Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.
The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.
Directed cell migration is a critical event occurring during different physiological and pathological processes, including development, immune response, tissue repair, and tumor initiation, progression, and dissemination1,2. This process relies on specific extracellular chemotactic signals sensed by their cognate receptors at the plasma membrane and then transduced into intricate intracellular signals. These pathways, in turn, activate cell migration through a series of responses, including local activation of the cytoskeleton, membrane trafficking and remodeling, and cell polarization3. Two distinct types of cell migration have been well characterized: mesenchymal and amoeboid4. Mesenchymal migration is relatively slow (<1 µm/min), relies on strong adhesion between cells and the extracellular matrix (ECM), and depends on metalloproteases-induced matrix degradation. In contrast, ameboid migration, a characteristic of immune cells responding rapidly to inflammatory cues, is faster (>10 µm/min) and utilizes weak adhesions to navigate through the ECM. Regardless of the modality, cell migration requires a constant remodeling of the plasma membrane resulting in the generation of protrusive structures at the leading edge of the cells, which are tightly coordinated with the retraction of the membrane at the rear1.
To unravel the molecular mechanisms underlying these sub-cellular processes, it is fundamental to visualize the dynamics of the membranes in migrating cells at an appropriate temporal and spatial resolution. Moreover, since membrane remodeling is strongly influenced by the properties of the tissue microenvironment surrounding the migrating cells, it is crucial to image this process directly in the native tissue, within live animals. This is accomplished by using intravital microscopy (IVM)5. The very first IVM imaging was performed ~200 years ago, when leukocytes extravasation was visualized using a simple trans-illumination microscope6 and thereafter IVM was used primarily on semi-transparent model organisms, such as Zebrafish and Drosophila7,8. In the last two decades, IVM has been successfully used to investigate cellular processes in mice, rats, and larger mammals such as pigs5,9. This was made possible due to 1) significant developments in confocal and multi-photon (MP) microscopy and 2) the raise of gene-editing technology such as CRISPR-Cas9, which has permitted the rapid engineering of a variety of animal models to express fluorescently tagged proteins and reporters. Besides, the visualization of individual cells and their microenvironment during processes such as tumor initiation, cell migration, and immune response has been made possible by both other forms of exogenous labelings, such as the systemic introduction of dyes to label the vasculature10,11, and the excitation of endogenous molecules, such as collagen through Second Harmonic Generation (SHG)10,12 and nerve fibers through Third Harmonic Generation (THG)11,13. Finally, an improvement in the techniques to minimize the motion artifacts due to heartbeat and respiration has led to the development of intravital subcellular microscopy (ISMic), which has allowed researchers to image and investigate several subcellular events directly in live animals at a level of resolution similar to those observed in vitro14,15,16,17. Examples of ISMic include investigating cytoskeletal dynamics during exocytosis14,15,16,17 and endocytosis15, cytoskeleton dynamic remodeling during cell migration17,18, mitochondrial localization and metabolism19,20, and calcium signaling in the brain21.
This protocol details the different steps and procedures to investigate cell membrane remodeling at a subcellular resolution during neutrophil migration in the ear skin of a live mouse using ISMic. This approach is based on previously described protocols22,23 and adapted to achieve higher spatial and temporal resolution.
All animal experiments and procedures were approved by the National Cancer Institute (National Institutes of Health, Bethesda, MD, USA) Animal Care and Use Committee (protocols LCMB-031 and LCMB-035) and were compliant with all relevant ethical regulations. Both male and female mice aged between 2 and 6 months were used for the experiments. mT/mG and wild-type (WT) host mice are in an FVB/NJ background while LyzM-Cre x mTmG mice are in a C57BL/6 background.
1. Materials and preparation of reagents and tools
2. Purification of neutrophils from a donor mouse
3. Neutrophil labeling
NOTE: To visualize the neutrophils, they were purified from LyzM-Cre mT/mG mice, in which myeloid cells express a membrane-targeted peptide fused with the GFP. In addition, purified neutrophils from wild-type (WT) mice were labeled in vitro. The following steps describe the procedure used to label purified neutrophils with a commercial cell-permeable green fluorescent dye and can be adapted to any other MPM compatible fluorescent probe of choice.
4. Neutrophil injection
NOTE: Anesthesia must be performed following the local institutional Animal Care and Use Committee guidelines.
5. Anesthesia for IVM
NOTE: A deeper anesthesia significantly improves the imaging quality by decreasing the motion artifacts due to heartbeat and respiration. Neutrophil-injected mice are subjected to chemical anesthesia, which provides deeper sedation than gas-induced anesthesia. Anesthesia must be performed following the local institutional Animal Care and Use Committee guidelines.
6. Imaging
NOTE: The animal setup and the imaging parameters described here are optimized for an inverted multiphoton microscope equipped with a single laser line to excite and simultaneously collect emissions from the different fluorophores.
7. Representative data analysis
NOTE: Data can be visualized and analyzed with either the microscope software, third-party software, or customized programs. The procedure and the tools utilized depend on the specific needs of the investigators. Shown here is one example of a workflow to quantify membrane curvature and local area changes at the leading edge and the rear of the migrating neutrophils.
Here, two different sets of results are presented to illustrate classical IVM and ISMic that provide cellular and sub-cellular resolution, respectively. In the first example, neutrophils were purified from a wild-type (WT) mouse, labeled with Cell Tracker Green to stain the cytoplasm, and injected into a transgenic mouse expressing a fluorescent protein targeting the plasma membrane (mTomato mouse, also known as mT/mG33, Figure 1 and Movie 1 and Movie 2). This recipient mouse enabled the visualization of structural features in the ear tissue such as blood vessels, resident cells, and hair follicles (Figure 1C and Movie 1) via IVM-based approach (step 6.8.1). Collagen fibers, revealed via SHG (detected at half the wavelength of the excitation), were arranged in an intricate network in the dermis, where neutrophils were injected. Along the edge of the hair follicles (HF), a layer of epithelial cells (i.e., keratinocytes) was observed. Occasionally, artifacts from residual hairs resulting in a local depression of the skin were visualized (H). The laser-induced injuries were easily visualized due to their strong auto-fluorescence detected in all the channels and by the alterations of the collagen arrangement (Figure 1D). A more complete view of the 3D architecture of the skin and the localization of the injected neutrophils can be appreciated in Movie 1, which portrays a Z-stack of the skin from the outer to inner layers and a 3D volume rendering. Time-lapse imaging showed the neutrophils sampling the ear skin and interacting with the ECM and host tissue (Movie 2). Imaging at this resolution and acquiring a Z stack every 30 s allows performing cell tracking, and measurement of motility parameters (i.e., speed and directionality), but a precise and detailed analysis of membrane remodeling is challenging at this resolution.
In the second example, membrane remodeling was assessed via the ISMic approach (step 6.8.2) using mGFP-expressing neutrophils purified from LyzM-cre mT/mG mice and injected into WT animals (Figure 2A, experimental flowchart). Using the ISMic protocol (Movie 3 and still images in Figure 2B) and upon laser injury, dynamic remodeling of the plasma membrane is observed during migration, and formation of membrane protrusions at the leading edge and the retraction of the rear of the cells are clearly visualized (Figure 2 and Movie 3). The time-lapse sequences highlighted in Movie 3 reveal the complexity of the interactions with the ECM. Indeed, neutrophils migrated through the interstitial space moving either along the fibers or in the spaces between them. Finally, quantitative aspects such as the changes in curvature and the area changes at the front and the back of the cells were quantified for each time point. Using the cell described in Figure 2B as an example, the local dynamics of the plasma membrane were analyzed using an algorithm pipeline (see step 7) based on the identification of 100 boundary points underlying the cell surface (Figure 3A). The changes in the local curvature (Figure 3B) and in the area underlined by plasma membrane protrusions (Figure 3C) were calculated for each boundary point and reported for each time frame as kymographs (Figure 3D,E). Both the front and the back of the cells maintain higher curvature than the side of the cells (Figure 3D); negative area changes (retractions, blue regions) are more apparent at the back of the cells than at the leading edge where the positive area changes are more prominent (protrusions, red regions) (Figure 3E).
Figure 1: IVM of neutrophils in the mouse ear skin. (A) Schematic drawing of the ear set-up on the microscope stage. (B) Experimental flowchart. (C) Representative image of the ear skin of a mTomato mouse injected with fluorescently labeled neutrophils, with different projections. Hair follicle (HF), Blood Vessel (BV), Epidermis (E), Dermis (D), Basal Cell Layer (BCL), and Hair artifact (due to a residual hair at the skin surface, H). (D) Representative image of mouse skin after a sterile laser injury: blue channel (right), green and red channels (middle), merged image (left). Injury is visible on the left of the image by a strong autofluorescence emission in both green and red channels as well as a disruption of the ECM observed by the formation of a hole in the collagen-I fibers. In C and D, Green: neutrophils; Red: mouse host tissue; Cyan: Collagen-I SHG. Please click here to view a larger version of this figure.
Figure 2: Time-lapse of mGFP neutrophil migrating in a WT mouse ear. (A) Experimental flowchart. (B) Representative still images from Movie 3. (C) Volume rendering of the same cell to visualize membrane 3D organization. The area of high membrane dynamics is illustrated by an arrow (red for retraction and blue for protrusion). (D) Close-up and tilted visualization of the rendered cell volume. Please click here to view a larger version of this figure.
Figure 3: Analysis pipeline for quantification of membrane dynamics collected using ISMic. (A) Cell contour determination and boundary points repartition (100 boundary points) between two consecutive frames for mGFP neutrophil described in Figure 2B. (B) Colored boundaries represent local membrane curvature determined for every boundary point. (C) Local area changes between two consecutive frames (current: blue, and next: red). The green areas represent the tracking results of the local membrane motion. Yellow arrows indicate the general direction of the membrane displacement. (D) Boundary curvature kymograph of the cell over time. The vertical axis represents the boundary points indices where 1 and 100 represent the rear of the cell according to its migration direction. (E) Local area change kymograph reflecting the membrane protrusion (red) and the membrane retraction (blue) of the cell over time. Please click here to view a larger version of this figure.
Movie 1: Green labeled neutrophils visualized in a mTomato mouse ear using IVM. Red: mTomato host mouse tissue. Cyan: Collagen-I SHG; Green: neutrophil. Please click here to download this Movie.
Movie 2: IVM of neutrophils migrating in a mTomato mouse ear skin. Videos are maximum-intensity projections of an image stack acquired for ῀27 min with a frame rate of 5 frames/s. Red: mTomato host mouse tissue. Cyan: Collagen-I SHG; Green: neutrophil. Please click here to download this Movie.
Movie 3: ISMic of membrane remodeling during neutrophil migration in a WT mouse ear. Videos are maximum-intensity projections of an image stack acquired for ῀8 min with a frame rate of 10 frames/s. Red: mTomato host mouse tissue. Cyan: Collagen-I SHG; Green: mGFP neutrophil; Light green: rendered neutrophil. Please click here to download this Movie.
Despite decades of advances in the field of cell migration and membrane remodeling, very few studies have employed IVM to visualize sub-cellular features in live animals. The procedures described in this protocol provide a powerful tool to gain novel insights into neutrophil migration in live animals and more specifically on plasma membrane remodeling during this process. This approach makes it possible to investigate neutrophil migration under physiological conditions considering the inherent complexity of the tissue microenvironment. Indeed, the use of MPM enables the visualization of multiple features within the host tissue by using a combination of mouse strains expressing selected fluorescent markers, exogenous tissue labeling, excitation of endogenous fluorescence, and signals generated through SHG or THG12,13.
One potential issue to consider in this procedure is photodamage. Even though MPM is generally safer than confocal microscopy with respect to photobleaching and phototoxicity, care must be taken when imaging live tissues34. Phototoxicity can drastically hamper the results by either creating imaging artifacts ranging from small bright speckles to larger areas of damaged tissue or inhibiting/stimulating a variety of intracellular pathways. To prevent phototoxicity, laser power should be kept at a minimum and the appropriate controls have to be designed to verify that physiological conditions are maintained (e.g., measuring blood flow, probes for oxidative stress)35,36. Moreover, in this specific procedure, which involves the skin, albino strains are highly recommended as the melanin present in dark-coated animals is more sensitive to phototoxicity36,37,38.
Among the main limitations of the procedure are the microscope system used and the nature of neutrophils. Although using MPM significantly increases the depth of tissue imaging when compared with other light microscopy techniques (e.g., confocal, spinning disk), the ability to visualize subcellular dynamics in the ear is restricted to the outmost layers of the tissue (80-100 µm). This is due to the intense light scattering produced by the thick ECM layer making it difficult to investigate migration in the deeper layers. Another limitation is the fact that neutrophils are very short-lived and cannot be kept in culture long enough to perform gene-editing techniques. This can be overcome by engineering mice to produce neutrophils lacking specific genes or harboring selected mutations, which of course increases the research costs and duration.
The procedures described here, although designed to investigate membrane dynamics, can be tailored to address any cell biological question not only in neutrophils but also in other immune cell types and migratory cells. The information gathered through low magnification IVM on cellular behaviors (e.g., migratory phenotype, cell speed, and directionality22) can be complemented and correlated with mechanistic information acquired through ISMic on organelle repositioning, protein secretion, endocytosis, nuclear dynamics, calcium dynamics, cytoskeleton organization, and netosis. The use of pharmacological and/or genetic manipulations can highlight the role of specific molecular pathways in the process of interest making this a unique and very powerful approach.
The authors have nothing to disclose.
This research was supported by the intramural research program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
1.5 mL tubes | USA Scientific | 4036-3204 | |
15 mL tubes | Corning | 430766 | |
1 mL syringe | Covidien | 8881501400 | |
27 G needle | Kendall | 827112 | |
27 G winged infusion set | Terumo | SV*27EL | |
30x objective | Olympus | UPLSAPO30XS | 1.05 NA, silicon oil immersion |
40x objective | Olympus | UPLSAPO40XS | 1.23 NA, silicon oil immersion |
60 mm dishes | Falcon | 353002 | |
6 mL syringe | Kendall | 8881516937 | |
Acepromazine (10 mg/mL) | Vet one | 13985-587-50 | |
ACK lysis buffer | Quality Biological | 118-156-101 | |
Balance | AND | EK-1200A | |
BSA | Sigma Aldrich | A9647 | |
Cell strainer 40 µm | Sigma Aldrich | CLS431750 | |
Fiji | ImageJ | N/A | Image visualization/analysis software |
Fluoview Software | Olympus | N/A | Acquisition software |
FVB mouse strain | Jackson | N/A | FVB background |
Gas Anesthesia system | Patterson veterinary | 07-8915712 | Link 7 model |
Green Cell tracker | Thermo | C2925 | Solubilized in cell culture grade DMSO to reach 1 mM concentration (1000x) |
Hair removal cream | Nair | N/A | |
HBSS (w/o Ca2+, Mg2+) | Gibco | 14175-095 | |
HEPES 1 M pH 7.3 | Quality Biological | 118-089-721 | |
Histopaque 1077 | Sigma Aldrich | 10771-100ML | |
Histopaque 1991 | Sigma Aldrich | 11191-100ML | |
Imaris | Bitplane | N/A | Image visualization/analysis software |
Isoflurane | Vet one | 13985-528-40 | |
Ketamine (100 mg/mL) | Vet one | 13985-584-10 | |
LyzM-cre x mT/mG | generated in the lab | N/A | C57BL/6J background |
Manual micromanipulator | WPI | M3301R | |
MATLAB | MatWorks | N/A | Analysis software |
mtomato mouse strain | generated in the lab | N/A | mT/mG, FVB background |
Multiphoton laser | Spectra Physics | Insight DS+ | |
Multiphoton Microscope | Olympus | MPE-RS | |
Nanofil 10 µL syringe | WPI | NANOFIL | |
Nanofil 33 G needle | WPI | NF33BV-2 | |
Objective heater | Bioptechs | N/A | |
Objective heater controller | Bioptechs | 150803 | |
Ophtalmic ointment | Major | NDC 0904-6488-38 | |
Oxygen concentrator | Caire | VisionAire 5 | |
PBS (w/o Ca2+, Mg2+) | Quality Biological | 114-058-131 | |
Saline | Quality Biological | 114-055-101 | |
Stage heater | Okolab | N/A | |
Stage heater controller | Okolab | H401-T | |
Surgical tape | 3M | 1538-1 | Hypoallergenic |
Syringe driver | Harvard Apparatus | PHD Ultra | |
Warming Pads | Parkland Scientific | A2789B | |
Warming Pump | Parkland Scientific | TP-700 | |
Xylazine (100 mg/mL) | Vet one | 13985-704-10 |