Protocol to locally express cxcl12a during zebrafish olfactory organ development by combining IR-LEGO with live imaging

Summary Temporal and spatial regulation of gene expression is crucial for proper embryonic development. Infrared laser-evoked gene operator (IR-LEGO) can provide information for various developmental processes. Here, we present a protocol to locally express cxcl12a during zebrafish olfactory organ development1 using a combination of IR-LEGO and live imaging. We describe steps for implementing IR-LEGO, biological sample preparation, live imaging, data collection, and analysis. This protocol can be applied to virtually any genetically modified experimental organism.


Highlights
Implementing infrared laser-evoked gene operator (IR-LEGO) for live imaging Performing IR-LEGO on live developing zebrafish telencephalon

Mounting zebrafish embryos to combine IR-LEGO technique with real-time imaging
Time-lapse of olfactory epithelium morphogenesis using a spinning-disk microscope SUMMARY Temporal and spatial regulation of gene expression is crucial for proper embryonic development.Infrared laser-evoked gene operator (IR-LEGO) can provide information for various developmental processes.Here, we present a protocol to locally express cxcl12a during zebrafish olfactory organ development 1 using a combination of IR-LEGO and live imaging.We describe steps for implementing IR-LEGO, biological sample preparation, live imaging, data collection, and analysis.This protocol can be applied to virtually any genetically modified experimental organism.

BEFORE YOU BEGIN
The protocol below describes a method of gene induction coupled with real-time imaging using stable lines of transgenic zebrafish expressing a gene of interest (here cxcl12a) under the control of a heat-sensitive promoter activated by an infrared (IR) laser.One of the main applications of IR-LEGO will be the analysis of gene function in vivo, where spatial and temporal control of gene expression are required. 2,3he use of IR-LEGO to induce targeted gene expression can also overcome the problems associated with gene expression in non-specific cells, as it is often the case with other techniques, such as mosaic analysis.Previous work has shown that the Cxcl12a-Cxcr4b chemotaxis signaling pathway is required for the assembly of the olfactory epithelium and the guidance of sensory axons during zebrafish olfactory system development. 1,4In this pathway, the ligand cxcl12a (also known as sdf1a) is expressed in the telencephalon as early as 12 hours post fertilization (hpf) and cxcr4b, the receptor, is expressed within the early olfactory neurons (EONs). 4,5In embryos mutant for cxcr4b or cxcl12a, the assembly of the olfactory epithelium is disrupted. 1Here, we established a protocol combining the IR-LEGO technique (which allows gene expression to be monitored in time and space, without photodamage) with live imaging, to create a zone of cxcl12a expression in the forebrain of zebrafish embryos in order to analyze its effect on olfactory morphogenesis.We first implemented IR-LEGO on a spinning disc confocal system.We then used real-time imaging to analyze the formation of olfactory epithelia following this perturbation.Finally, this protocol was used to rescue genetic mutants with disrupted Cxcl12a signaling and olfactory morphogenesis, in order to restore normal morphogenesis (Zilliox, Letort et al., in preparation).
Using the IR-LEGO technique, we aim at targeting the expression of cxcl12a within the telencephalon at 12 hpf, and address its effect on the morphogenesis of the olfactory epithelium by live imaging.For this purpose, we use the Tg(hsp70L:mCherry-cxcl12a) transgenic line 6 that expresses cxcl12a under the control of a heat shock promoter (hsp70) and the Tg(-8.0cldnb:lynGFP)transgenic line 7 to label the cellular plasma membrane.We crossed these transgenic lines to obtain Tg(hsp70L:mcherry-cxcl12a); Tg(-8.0cldnb:lynGFP)double transgenic embryos.
1.The following protocol describes a procedure to trigger gene expression at a specific time and position (i.e., the telencephalon) in the zebrafish embryo using an infrared (IR) laser.A detailed procedure for the implementation of this previously described technique 2,3 on a spinning disc is provided to perform real-time live imaging.Here we outline how this protocol may be used to control chemokine gene expression (cxcl12a) within the telencephalon of the embryo to study olfactory epithelium morphogenesis.2. A facility for raising adult zebrafish with the desired genotype and approval for animal experimentations is required to carry out this protocol.The IR-LEGO technique requires a specific transgenic zebrafish line expressing the gene of interest under the control of a heat inducible promoter.
Ideally, the gene of interest should be fused with DNA encoding a fluorescent tag or fluorescent protein to allow microscopy monitoring of infrared laser activation during imaging.

Institutional permissions
All procedures involving zebrafish were carried out in accordance with relevant national and international guidelines and approved by the French veterinary services and the local ethical committee (ID: E31555011, APAPHIS #34368-2021121409357964-v6).Users of this protocol should note that approval for conducting experiments on zebrafish must be acquired in advance from the relevant institutions.

STEP-BY-STEP METHOD DETAILS
Infrared laser induced heating implementation and laser heating theoretical estimation

Timing: 1 to 4 days
This step describes adaptation of an imaging system (spinning disk) with an infrared laser to irradiate a sample (Figure 1A).The in vivo irradiation conditions required to allow expression of a heat shock promoter are described in step 19.The most challenging point is to reduce the cost to obtain a full optically corrected spectral range from 450 to 1470 nm, to combine both live imaging and IR irradiation.There are two ways to implement focalized laser scanning for IR heat.Firstly, we can have a passive optical path (the laser will be in a fixed position at the center of the field of view) with a motorized stage.The second is to use a scanning head (usually consisting of a galvanometric mirror) to change the position of the focused laser.
In both cases, the best way to connect any microscope is to use the corrected optical input at the rear of the microscope without an intermediate optical lens.These lenses are generally optimized for visible light.In fact, it is possible to connect to the tubular lens at the output of the microscope, but generally in the visible range.
1. Use a spinning disk microscope dedicated to confocal fluorescence imaging in the visible range, coupled to an infrared photo-irradiation device (Rapp OptoElectronic GmbH) (Figure 1A).
Optional: A widefield system can be used for smaller samples (bacteria, cell culture, etc.).
2. Couple the IR-LEGO module (Figure 1A, light red rectangle) to the infinity optical corrected input of the inverted microscope (Figure 1A, gray rectangle).

Note:
The near infrared laser diode (light source DL-1470/2500/RSP2 Rapp OptoElectronic GmbH) with centered wavelength at 1470 nm leads to a 17 C temperature increase as described in step 3.
Note: A galvanometric head mirror (UGA-42 Geo Rapp OptoElectronic GmbH) composed of silver mirror, is conjugated to the back focal plane of the lens to align the laser in the center of the field.
Note: Synchronization between the two modules, IR-LEGO and microscope, during IR irradiation (Figure 1D) or real-time image acquisition (Figure 1E) is performed by the IR laser supplier according to the experimenters' needs.
Note: IR-LEGO set up (IR laser of 1470 nm) was added on a spinning device to avoid photodamage and photobleaching during live imaging.
Note: Contact your IR laser supplier to check that your microscope is compatible with the addition of a LEGO IR device.They can also help you define what you need to order specifically to add to your spinning disc confocal microscope in order to carry out this protocol.3. Check in vivo (Figure 1B) IR irradiation condition using a dedicated tool (Calibration sample: Phosphor Micro Upconverting Particles, Rapp OptoElectronic GmbH 8 ) at the end of the installation, to ensure that the IR-LEGO set up hardware (Figure 1A) is operational.
Note: In our configuration (step 19), using a laser intensity of 90%, we estimate the IR irradiation radius to be approximately 17 mm (Figure 1C).In our experimental setup the laser power in the back pupil plane of the microscope is around 100 mW.This corresponds to a surface irradiation density of 5.5 kW/cm 2 .Hence, in 1 min, the sample receives 330 Kilojoule (KJ).Depending on your biological sample, these values will be adapted.We advise to start with a test experiment depending on the laser power and time of exposure.
Note: With conventional laser (between 100 mW and 200 mW and wavelength between 670 nm and 980 nm compatible in conventional microscopy), the maximal temperature increase with a very high numerical aperture, is only about 5 C.This conclusion is based on the laser-induced heating model developed by Peterman and co-workers. 9The only way to achieve an increase from 21 to 37 degrees, and to be able to activate a heat-shock promoter without photodamage, is to increase the wavelength to 1470 nm.Here, we refer to the analytical model introduces by Peterman 9 to achieve an increase of 17 degrees for 10 cells.

Note:
The heat power transferred across two infinitesimally separated concentric cylindrical surfaces with radius, r; is given by the first Equation 1: where q is the heat power absorbed by water along the distance L of the laser-light path inside the chamber, K is the thermal conductivity of water, r and L respectively the radius and the length of the cylinder, and dT is the temperature difference between the two cylindrical surfaces from the focal point of the laser to the cover slide.By rearranging Equation 1 and integrating dr, the temperature change between two finitely separated cylindrical surfaces can be obtained in Equation 2: Where r' is the radius of the outside cylinder at the surface of cover slide.The absorbed power over the distance L is very small (Pout/Pin close to 1, for L = e, where e is the distance between coverslip end the focalized laser, 170 mm).
Note: Therefore, by using Lambert-Beer's law, the heat power, q, is given by: q = P in À P out = P in À P in expð À a water eÞ qzP in a water e a water is the absorption coefficient of water.
r' is equal to 17 mm based on the measurements (Figure 1C).r, for an over filling of the objective around 30%, is close to e = 170 mm.
By using r equal to 17 mm, r' around 170 mm, Pin(100 mW) a water = 2.81 cm-1 (at l = 1470 nm and K = 0.60 W.m-1.K-1).Thanks to Equation 2, the difference is around 17 C between the center of the focal plane and the temperature at 21 C on the coverslip.Alternatives: A measurement using a heat camera to characterize the system experimentally could be envisaged.

Preparation of experimental zebrafish embryos
Timing: 17 h

Day 1
Preparation of the desired fish lines for IR-LEGO and live imaging.
Note: Fishes are maintained at the Centre de Biologie Inte ´grative zebrafish facility in accordance with the rules and protocols in place.The cxcl12a t30516 mutant line has previously been described. 10te: Embryos were obtained through natural crosses and staged according to. 11ternatives: Different transgenic lines with an appropriate genetic background (i.e., gene of interest fused with a fluorescent protein DNA under the control of an hsp promoter) can be used.
Note: Handling adult zebrafishes could cause stress which may prevent them from mating.For this reason, breeders are placed in the breeding tanks in the evening before starting the mating process the next morning (see Figure 2A for a timeline overview).
Note: Select the transgenic zebrafish lines whose embryos will be used in the experiment.The transgenic fishes (Tg) breeders used in this study: Tg(hsp70l:mcherry-cxcl12a) and Tg(-8.0cldnb:lynGFP).
Note: In the slope breeding tank with a temporary divider and the internal tank with perforated bottom (Figure 2B), gently place the female breeders on one side and the male zebrafishes on the other (Figure 2C).

Note:
The number of fish per breeding tank is determined by the size of the tank.We aim for a male: female ratio between 1:2 and 1:3.By placing one male with several females, a higher number of embryos can be harvested the next day.Adjust the number of adults to obtain a sufficient number of embryos the next day, as you will need at least 30 embryos per experiment (maximum number of embryos that could be mounted per experiment which will be selected according to their fluorescence intensity).A couple produces around 150 embryos.For additional information on standard zebrafish husbandry practices, please refer to. 12y 2 Timing: 7 h 5. Remove the divider between breeders after the light is switched on in the morning to allow males and females to breed (Figure 2D).
Note: Depending on the required stage for embryo collection, this step can be delayed and adjusted according to experimental needs.Manipulate the embryos staging by controlling the start of the fish mating through removing the divider between males and females around 10 am (Figure 2A).
Note: External fertilization occurs approximately 30 min after removing the divider.The stage of an embryo is referred to by using the acronym hpf (hours post fertilization).
Alternatives: Crosses can be performed at any time in the day.However, the next steps should follow the indicated timeline.
CRITICAL: Once you have removed the divider and the adults have laid eggs, you cannot stop the experiment, but you can slightly adjust the timeline (see step number 9 below).
6. 30 min after removing the divider, collect embryos using an egg strainer.7. Transfer them into a Petri dish filled with fish water (Figure 2E).This ensures a homogeneous staging population of fertilized embryos.8. Place embryos in a 28.5 C incubator and grow to 11 hpf for IR-LEGO and time lapse imaging (Figure 2A).
Note: Dead embryos and unfertilized eggs should be removed.
Note: Keep the fish water clean by changing it regularly (at least twice a day) until the embryos have been processed.

Note:
The optimal density of embryos is 50-100 per 90 mm diameter Petri dish.Use a stereoscopic microscope equipped with a transmitted light source to check the embryos' state.
CRITICAL: Embryos used for imaging must be handled with care and changed at least once a day with fresh fish water.
Note: When the embryo reaches the shield stage i.e., 6 hpf, place the embryos in a 21 C incubator from 4 pm to 10 am to slow down their development and get the stage of interest to perform IR-LEGO the next morning (Figure 2A).
Note: Following our timeline, the embryos are at shield stage around 4 pm.At this time, we transfer them into a 21 C incubator to reach the 12 hpf stage the next day at 10 am (Figure 2A).
Note: Transfer the selected embryos to a Petri dish with fish water.
10.At the desired stage (here 12 hpf) remove the chorion under a stereoscopic microscope equipped with a transmitted light source.a.Using dissecting tweezers, gently make a tear in the chorion.b.Turn it upside down so that the embryo falls out.
Note: Following the timeline the embryos reach 12 hpf at 10 a.m., removing the chorion will result in better imaging and allowing the embryos to favorably unwrap itself and elongate during the time-lapse movie.
Alternatives: To dechorionate the embryos, a Pronase solution can be used according to ''The Zebrafish book'' recipes.
CRITICAL: Dechorionated embryos will stick to plastic, therefore, use glass pipettes and glass dishes.
11. Prepare 0.7% low melting (LM) agarose mounting solution.Each 35 mm glass-bottom dish required 1 mL of LM agarose.a. Weight out the appropriate amount of LM agarose.b.Add sterile fish water and stir to form a homogeneous solution.c.Microwave the solution until the LM has completely dissolved in the fish water (10 mL will take about 4 min at maximum power).d.Place dissolved LM agarose in a pre-equilibrated 32 C water bath to cool for about 20 min.12. Add 120 mL of 0.7% LM agarose in fish water heated at 32 C into one glass Petri dish (Figures 3A  and 3B).Note: The size of the droplet depends on the size of the glass Petri dish which should be adapted to the microscope setup.We use 120 mL of 0.7% LM agarose on a 35 mm bottom glass Petri dish adapted for an inverted microscope.This system could be adapted to an upright microscope.
Note: As required, place the 2 mL tube containing the 0.7% LM agarose solution (stored at 4 C) in a dry bath (90 C for 15 min) to melt the agarose, then transfer the tube to a dry bath at 32 C for a maximum of 45 min, otherwise the agarose will solidify.
CRITICAL: Be careful to minimize boiling over to reduce evaporation.
Note: When the 4 C aliquoted 0.7% LM agarose melted do not return it to 4 C but discard what remains if it has not been completely used.
Note: 0.7% LM agarose should be melted at least 45 min prior mounting embryos in order to allow it to melt and adjust to the appropriate temperature in the 32 C dry bath.0.7% LM agarose in fish water is used to immobilize embryos for live imaging.
CRITICAL: Use a traceable thermometer to monitor agarose temperature.Be aware that temperatures above 35 C will activate the heat shock promoter in the whole embryo during mounting.This would cause activation of gene expression throughout the embryo independently of exposure to the IR laser and therefore without spatial control.
13. Use a fine tip glass Pasteur pipet to transfer the embryos in the agarose droplet (Figure 3C).Troubleshooting, problem 1. 14. Before the 0.7% LM agarose solidifies, position the embryos dorsally by using dissecting forceps.
Note: Embryos should be mounted with the somites and trunks at the top and the olfactory system (region of interest, ROI) near the glass slide at the bottom for observation (Figure 3D) on the inverted spinning disk microscope.
CRITICAL: There is a short time window of less than 2 min to correctly position the embryos before agarose solidifies.Work quickly but gently during mounting.
15.When all the embryos have been properly oriented, wait for agarose to solidify then fill the whole glass Petri dish glass bottom (homemade device using 35 mm glass Petri dish and 14 mm glass diameter coverslip (Figures 3B and 3C) with 1 mL of 0.7% low melting agarose to obtain a stable and stiff mounting device.Troubleshooting, problem 2. 16.Finally, cover the embryos trapped in the agarose with 2 mL of sterile fish water.17.Annotate the position of each embryo on the lid of the glass Petri dish (Figure 3E) so that they can later be tracked specifically during live-imaging.
CRITICAL: To achieve high resolution imaging, all the embryos should be perfectly oriented the same way when the agarose polymerizes.This way you avoid acquiring thick z-stacks and thus reduce photodamage.
CRITICAL: Preparing high quality LM agarose glass Petri dishes filled with fish water is the key step for live imaging of zebrafish embryos.In zebrafish, EONs are localized as early as 12 hpf at the edge of the neural plate, all around the telencephalon, 5,13 and then actively converge from 18 to 24 hpf to form two ellipsoidal neuronal clusters on either side of the telencephalon at 24 hpf.We aim to perform IR-LEGO at 12 hpf to trigger cxcl12a expression in the center of the telencephalon.Here, we describe the IR-LEGO irradiation protocol, as well as, how the imaging settings are chosen for real-time imaging.For analysis purposes, the sample is imaged before and after IR irradiation.
18. Set up the noise to signal conditions before using the IR-LEGO technique.4A).d.Use white light to localize the anterior brain of the first embryo.Troubleshooting problem 3. e.On Metamorph, set the Z stack.Choose the range of your Z-stack, select ''Range around current'' and place the stage in the central plane of the Z-stack (Figure 4B).f.Choose ''Multiposition'' and mark the position (Figure 4B).g.For every embryo repeat step 18d and 18.f h.Set the wavelength parameters to visualize: i.In green channel, Tg(cldnb:lyn-GFP), the cell membranes.Use an excitation wavelength of 488 nm and a band emission filter 510-540 nm (or other appropriate excitation and emission parameters for the fluorophore).Use a low laser power ($2% of 150 mW laser, power measured at fiber tip, corresponding to approximately 0.188 mW measured at lens output and 0.368 W/cm 2 on the sample in our system) and a short exposure time (50 ms) to minimize photobleaching.ii.In red channel, Tg(hsp:mcherry-cxcl12a), mcherry-cxcl12a expression.Use an excitation wavelength of 561 nm and a band emission filter 589-625 nm (or other appropriate excitation and emission parameter for the fluorophore).Use a low laser power ($8% of 100 mW laser, power measured at fiber tip, corresponding to approximately 0.55 mW measured at lens output and 1.07 W/cm 2 on the sample in our system) and an exposure time of 300 ms.i. Acquire a stack at both wavelengths with 143 planes every 0.7 mm and automated stages for multiple position acquisition.
Note: Use minimal laser power and exposure time to minimize phototoxicity.We choose to acquire 143 planes at 0.7 mm of each other since the size of the fully formed olfactory epithelium at 24 hpf is around 100 mm.0.7 mm thickness interval allows us to obtain high quality images.Troubleshooting, problem 2. coordinates of the multi-positions, depositing enough oil on the bottom of the dish to image a large number of samples correctly.
Optional: The use of an objective suitable for both irradiation at 1470 nm and imaging could simplify this step.
CRITICAL: IR-Laser is a very dangerous invisible laser that can induce irreversible eye damage, including blindness.At the end of the IR-Laser activation, be sure to close both IR laser shutter (SysCon software) and source.Troubleshooting, problem 5.

Timing: 24 h
The early morphogenesis of the olfactory epithelium occurs in the forebrain and starts with the birth of EONs all around the telencephalon at 12 hpf.Between 12 hpf and 18 hpf, EONs complete their initial migration, in which they migrate towards the middle of the telencephalon. 1 About 6 h later, EONs compact into spheroidal clusters at each side of the telencephalon to form at 24 hpf the olfactory epithelium. 14This step describes the live imaging section of the protocol from 12 hpf to 24 hpf.

EXPECTED OUTCOMES
Using IR-LEGO on live zebrafish embryos, we can control the expression of a gene at a specific time and location.After being born, zebrafish embryos development can be slowed down or accelerated by temperature switches, which makes it easy to control time course experiments.However, managing the spatial activation of a gene is more complicated, thus we propose the IR-LEGO technique.Using this protocol, we can activate a gene of interest by heating a targeted area in the embryo using an IR laser.In a genetically modified embryo mutant for the chemotaxis cytokine Cxcl12a, we activate production of mCherry-Cxcl12a in a 17 mm radius region of interest in the middle of the zebrafish embryos brain at 12 hpf, at the start of the olfactory epithelium morphogenesis.Using in vivo live imaging, we are able to visualize the area of mCherry-Cxcl12a activation and follow the olfactory epithelium morphogenesis in response to local expression of the chemotaxis source.We found that mCherry-Cxcl12a activated at 12 hpf is detected from 16 hpf to 22 hpf, with strong expression at 18 hpf (Figure 5).providing plasmids; Ste ´phanie Bosch and Brice Roncin and the Toulouse RIO imaging platform; and Aurore Laire for taking care of the fish.We also thank EMBO Workshop MMM2017, Magali Suzanne, Christian Mosimann, Nicolas Minc, and members of the Blader laboratory for advice experiments and comments on the manuscript.

Figure 1 .
Figure 1.IR-LEGO experimental setup combined with confocal spinning disk for live imaging (A) IR-LEGO system schematic.The infrared photo-irradiation device (IR-LEGO module in light red) is coupled to a spinning disk microscope dedicated to fluorescence live imaging (spinning disk confocal module in gray).IR-LEGO module consists in an infrared laser diode, a galvanometric head mirror to align the laser in the center of the field, a mirror, a dichroic mirror D1 and an IR compatible objective lens, to focalize the laser on the sample.Spinning disk confocal module contains an inverted microscope equipped with an imaging laser combiner composed of 4 diode lasers, a Nipkow spinning head (Yokogawa CSU X1), a fast SCMOS camera and a galvanometric stage for fast image acquisition.(B) A schematic of the sample setup for IR-LEGO using a 40 X APO IR water objective.The magnification shows the laser target in the middle of the zebrafish embryo telencephalon required here for the experiments.(C) IR irradiation of "upconverted phosphor particles" imaged in transmission."Upconverted phosphor particles" were irradiated with a power of 90% IR laser, inducing an emission wavelength of 545 nm.The irradiation diameter on the calibration sample is estimated to 34 mm (black outlined circle, grey line shows the diameter).

Figure 1 .
Figure1.Continued (D and E) Experimental system for synchronization of IR irradiation (D) and live imaging (E) conditions.Synchronization timings of all laser-induced heating components followed by (E) imaging chromatogram for each component to reduce phototoxicity.These timings are driven by Metamorph ("synchronization card").

Figure 2 .
Figure 2. Mating of adult zebrafish and collecting of synchronized embryos (A) Timeline of the 4-day protocol with hours on the y-axis and days in the x-axis.(B) Material used in the fish facility to breed adult zebrafishes and collect embryos.(a) 1.7 L Breeding Tank.(b) Beach breeding compartment.(c) Fishing net.(d) Small strainer.(e) Breeding Tank lid.(f) Divider.(g) Petri dish filled with fish water.(C) Male and female are placed separately on each side of the divider.(D) Remove the divider to start the mating.

Figure 2 .
Figure 2. Continued (E) Collect the embryos with the small strainer (left).Turn the strainer upside down and gently press the embryos in the Petri dish filled with fish water (middle).The embryos are in the Petri dish (right).(F) Frontal and anterior view of the selection of embryos at stage 11.3 hpf in white light (brightfield) and GFP.The anterior part of the head is shown in yellow dotted line.

CRITICAL: 3 Timing: 1 h 9 .
Embryos will not develop properly if the temperature is below 21 C. Colder temperatures would lead to high mortality.Do not slow down the embryos before they reach the shield stage because gastrulation has not yet occurred.11Preparation of agarose Petri dish and mounting zebrafish embryos Day Sort the proper staged embryos for fluorescent expression under a stereo zoom microscope (SMZ18 Nikon).

Figure 3 .
Figure 3. Mounting the zebrafish embryos in 0.7% low melting agarose (A) Material used for mounting embryos.(a) Stereoscopic microscope equipped with a transmitted light source.(b) Heating block set at 32 C. (c) Small aliquot of 0.7% LM agarose in fish water.(d) Small Petri dish.(e) 200 mL pipette.(f) Fine tip glass Pasteur pipet.(g) Dissecting tweezers.(B) Pipet 120 mL of heated 0.7% LM agarose in fish water and drop it in the center of the small Petri dish.(C) Transfer the dechorionated embryos to the agarose drop using a glass Pasteur pipet.(D) Picture showing the orientation of the embryos (black arrowhead points towards the anterior part, the head, of the embryo).(E) Embryos positions are annotated on the lid.

Optional:
Mount one embryo per glass Petri dish to optimize imaging later on.STAR Protocols 4, 102538, September 15, 2023 Protocol IR-LEGO irradiation and imaging settings Timing: h

Figure 4 .
Figure 4. IR-LEGO setup on the Spinning-Disk microscope (A) Placing the sample under the microscope.(B) Metamorph settings.(a) Multi-position pathway; (b) Laser intensity; (c) Z-acquisition scheme.(C) Rapp OptoElectronic settings.(a) Laser 1470 nm; (b) Center the laser; (c) Start of IR irradiation; (d) Adjustment of IR time and intensity.
20. Live imaging of mCherry-cxcl12a expression and EONs migration during olfactory epithelium morphogenesis.a. Change to the APO 403/1.3 oil objective for live acquisition.b.Repeat steps 19.d to 19.i.c.Set time lapse parameters for an automated acquisition of both wavelengths (with 143 planes at 0.7 mm interval and automated stages for multiple positions) every hour during 12 h (Figure4B).d.Launch the 24 h acquisition (Figure2A).Note: Do not select embryos that are close to each other to image, as this will affect their development and imaging quality.e.The next morning, at the end of the time-lapse acquisition, remove your sample and shut down the system.

Figure 5 .
Figure 5. Live imaging after IR irradiation (A and B) Left, image showing the IR laser target area (17 mm radius circle in the middle of the telencephalon) at 12 hpf and Right, imaging over time from 12 hpf to 24 hpf in a (A) Tg(cldnb:lyn-GFP), control, or (B) Tg(cldnb:lyn-GFP); Tg(hsp:mCherry-Cxcl12a) double transgenic embryos.Scale bar represents 100 mm.(C and D) Plot profiles of mCherry-Cxcl12a expression (Gray value) visualized in (A-B, dashed white rectangle) respectively using Fiji.(E)Visualization of mCherry-Cxcl12a activation at 16 hpf using the Imaris ''Surface'' tool.The picture in Figure5Bat 16 hpf was used to represent mCherry-Cxcl12a expression.mCherry-Cxcl12a surface is shown in yellow and cldnb:lyn-GFP expression in green.

TABLE REAGENT
2,3Samples are imaged using an inverted spinning disk confocal microscope (Leica inverted DMi8 microscope, equipped with a CSU-X1 spinning disk head), a sCMOS camera (Hamammatsu Flash4 V2+ camera), and a 403 oil-immersion objective.a. Power on the Leica Spinning Disk confocal microscope, the computer and open Metamorph and SysCon (Rapp OptoElectronic GmbH microscopy photomanipulation) softwares.Load the correct journal in the Metamorph software to connect with the SysCon software.b.Select the APO 403/1.3 oil objective.c.Place the 35 mm Petri dish with the mounted embryos on the microscope stage.(Figure