number	Comments
Plasticwares
1.7 L Breeding Tank - Beach style Design	Tecniplast	1.7L SLOPED	Breeding tank
Capillaries GC100F-10	Harvard apparatus	30-0019	One-cell stage embryo microinjection
Cell and Tissue Culture Plates - 12 well	BIOFIL	TCP011012	Embryo collection and treatment
Cell and Tissue Culture Plates - 6 well	BIOFIL	TCP011006	Embryo collection and treatment
Cell Culture Dish	SPL Life Sciences	20100	Embryo collection
Nunc Glass Dishes 12mm	Thermo Fisher	150680	Embryo FRET spectral imaging
Pipette Pasteur	Corning	357524	Embryo transfer
Protein Lobind Tubes 2ml	Eppendorf	30108450	IHC assay
Reagents and others
Caviar 500-800 µm	Rettenmaier Italia	BE2269 (500-800)	Dry fish food
Great Salt Lake Blue Artemia Cysts	Sanders	00004727	Live fish food
Instant Ocean salt	Tecniplast	XPSIO25R	Dehydrated sea salt for live food preparation
Tg(EF1a:ERK Biosensor-nes) (Teen)	Contacts for ordering*: National BioResource Project Zebrafish, Support Unit for Animal Resources Development, RRD, RIKEN Center for Brain Science, Japan. https://shigen.nig.ac.jp/zebra/index_en.html *upon MTA signature.	-	Supplier of ERK Reporter zebrafish line. Fish embryos can be obtained upon MTA signature from National BioResource Project of Japan for Zebrafish (RIKEN, Japan). The zebrafish line is deposited by Nara Institute of Science and Technology (Takaaki Matsui) and the National Institute of Genetics (NIG/ROIS) (Koichi Kawakami, patent for Tol2 system) (Wong et al., 2018, Urasaki et al., 2006, Okamoto and Ishioka, 2010).
6x loading dye	Cell Signaling	B7024S	Gel Elecrophoresis
100 bp DNA ladder	NEB	N3231S	Gel Elecrophoresis
Agarose	Sigma-Aldrich	1,01,236	Gel Elecrophoresis
Agarose, low gelling temperature	Sigma-Aldrich	A9414-10G	Embryo mounting for FRET spectral imaging and IHC assay
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A8022	IHC assay
Calcium chloride	Sigma-Aldrich	223506	E3 medium component
Calcium nitrate	Sigma-Aldrich	237124	Danieau stock solution component
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D8418-100ML	IHC assay
EDTA	Sigma-Aldrich	E9884	TBE buffer component for gel preparation
Ethanol 99%+	Fisher Scientific	10048291	In vitro RNA purification
Formaldeide 16%	Thermo Fisher	28908	Embryo fixation
Formamide	Sigma-Aldrich	F9037	Gel Elecrophoresis
Gel Loading Buffer II (Denaturing PAGE)	Thermo Fisher	AM8546G	In vitro RNA transcription
Glacial Acetic Acid	Sigma-Aldrich	695092	TBE buffer component for gel preparation
Glycerol	Sigma-Aldrich	G6279-1L	IHC assay
Goat anti-mouse Alexa Fluor 488	Thermo Fisher	A11001	IHC assay
Goat anti-rabbit Alexa Fluor 633	Thermo Fisher	A21070	IHC assay
HEPES	Sigma-Aldrich	H3375	Danieau stock solution component
KpnI - HF (Enzyme + rCutSmart Buffer)	NEB	R3142	Plasmid linearization
Magnesium sulfate	Sigma-Aldrich	230391	E3 medium component/Danieau stock solution component
Millennium RNA Markers	Thermo Fisher	AM7150	Gel Elecrophoresis
Monarch Genomic DNA purification Kit	NEB	T3010L	Plasmid linearization
Mouse monoclonal p44/42 MAPK	Cell Signaling	4696S	IHC assay
mMACHINE SP6 Transcription Kit	Thermo Fisher	AM1340	In vitro RNA transcription
Normal Goat serum (NGS)	Sigma-Aldrich	G9023	IHC assay
Nuclease-free water Ambion	Thermo Fisher	AM9937	In vitro RNA transcription
PD0325901	Sigma-Aldrich	PZ0162	MEK inhibitor
Phenol Red solution	Sigma-Aldrich	P0290	Microinjection mix component
Poly A Tailing Kit	Thermo Fisher	AM1350	In vitro RNA transcription
Potassium chloride bioxtra	Sigma-Aldrich	P9333	E3 medium component/Danieau stock solution component/PBS stock solution component
Potassium dihydrogen phosphate	Sigma-Aldrich	P0662	PBS stock solution component
Proteinase K	Sigma-Aldrich	P2308	IHC assay
Rabbit polyclonal phospho-p44/42 MAPK	Cell Signaling	4695S	IHC assay
SYBR safe DNA gel staining	Thermo Fisher	S33102	Gel Elecrophoresis
Sodium Chloride	Sigma-Aldrich	31434-M	E3 medium component/Danieau stock solution component/PBS stock solution component
Sodium phosphate dibasic	Sigma-Aldrich	71643	PBS stock solution component
Trizma base	Sigma-Aldrich	T1503	TBE buffer component for gel preparation
Triton X-100	Sigma-Aldrich	T8787	PBSTr buffer component
Equipment
Alliance Mini HD9	Uvitec	-	Imaging system
Centrifuge 5430 R	Eppendorf	5428000205	Microcentrifuge
Eppendorf ThermoMixer C	Eppendorf	-	Embryo mounting
FemtoJet 4x	Eppendorf	-	Microinjection system
Infinite M Plex	Tecan	-	Multimode plate reader
Leica M205FA	Leica Microsystems	-	Fluorescence stereo microscope
Leica TCS-SP8X equipped with incubator (OkoLab)	Leica Microsystems	-	Confocal microscope
Mini-sub Cell GT Horiziontal Electrophoresis System	Bio-Rad	1704406	Gel Elecrophoresis
PC-100 Vertical puller	Narishige	-	Needle puller
PowerPac Universal Power Supply	Bio-Rad	1645070	Gel Elecrophoresis
Stellaris 5	Leica Microsystems	-	Confocal microscope
Vortex MiniStar silverline	VWR	-	Plasmid preparation
Softwares
Biorender	Biorender	CC-BY 4.0 license	Cartoon elaboration for Figures
Excel	Microsoft Office Professional Plus 2019	-	Data analyses
Fiji software	ImageJ	15.3t	Imaging rendering and quantitative analyses (FRET signals measurements, ERK fluorescence intensity in IHC assay, embryo axes lenght)
GraphPad Prism	GraphPad Software LLC	v. 9	Statistical data analyses
iControl spectrophotometer software	Tecan	v. 2.0	RNA quantification
Illustrator	Adobe	26.0.3 (64-bit)	Figure assembling
LASX software	Leica Microsystems	v. 4.5 (Stellaris 5), v. 3.0 (M205FA), v. 3.5 (TCS-SP8X)	Imaging acquisition for spectral FRET experiments and embryo imaging for axes lenght measurements
Q9 Mini 18.02-SN software	Uvitec	-	Gel image acquisition