We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.
Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.
Platelets are produced by megakaryocytes-specialized cells located in the bone marrow. The precise way megakaryocytes release platelets in the circulation has long remained unclear owing to the technical challenge in observing real-time events through the bone. Two-photon microscopy has helped overcome this challenge and led to major advances in understanding the platelet formation process. The first in vivo megakaryocyte observations were made by von Andrian and colleagues in 2007, with the visualization of fluorescent megakaryocytes through the skull1. This was possible because the bone layer in the frontoparietal skull of young adult mice has a thickness of a few tens of microns and is sufficiently transparent to allow visualization of fluorescent cells in the underlying bone marrow2.
Ensuing studies applied this procedure to evaluate proplatelet formation under various conditions and to decipher the underlying mechanisms3,4,5,6. These studies provided definitive evidence that megakaryocytes dynamically extend protrusions, called proplatelets, through the endothelial barrier of the sinusoid vessels (Figure 1). These proplatelets are then released as long fragments that represent several hundred platelets in volume. The platelets will be formed after the remodeling of the proplatelets in the microcirculation of downstream organs, notably in the lungs7. To date, however, the precise process and molecular mechanisms remain subject to debate. For instance, the proposed role of the cytoskeletal proteins in the elongation of proplatelets differs between in vitro and in vivo conditions3, and differences in proplatelet formation have been demonstrated under inflammatory conditions6. Complicating things further, a recent study disputed the proplatelet-driven concept and proposed that in vivo, platelets are essentially formed through a membrane-budding mechanism at the megakaryocyte level8.
This paper presents a protocol for the observation of megakaryocytes and proplatelets in the bone marrow from the skull bone in living mice, using a minimally invasive procedure. Similar approaches have been previously described to visualize other marrow cells, notably hematopoietic stem and progenitor cells9. The focus here is on the observation of megakaryocytes and platelets to detail some parameters that can be measured, notably proplatelet morphologies and platelet velocity. This protocol presents how to insert a catheter into the jugular vein to inject fluorescent tracers and drugs and observe through the skull bone. The calvarial bone is exposed using minor surgery so that a ring is glued to the bone. This ring serves to immobilize the head and prevent movements due to breathing and form a cup filled with saline as the immersion medium of the lens. This technique is well suited to i) observe in real time the sinusoid geometry and megakaryocytes interacting with the vessel wall; ii) follow megakaryocytes in the process of proplatelet formation, elongation, and release; and iii) measure platelet movements to monitor the complex sinusoid blood flow. Data obtained using this protocol have been recently published3.
All animal experiments were performed in accordance with European standards 2010/63/EU and the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg, Animal Facility agreement N°: G67-482-10, project agreement N°: 2018061211274514).
1. Preparation of mice and insertion of a catheter in the jugular vein
NOTE: Here, male or female, 5-7-week-old mTmG reporter mice were used (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo10) crossed with Pf4-cre mice11, allowing intense green fluorescence labeling in megakaryocytes and platelets12. Before beginning the experiment, preheat the heating chamber of the microscope for a few hours.
2. Surgery and installation of the cranial ring
NOTE: The full support for mouse installation comprises 4 pieces, a block and a plate, the ring to hold the mouse head, and a screw to fix the ring to the block (Figure 3A). All elements of the support have been obtained from i.materialise.com by 3D printing. The plate is made of acrylonitrile butadiene styrene (ABS) polymer, the block and screw of stainless steel, and the ring of high-grade stainless steel (high-detailed stainless steel) (See Supplemental Figure S1 for dimensions and Supplemental Materials for the 3D printing files). The block is fixed permanently to the support, either screwed or glued. Once the ring is fixed on the mouse skull, it should be screwed to the block holder (Figure 3A).
3. Follow-up of the anesthetized mice until the end of the experiment
NOTE: Anesthesia is re-induced every 35 min by alternating subcutaneous (s.c.) injections of ketamine (25 µg/g) in a volume of 5 µL/g body weight and a mixture of ketamine (50 µg/g) and xylazine (5 µg/g) (1.2 µL/g). As it is not possible to open the heated microscope chamber during acquisition, anesthetic monitoring is performed before and after re-administration of the anesthetic by toe pinching. Similarly, toe pinch is performed before beginning each new video recording.
4. Two-photon imaging
NOTE: See the Table of Materials for details about the microscope and related equipment. Images were recorded with a resonant scanner (12 or 8 kHz). The bidirectional mode was set up to increase speed acquisition as pixels are recorded in both directions; hence, any mismatch in the phase must be corrected with the control panel "phase correction." Finally, an adapted averaging was set up as a compromise between speed of acquisition and signal-to-noise ratio.
Using this protocol, the fluorescent tracer, Qtracker-655, was intravenously administered to image anastomosed marrow sinusoid vessels in the skull bone marrow and the flow direction as depicted by the arrows (Figure 4A, left). Using mTmG mice, eGFP-fluorescent platelets were recorded over 20 s in each vessel branch, and their velocity was measured using ImageJ and GNU Octave software (Figure 4A, right). Note the heterogeneity in flow velocity and direction. Sinusoid vessels present complex flows due to the anastomoses, with the presence of flow-reflow and even stasis, as shown in the bifurcation recorded in Video 1. The same bifurcation is shown in Figure 4B (left), with the red and blue arrows highlighting opposite flows. Platelet velocity in this bifurcation has been measured and reported in the graph (Figure 4B, right). The red tracing corresponds to the velocity in the left vessel branch and the blue tracing to that in the right vessel branch. This shows irregularity over time in each vessel branch, with phases of acceleration, stasis, and deceleration. It is important to note that in some vessels, blood flow was too rapid for reliable analysis under the present acquisition conditions.
Proplatelet elongation recording allows the visualization of their various morphologies over time (Figure 5). Some proplatelets elongate with irregular morphologies (Figure 5Ai and Video 2). Others, usually thinner ones, may extend over long distances necessitating the movement of the acquisition window to follow their extension (Figure 5Aii and Supplemental Video 1). Others are shorter and thicker and usually elongate slowly (Figure 5Aiii and Supplemental Video 2). When megakaryocytes extend proplatelets in areas of complex flows, such as the bifurcation shown in Figure 4B, proplatelets are tossed from one vessel branch to the other according to flow direction (Figure 5B and Video 3). The importance of hemodynamic forces for proplatelet elongation in the flow direction was evidenced when the mouse unexpectedly underwent cardiac arrest. Stopping blood flow relaxed the proplatelets extended by the megakaryocyte (Figure 5C and Video 4).
When the quality/resolution of acquisition is appropriate, morphological parameters can be measured, such as the length of proplatelets until detachment, their width at the base of the proplatelet or any other defined location, or the size of the buds (Figure 6A). A mean maximal proplatelet length of ~185 µm (range 41.5-518.5 µm) and a mean proplatelet width of 5.2 µm (range 2.8-8 µm) was calculated (Figure 6A). Plotting proplatelet length as a function of time allows the visualization of the behavior of proplatelets during phases of elongation, stasis, or even retraction (Figure 6B). Measurement of proplatelet elongation velocity is an important parameter, which directly reflects the elongation, stasis, or retraction behavior of proplatelets3.
Proplatelet elongation velocity can be measured if the proplatelet remains attached to its mother cell; once detached, it will be immediately carried away by the flow and disappear from the visualization window. In wild-type (WT) mice, the mean proplatelet elongation velocity was ~10 µm/min (Figure 6C), in agreement with previous publications1. Finally, it is possible to inject drugs through the catheter inserted in the jugular vein. For instance, it has been observed that intravenous administration of vincristine, a drug that depolymerizes microtubules, led to the retraction of proplatelets from WT mice but had no effect on proplatelets from myosin IIA-deficient mice(Myh9-/-) (Video 5 and Video 6)3.
Figure 1: Schematic representation of proplatelet formation in the bone marrow. After differentiation from hematopoietic stem cells, large megakaryocytes align along sinusoid vessels and extend cytoplasmic projections, called proplatelets, through the endothelial barrier. Proplatelets elongate and detach under the influence of hemodynamic forces to further remodel into platelets in the downstream microcirculation. Please click here to view a larger version of this figure.
Figure 2: Insertion of the catheter. (A) An incision is made to expose the jugular vein and the top of the pectoral muscle. (B) The catheter filled with saline is inserted in the jugular vein by passing through the muscle, creating a compression point. (C) The mouse is carefully turned so that it lies ventral surface downward with the catheter well-positioned. Please click here to view a larger version of this figure.
Figure 3: Experimental setup used for two-photon imaging through the skull bone. (A) The 3D printed stainless steel block is fixed on the ABS polymer support (see Supplemental Figure S1 and Supplemental Materials for the 3D designs). The block and the skull ring are designed so that the ring can be screwed to the block. Note that, for ease of use, the screw head has been embedded in Epon. (B) Schematic showing the positioning of the ring on the exposed skull bone. (C) Photographs illustrating the mouse with the ring glued to the skull, the head on a tissue pad, and (D) the positioning under the microscope objective. Abbreviations: 3D = three-dimensional; ABS = acrylonitrile butadiene styrene. Please click here to view a larger version of this figure.
Figure 4: Measure of platelet velocity in skull bone marrow anastomosed sinusoid vessels. (A) Left, two-photon imaging of sinusoid vessels labeled by Q-Dot injection. z-projection showing the anastomosis of the sinusoid vessels in the skull bone marrow. Arrows indicate the direction of flow, illustrating the complexity of the flows in sinusoids. Right, mean flow velocity estimated by the measurement of the platelet velocity in each vessel branch. The numbers on the x-axis correspond to the arrow numbers in the image on the left. (B) Left, sinusoid bifurcation delineated by dotted lines, showing platelets flowing in opposite directions (red and blue arrows) (corresponding to Video 1). Two-photon single plane image. Right, a graph showing the flow velocity in each portion of the vessel as a function of time (red line, left side; blue line, right side), showing phases of stasis, acceleration, and deceleration. Scale bars = 20 µm. Please click here to view a larger version of this figure.
Figure 5: Various proplatelet morphologies observed in the skull marrow by two-photon microscopy. (A) Three representative in vivo proplatelets: z-projection images from time-lapse experiments (shown in Video 2, Supplemental Video 1, and Supplemental Video 2) showing the various morphologies of proplatelets (arrows) extending within bone marrow sinusoids. Proplatelets and megakaryocytes are in green; sinusoid vessels are in red. (B) Time-lapse images of a proplatelet in the same bifurcation as in Figure 4B, oscillating according to the direction of flow (also shown in Video 3). (C) z-projection time-lapse images showing relaxation of 3 proplatelets after cessation of blood flow due to cardiac arrest of the mouse (shown in Video 4), previously aligned in the same flow line (indicated by the white, yellow, and blue arrows). Scale bars = 20 µm. Please click here to view a larger version of this figure.
Figure 6: Proplatelet morphological analyses. (A) Upper, z-projection image depicting the site where the proplatelet width was measured along with maximal length; below are graphs representing the maximal length of 25 individual proplatelets and the mean proplatelet width of the same proplatelets (same gray code) throughout elongation. i) The proplatelet width was measured by drawing a perpendicular line at a fixed position close to the base of the proplatelet, and the profile intensity was recorded at this position every minute. A Gaussian curve was fitted to this profile, and the FWHM was considered to represent the width of the proplatelet. The mean width was then calculated by averaging the widths of the proplatelet measured at each time point at the fixed position. Bars are mean ± sem of the width measured every minute during acquisition; ii) the maximal proplatelet length was also determined, from the base of the proplatelet to its tip. (B) Individual tracings showing the elongation behavior of WT proplatelets, some of them presenting pause and retraction phases. To better highlight the pauses and retractions upon the process of proplatelet growth, the length measured every 10 s was represented as a percentage of the maximal length of each proplatelet. (C) Scatter plot representing the proplatelet elongation speed. The net mean proplatelet elongation speed (including pauses and retractions) was calculated as the change in proplatelet length at 1 min intervals, averaged over the whole time sequence. Individual values and mean ± sem. Abbreviations: FWHM = Full Width at Half Maximum; sem = standard error of the mean; WT = wild-type.
Video 1: Reverse flow within sinusoid vessels. Circulating platelets (green) within the sinusoid vessels (red, Qtracker) (single confocal z-plane). An anastomosed sinusoid vessel is shown presenting bifurcations where the flow is unstable and displays phases of stasis, acceleration, and deceleration. Acquired with a Leica SP5 microscope equipped with a 12 kHz resonant scanner and a 25x water objective with a numerical aperture of 0.95 (Leica), one plane, 256 x 90 pixels, bidirectional acquisition, line averaging 2, 133 frame/s. Please click here to download this Video.
Video 2: Elongating proplatelets display various morphologies. Representative time-lapse video recording (z-projection) in a WT mouse with megakaryocytes extending proplatelets with various morphologies (green) within bone marrow sinusoids (red, Qtracker). Acquired with a Leica SP5 microscope equipped with a 12 kHz resonant scanner and a 25x water objective with a numerical aperture of 0.95 (Leica), z-stack acquisition at 10 s intervals, z-depth of 4 µm, x-y resolution 384 x 384 pixels, line averaging 8. Whenever necessary, the profiles were aligned using the ImageJ template matching plug-in to obtain an average z-projection. Depending on the image quality, the background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Video 3: Proplatelets are tossed by changes in the flow direction. Time-lapse video showing a wild-type megakaryocyte in the sinusoid bifurcation shown in Video 1, in the process of extending a proplatelet that oscillates in one vessel branch depending on the direction of flow (z-projection). Acquired with a Leica SP5 microscope equipped with a 12 kHz resonant scanner and a 25x water objective with a numerical aperture of 0.95 (Leica), z-stack acquisition at 10 s intervals, z-depth of 4 µm, x-y resolution 384 x 384 pixels, line averaging 8. The profiles were aligned using the ImageJ template matching plug-in, and an average z-projection was obtained. The background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Video 4: Impact of flow on proplatelet alignment and tension. Time-lapse video recording of a proplatelet before and after cardiac arrest. The video shows three proplatelets (green, labeled with AF-488 anti-GPIX antibody derivative) in the same flow line before cardiac arrest, thus poorly individualized at the resolution of the two-photon microscopy. Following cardiac arrest, the three proplatelets separate and become relaxed in the absence of blood flow. Video acquired with a Leica SP8 confocal microscope equipped with an 8 kHz resonant scanner, image with a 25x water objective with a numerical aperture of 0.95 (Leica). The profiles were aligned using the ImageJ template matching plug-in, and an average z-projection was obtained. The background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Video 5: Impact of vincristine drug injection on the proplatelet behavior of WT mice. Time-lapse video recording of a proplatelet before and after administration of vincristine (1 mg/kg) (z-projection). The elongating proplatelet (green) within the sinusoid vessels (red) starts to retract after intravenous administration of vincristine. Video was acquired with a Leica SP8 confocal microscope equipped with an 8 kHz resonant scanner, image with a 25x water objective with a numerical aperture of 0.95 (Leica). The profile was aligned using the ImageJ template matching plug-in, and an average z-projection was obtained. The background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Video 6: Impact of vincristine drug injection on the proplatelet behavior of myosin-deficient mice. Time-lapse video recording of a proplatelet before and after administration of vincristine (1 mg/kg) (z-projection). The elongating proplatelet (green) within the sinusoid vessels (red) continues to elongate in the Myh9-/- mice. Video was acquired with a Leica SP8 confocal microscope equipped with an 8 kHz resonant scanner, image with a 25x water objective with a numerical aperture of 0.95 (Leica). The profile was aligned using the ImageJ template matching plug-in, and an average z-projection was obtained. The background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Supplemental Figure S1: Design for the 3D printing of the skull ring and support. This design was used to 3D print (A) the skull ring in high-quality stainless steel, (B) the screw, (C) the block in stainless steel, and (D) the support in ABS polymer. Abbreviations: 3D = three-dimensional; ABS = ABS = acrylonitrile butadiene styrene. Please click here to download this Figure.
Supplemental videos:
Supplemental Video 1: Representative time-lapse video recording (z-projection) in a wild-type mouse with megakaryocytes extending proplatelet (green) within bone marrow sinusoids (red, Qtracker). Acquired with a Leica SP5 microscope equipped with a 12 kHz resonant scanner and a 25x water objective with a numerical aperture of 0.95 (Leica), z-stack acquisition at 10 s intervals, z-depth of 1.34 µm, x-y resolution 384 x 384 pixels, line averaging 8. Whenever necessary, the profile was aligned using the ImageJ template matching plug-in to obtain an average z-projection. Depending on the image quality, the background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Supplemental Video 2: Representative time-lapse video recording (z-projection) in a wild-type mouse with megakaryocytes extending proplatelet (green) within bone marrow sinusoids (red, Qtracker). Acquired with a Leica SP5 microscope equipped with a 12 kHz resonant scanner and a 25x water objective with a numerical aperture of 0.95 (Leica), z-stack acquisition at 10 s intervals, z-depth of 1.34 µm, x-y resolution 384 x 384 pixels, line averaging 8. Whenever necessary, the profile was aligned using the ImageJ template matching plug-in to obtain an average z-projection. Depending on the image quality, the background was subtracted, the image was smoothed, and the brightness and contrast adjusted. Please click here to download this Video.
Supplemental Materials:
Coding File 1: 3D printing-Block. 3D object file for the printing of the block. Please click here to download this Coding File.
Coding File 2: 3D printing-Skull ring. 3D object file for the printing of the skull ring. Please click here to download this Coding File.
Coding File 3: 3D printing-Support. 3D object file for the printing of the support. Please click here to download this Coding File.
Coding File 4: 3D printing-Screw ring. 3D object file for the printing of the screw. Please click here to download this Coding File.
The mechanisms of platelet formation are highly dependent on the bone marrow environment. Hence, intravital microscopy has become an important tool in the field to visualize the process in real-time. Mice with fluorescent megakaryocytes can be obtained by crossing mice expressing the Cre recombinase in megakaryocytes with any floxed reporter mice containing a conditional fluorescent gene expression cassette. Here, mTmG reporter mice were used (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo10) crossed with Pf4-cre mice11, allowing intense green fluorescence labeling in megakaryocytes and platelets12. It is important to note that some leakage has been reported with Pf4-cre mice in leukocytes, including monocytes/macrophages and fibroblasts12,13.
However, megakaryocytes are easily recognized by their large size and nucleus. Experimenters new to the field could compare observations in [mTmG; Pf4-cre] mice with those obtained after megakaryocyte-specific labeling. This is possible through intravenous administration of an Alexa-fluor-labeled antibody directed against the platelet-specific protein, GPIX (CD42a)3,14. This latter approach is also beneficial for imaging fluorescent megakaryocytes from any genetically engineered mouse without performing tedious and time-consuming mouse crossings with fluorescent reporter mice. Here, 5-7-week-old mice were used as younger mice present with a thinner bone that is more translucent and facilitates imaging compared to older mice. However, mice that are too young may present difficulties in the installation of the cranial ring without leakage.
In this study, intravital imaging was performed using a Leica microscope equipped with a 25x water objective with a numerical aperture of 0.95 (Table of Materials). The objective should be conical so that it can enter the cup formed by the cranial ring with an optimal working distance. Images were recorded with a resonant scanner (12 or 8 kHz). The overall settings will depend on the type of scientific question to be answered and should be optimized depending on the magnification, resolution, and acquisition speed needed. For instance, with a 12 kHz resonant scanner, a 25x objective would be better suited to measure platelet velocity, whereas an 8 kHz scanner and a lower magnification could be sufficient to record proplatelet elongation.
The most critical step of the protocol is the correct attachment of the ring to the skull bone so that no leakage occurs, and so that it does not detach during recording. For that, it is essential that the skull bone be dry just before applying the ring. Once the ring is glued, rapidly re-humidify the bone. Furthermore, due to the force exerted by the weight of the head on the ring, the ring is subjected to a tension that may cause it to detach partially during the recording. This may cause leakage and thus, dryness of the bone, leading to both poor-quality images and undesirable inflammatory reactions. This problem can be easily avoided by adding a folded compress under the nose of the mouse to support the head (Figure 3C). Another critical point is to minimize bleeding and the presence of blood inside the ring as it may lead to blurred images. This must be kept in mind when studying mice that present defective hemostasis and are prone to bleeding. Some tracers, such as dextran, may present leakage in the bone marrow cavity if injected in a concentrated form, which worsens vessel imaging, though not the green fluorescence of megakaryocytes. Qtracker-655 does not leak much but is cleared from the circulation within 30 min so that new injections of the tracer are required to visualize vessels over longer periods.
One limitation of this method described here is the necessity to stop acquisition every 35 min to re-inject anesthetics. This limitation can be overcome if an anesthesia machine is available. In that case, anesthesia can be induced similarly by i.p. injection of a mixture of ketamine and xylazine, which is the easiest way to perform the insertion of the catheter and the minor surgery. Once positioned below the microscope objective, anesthesia is then maintained by inhalation of gases (mixture of oxygen, anesthetics such as isoflurane and ambient air). In this way, observations can be made over several hours without interruption. However, for ethical reasons, observations were limited to 3 h in this study. Another limitation in using ketamine/xylazine mixture could be its potential deleterious impact on platelet functions16 that could require alternative anesthesia schemes.
Overall, the principal merit of this well-established protocol is its noninvasive aspect with only minimal surgery. Other protocols have been developed to perform time-lapse intravital imaging in long bones. These rely on either invasive surgery that requires muscle tissue removal and bone abrasion17,18, implanted endoscopic probes close to the femur head19,20, or installing a window chamber in the femur21. All these procedures result in major trauma and varying degrees of inflammation. Inflammation is not only detrimental to the mouse, but it has also been reported to modify the process of platelet formation6 so that uncontrolled inflammatory conditions might lead to discrepancies and misinterpretation of the data. That is why this procedure was chosen to avoid inflammatory reactions. However, depending on the scientific question, experimenters may prefer to have a deeper observation field and higher resolution (less scattering), which is possible by using a skull-thinning-based approach at the expense of a low degree of inflammatory reaction.
Because of its noninvasive nature, the major limitation of this method is the maximal depth that can be reached. Due to the density of the bone and its scattering properties, it is possible to image regions within a depth of only a few hundred micrometers, preventing observations of the whole calvarial marrow. The development of three-photon microscopy, which relies on even higher infrared excitation wavelengths, seems to be a promising approach with superior deep-tissue resolution. It has already been successfully used to image deep into the brain even through the bone22,23, opening up the exciting possibility to image bone marrow within long bones without having to thin the bone or implant a surgical window.
In summary, this method is increasingly used to study the behavior of megakaryocytes in the bone marrow and visualize the extension of proplatelets.In addition, by allowing the visualization of the outer calvarial compact bone as well as part of the underlying marrow, this method has already been used in many applications outside the platelet field. It has been used for the study of both bone and marrow cell dynamics in their environment, including osteoblasts and osteoclasts, leukocyte trafficking and marrow exit, endothelial cells and microvasculature architecture, blood flow dynamics in marrow microvessels, or cell homing24.
The authors have nothing to disclose.
The authors would like to thank Florian Gaertner (Institute of Science and Technology Austria, Klosterneuburg, Austria) for his expert advice on two-photon microscopy experiments at the time when we established the technique in the lab, and Yves Lutz at the Imaging Center IGBMC /CBI (Illkirch, France) for his expertise and help with the two-photon microscope. We also thank Jean-Yves Rinkel for his technical help and Ines Guinard for the drawing of the schema in Figure 1. We thank ARMESA (Association de Recherche et Développement en Médecine et Santé Publique) for its support in the acquisition of the two-photon microscope. AB was supported by post-doctoral fellowships from Etablissement Français du Sang (APR2016) and from Agence Nationale de la Recherche (ANR-18-CE14-0037-01).
GNU Octave software | GNU Project | https://www.gnu.org/software/octave/ | |
Histoacryl 5 x 0, 5 mL | Braun | 1050052 | injectable solution of surgical glue |
HyD hybrid detectors Leica Microsystems 4tunes | Leica Microsystems | ||
ImageJ | GNU project | Minimum version required | |
Imalgene/Ketamine 1000 fl/10 mL | Boehring | 03661103003199 | eye protection |
Leica SP8 MP DIVE microscope equipped with a 25x water objective, numerical aperture of 0.95 | Leica Microsystems | simultaneous excitation of AlexaFluor-488 and Qtracker-655 | |
Matlab | MathWorks | https://www.mathworks.com/ | |
Ocrygel 10 g | Laboratoires T.V.M. | 03700454505621 | Silicon dental paste blue and yellow |
Picodent twinsin speed | Rotec | 13001002 | |
Qtracker 655 vascular label | Invitrogen | Q21021MP | injectable solution |
Resonant scanner, 8 or 12 kHz | |||
Rompun Xylazine 2% fl/25 mL | Bayer | 04007221032311 | |
Superglue gel | to glue the ring to the bone | ||
Surflo IV catheter – Blue 22 G | Terumo | SR-OX2225C1 | |
Ti:Saph pulsing laser (Coherent) (femtosecond) | Coherent |