The overall goal of this procedure is to demonstrate a technique for introduction of microparticles into the circulatory system of adult zebrafish by injection into the fish kidney. The introduced microparticles are further visualized in the vasculature by a simple technique of intravital imaging in the fish gills. This procedure can be used, for example, to carry out repeated measurements of fish blood pH in vivo by the introduction of microencapsulated fluorescent probes for pH.
To do this, at the first stage, the pH-sensitive dye SNARF-1 conjugated with dextran is encapsulated in the semi-permeable polyelectrolyte shell using layer-by-layer approach. The fluorescent dye is co-precipitated into porous calcium carbonate microcores, and the cores are coated with multiple layers of oppositely charged non-biodegradable polymers and top-coated with a biocompatible polymer containing polyethylene glycol. Next, calcium carbonate cores are resolved to obtain whole microcapsules enclosing SNARF-1.
At the second stage of the fish anesthesia and fixation, prepared microcapsules are injected directly into the kidney of the animal to deliver encapsulated dye into the fish circulatory system. Next, minimal surgery is needed to remove gill cover and denude capillaries of fish gills. Then, visualization of microcapsules in the blood and recording of fluorescent spectrum can be done in vivo by intravital microscopy to monitor blood pH.
When the injection performed right, it is possible to observe fluorescent microcapsules in the fish gills immediately after the procedure. The spectrum of florescence of the encapsulated probe can be recorded using a spectrometer coupled to a fluorescent microscope. SNARF-1 has two emission peaks, and the ratio between two wavelength corresponding to those peaks can be used to measure pH.
The proposed procedure allows to deliver fluorescent microcapsules into the fish circulatory system and to monitor remotely the fluorescence in fish blood in vivo. In case of physiological research, the main advantage of this method is repeated measurements of blood parameters in small laboratory animals, which are usually difficult to perform. If a light-sensitive fluorescent probe is used, it is advisable to perform the whole procedure in a light-reduced room.
Mix two milliliters of solution of a chosen polymer-bound fluorescent dye here, SNARF-1-dextran, with 0.6 milliliter of one molar calcium chloride solution and 0.6 milliliter of one molar sodium carbonate solution under fast stirring. At this step, porous calcium carbonate microcores enclosing the fluorescent dye are synthesized. After five, 10 seconds of agitation, transfer the suspension to two milliliters microtubes and centrifuge for 15 seconds to pellet calcium carbonate microcores.
Discard supernatant, wash the cores with about two milliliters of deionized water, and shake up the suspension. Repeat centrifugation and washing procedure three times. Incubate the suspension for one minute in an ultrasonic bath to reduce aggregation of the microcores.
Resuspend the calcium carbonate microcores in approximately two milliliters of a four microgram per milliliter solution of the cationic polymer PAH to deposit the first polymeric layer on the templates. Keep the microcores in the pH solution for about five minutes with constant shaking. After 15 seconds centrifugation, discard supernatant with unbound PAH and wash the microcores with water three times by multiple centrifugation and washing steps.
Repeat the same procedure with four milligram per milliliter solution of anionic polymer PSS to deposit the second polymeric layer on the templates. Right before adding next layer, it is advisable to apply one minute incubation in ultrasonic bath. Repeat sequentially the deposition of cationic and anionic polymers six times to obtain a 12-layer microcapsules shell.
Next, incubate microcores with shells in the polyethylene glycol-grafted poly-L-lysine for at least two hours. Then, wash the covered microcores once with water by sequential centrifugation and resuspension. Finally, dissolve calcium carbonate templates in two milliliter of 0.1 molar EDTA solution, adjusted to pH approximately 7.1, to obtain hollow microcapsules.
Discard supernatant after 45-second centrifugation, and repeat washing with EDTA twice. Discard supernatant after 45-second centrifugation, and add two milliliters of saline. Repeat washing centrifugation steps with saline three times.
Investigate size distribution and concentration of the prepared microcapsules in a hemocytometer under a fluorescent microscope. Use ImageJ or equivalent software for size analysis of the particles. Store obtained encapsulated probe in the dark.
To prepare optic system, place the required set of fluorescent filters to the fluorescent microscope according to the characteristics of the applied fluorescent dye, and turn on the fluorescent lamp. Pull out the lever to the eyepieces. Connect the optical fiber from one end to the spectrometer and on the other end to a collimator.
Using adapters, play the collimator in the focus of the camera tube or other available port of the fluorescent microscope. For calibration of the prepared microcapsules, place about five microliters of the microcapsule suspension on a microscope slide, and dry the drop in a dark place. Turn on the spectrometer.
Run the spectrometer control program, and prepare the spectrometer for measurements. To calibrate spectral characteristics of the microencapsulated SNARF-1, use series of buffers with different pH. Drop about 10 microliters of sodium buffer onto the dried microcapsules with SNARF-1, and cover it with a coverslip.
Place a glass slide on the microscope stage. Locate the microcapsules using a magnification about 400. Turn the microscope lever to the camera port.
Register the fluorescence with the spectrometer. Make sure the spectral signal is far beyond the background level, and observe microcapsules are not in a bubble. Avoid prolonged illumination of the same microcapsules as SNARF-1 is sensitive to photobleaching.
Turn the lever back to the eyepieces. Calculate the ratio of fluorescence intensity of encapsulated SNARF-1 at the wavelength corresponding to the emission peaks of the protonated and deprotonated dye. Build a calibration curve of the ratio dependence on the pH of the medium.
Collect about 10 microliters of blood from euthanized fishes. Determine its pH by pH meter with a microelectrode. Then, drop blood onto slide with dried microcapsules, and register the ratio of the fluorescence intensity as described for calibration buffers.
Since you should take into account influence of blood components on readout of encapsulated SNARF-1, adjust linear coefficient of the calibration curve to make the curve match the measurements in fish blood. To prepare needle for injection, release a steel needle from the tip of the insulin syringe by removing plastic with a sharp lancet. Insert the needle halfway into the glass microcapillary.
Quickly and gently solder it by a gas torch. Connect glass microcapillary to microinjector, and flush it with sterile water for three times. Ensure that the liquid flows through the needle.
Fill the system with water. Make sure there are no bubbles in the system. On the day of the experiment, take the prepared suspension of microcapsules in sterile saline containing from one half to six millions of microcapsules per microliter.
Resuspend it by the ultrasonic bath for one minute. During the following injection, shake the vial periodically to resuspend microcapsules and prevent their aggregation. Using a spoon, get the fish out of anesthetic, and gently place it on a damp sponge in a lateral position with the head towards the left if you are right-handed or towards the right if you are left-handed.
Just before the injection, suck one to two millimeters of air into the glass capillary connected with microinjector. Then, load it with approximately two microliters of the dispersed microcapsules. Gently stabilize the body of the fish on the sponge with your non-dominant hand.
Find the fish lateral line. Put the needle one milliliter below of the midpoint of an abdominal segment of the lateral line. Then, by a scraping movement, move the fish scale aside, and make a puncture.
Insert the needle into the body at an angle of 45 degrees to the table surface. Push the needle toward the spine until it carefully rests against the spine. Then, slowly release about one microliter of the microcapsule suspension into the kidney, and slowly withdraw the needle.
Rinse the fish from head to tail with a stream of water to remove spilled microcapsules at the injection site. Use dissection scissors to remove the gill cover from the fish head and denude fish gills. Rinse gills with water.
Using a spoon, transfer the fish to a microscope slide, and place it on the stage of a fluorescent microscope. When you perform following procedures, make sure that the gills of the fish do not dry out. To do this, periodically moisten them with water using a Pasteur pipette.
Darken the room and using low magnification inspect the gills to find fluorescent microcapsules. When you find it, switch the lens to a higher magnitude, and position it in the center of the field of view. Turn the lever to the port with a connected spectrometer.
Record the spectral signal. Turn the lever back to the eyepieces. Repeat the measurements for different microcapsules several times.
Transfer the fish to the aquarium with proper aeration for recovery. The measurement procedure can be repeated for one individual for several times with the use of repeated anesthesia or another method of fixation. The picture shows a representative example of in vivo measurements of zebrafish blood and the hypercapnia by encapsulated fluorescent dye SNARF-1.
In control conditions, blood pH remains stable during four hours after injection of microcapsules, while five minutes exposure under elevated carbon dioxide causes acidification of fish blood. While carrying out the procedure, it is important to minimize animal stress caused by handling and surgery. With some practice, both injection and signal registration on average take about two, three minutes, so this protocol allows to complete manipulations before the fish awakens from mild anesthesia.
During the procedure, make sure that the gills of the fish are always moistened. After watching this video, you should have a good understanding of how to perform simple and effective implantation of microparticles into the circulatory system of small fish. As demonstrated, this technique is very convenient for repeated in vivo recordings of blood pH in adult zebrafish using microcapsules filled with a fluorescent probe.
