This work was supported by the Research Foundation Flanders (PhD grant fundamental research 11L7222N to M.C.), the Boehringer Ingelheim Foundation (PhD Fellowship to C.L.G.J.S), an EMBO postdoctoral fellowship (grant ALTF 1035-2020 to C.L.G.J.S.), and the Doctor Josef Steiner Award (to J.v.R).

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The authors have nothing to disclose

The R.MIW allows longitudinal imaging of the healthy and diseased mammary gland in its native&#160;and minimally disrupted environment and permits repeated visualization of the mammary gland at diverse developmental stages. The R.MIW design allows the window to be opened at any point during the experiment. Long-term visual access to the tissue of interest may be hindered, for example, by the accumulation of cell debris on the coverslip. In such cases, the R.MIW can be opened before or immediately after an imaging session to allow cleaning of the entire visible tissue area and lid. The removable lid also allows manipulations of the tissue as well as local application of substances, such as specific inhibitors and therapies, labeling dyes, or specific cell types of interest.
This method overcomes the limitation of the previously described mammary skinflap procedures4,7,8,13, which are limited to one imaging session, and can visualize processes such as branching morphogenesis (Figure 5), homeostatic tissue turnover (Figure 6), or tumor growth at a cellular level (Figure 3B). Yet, at the same time, the R.MIW permits local manipulation of the tissue in between imaging sessions, which comprises a great asset of the R.MIW compared to the previously published MIW3,18 and all other imaging windows20,22. When opening the R.MIW, it is important to maintain aseptic conditions at all times to prevent any source of infection. The ability to open the R.MIW in an aseptic environment allows optimizing the imaging conditions prior to every imaging session, which greatly improves long-term visual access to the tissue of interest. Specifically, in the mammary gland, which is embedded in an adipocyte-rich stroma, this is of great value. Moreover, the replaceable lid could uniquely enable the local administration of therapeutics, different cell types, labeling dyes, image-guided microdissection, or any other local manipulation of the tissue without the need to terminate the IVM experiment. For instance, to study tumor initiation, specific cancer cell populations could be injected directly in the ductal tree or stroma at a determined IVM time-point and precise mammary gland location, as long as this ROI is accessible through the R.MIW ring. To study tumor progression, specific cancer cell populations (at a specific location, in a specific micro-environment, or with a specific behavior) could be photo-marked during the IVM session and subsequently microdissected using a fluorescent dissection microscope after the opening of the R.MIW. Isolated cells could be further processed for downstream analyses, such as (single-cell) mRNA sequencing. By using this approach, one could couple in vivo cell behavior to molecular expression profiles. The advantage of local drug administration enabled by the R.MIW allows the tissue to be imaged prior to and directly after treatment. The interval needed to remove the mouse from the imaging box and to subsequently perform local drug administration after opening the R.MIW lid can be performed in minutes, which allows the immediate phase capture of fast-acting drugs.
We show that IVM of the mammary gland through the R.MIW is compatible with many different fluorescent reporter mouse models. The adipose-rich environment is challenging to image, and therefore the use of bright fluorophores is recommended. However, as shown here, even less bright fluorophores such as mCFP can be visualized through the R.MIW in optimal imaging conditions. Inevitably, the fat pad will preclude the imaging of the deeper ductal structures, and limit the imaging to the more superficial ducts. A low-resolution overview image at the start of each IVM experiment will help to identify the ductal structures of interest that are sufficiently superficial for high-resolution imaging. Local tissue manipulation, removal of connective tissue, or repositioning of the adipose tissue after opening the R.MIW can optimize the IVM for specific ROIs that are overlaid by adipose tissue. This is an important advantage over all previous window designs, which do not allow to perform these manipulations and would require complete removal of the window itself. Specifically, for visualization of the inguinal lymph node, it is recommended to gently remove the overlying fatty tissue, which reduces light scattering and enables high-resolution imaging. When manipulating the tissue, always keep aseptic conditions and prevent bleeding or serious damage to the tissue, as they may affect the processes being studied during the IVM experiment.
The R.MIW is made of titanium, a material that is commonly used in clinical practice to replace hard tissues such as joints or bone plates. Titanium has several advantages over steel windows, including its lightweight and inert character21. Recently, several other materials were used to generate novel imaging window types, including the flexible silicon window20. In contrast to the R.MIW, the flexible window does not require any sutures for implantation and is suitable for nearly any anatomical position, specifically in the case of soft and fragile tissues. Silicon windows have a minimal impact on animal motility due to their lightweight and deformable nature and maybe more suited in experiments studying rapid tissue expansion and growth20. Another advantage over the titanium version is that silicon windows are compatible with other imaging modalities, including magnetic resonance imaging20,27. However, it will be important to keep in mind that objectives are optimized for 0.17 mm glass coverslips. Moreover, mammary tissue is susceptible to breathing movements, which are hard to restrict using the flexible window, especially when using an inverted microscope. Breathing artifacts are minimized by the R.MIW design and the fixation of the R.MIW in the inlay of the imaging box. As a result, images acquired using the proposed R.MIW setup are not distorted due to breathing artifacts. However, minor drifts in tissue localization can occur, which are usually gradual and can be corrected by using post-acquisition motion correction software28. With the increasing toolbox of IVM technologies2,20, the specific requirements for each experiment will eventually determine the best way of in vivo visualization of the tissue of interest. Different window designs have different advantages and disadvantages and depending on the research question, the available microscopy setup, the required spatial and temporal resolution, and the total time span of the studied process, the optimal approach needs to be determined.
In summary, the R.MIW facilitates high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease over multiple days to weeks.

The mammary epithelium is a unique secretory organ present in mammals, which produces and secretes milk to nourish offspring. Throughout life, the mammary gland undergoes multiple rounds of development and growth, which are accompanied by structural and functional changes of the tissue1. Depending on the developmental stage, the cell types contributing to tissue remodeling are different, as well as the location within the ductal tree.
Multiphoton intravital microscopy (IVM) allows the study of mammary cell dynamics in vivo in the native and minimally perturbed setting2,3,4. To obtain visual access to the mammary gland, several temporary ex vivo or skin flap imaging techniques have been published during different stages of mammary gland development, including puberty4,5,6,7, adulthood2,8, lactation9,10,11,12, and tumor dynamics13,14,15. Although these techniques result in high spatially and temporally resolved imaging of mammary cell dynamics, the time frame is limited to hours whereas most mammary gland remodeling processes take days to weeks. Therefore, methods that allow optical access to the tissue of interest for extended time frames are required. Over the years, several permanent imaging windows have been developed for mammary tumor imaging15,16,17,18, including a titanium mammary imaging window (MIW)2,3,19. Although very useful to study mammary tumor growth, visualization of the healthy mammary gland structure remained limited to a few days. Recently, a flexible silicon imaging window was developed, which allows visualization of the pubertal mammary gland over multiple weeks20. However, the mammary gland is embedded in an adipocyte-rich fat pad, which leads to extensive light-scattering and as a result limited visibility of the mammary ductal structures. Therefore, superior imaging conditions are required at all times to visualize tissue dynamics over prolonged periods of time in the mammary gland. Neither the classical MIW, nor the flexible silicon window&#160;allow for tissue manipulation or optimization of physical tissue location before imaging, as the window forms a closed system after surgery and implantation. As a result, optimal optical access to the underlying mammary tissue is likely to become precluded over longer time periods. In contrast, the skin flap technique does allow for optimization and repositioning of the tissue during the imaging session and a skin flap can be repeated multiple times2. However, repeated imaging sessions through a skin flap are only possible when sufficient time (at least 7 days) is allocated in between surgeries to allow recovery of the skin and is therefore mostly suited to study processes at longer time scales. Moreover, it is advisable to not perform this procedure many times because of its invasive nature and large risk of infections and scarring upon wound closure.
To overcome these limitations, i.e., to ensure optimal imaging conditions for a prolonged period of time at a high frequency&#160;and at the same time allow tissue manipulation, an improved titanium version of the MIW with a replaceable lid (R.MIW) was designed to visualize the healthy and diseased mammary gland over multiple days to weeks2 (Figure 1A,B). The R.MIW was custom designed to provide optimal tissue access, enabling direct tissue manipulation over the entire duration of the IVM experiment, and thereby permits visualization of the mammary gland at the right time and place over prolonged periods of time. When closed, the R.MIW forms an airtight system comparable to the classical MIW (Figure 1C). When opened under aseptic conditions, the R.MIW allows local tissue manipulation to improve optical access and also enables local administration of substances, such as pathway inhibitors or agonists, injection of different cell types of interest, such as cancer cell or immune cell populations, or addition of tissue labeling-dyes. The lid can be opened at any moment in between imaging sessions without causing&#160;damage to the underlying tissue.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63326/63326fig01.jpg" /> 
Figure 1: Design of the mammary imaging window with a replaceable lid. (A) Top view and side view of the replaceable lid of the mammary imaging window&#160;with a 10 mm coverglass glued to the ring. (B)&#160;Top view and side view of the mammary imaging window, which consists of an outer ring and inner ring&#160;with a groove in between to secure the window within the skin of the mouse using a purse-string suture. The outer ring has a small groove that fits the four projections (arms) of the lid. (C) Cartoon and pictures demonstrating the opening and closing mechanisms of the lid. The slanting projections make sure the lid is fixed inside the window frame. This figure has been modified from Messal et al. 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63326/63326fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol describes the design and implantation procedure of the R.MIW, as well as a longitudinal IVM strategy to revisit the same mammary ducts and their visualization at a cellular resolution. The R.MIW allows following cell divisions and morphological changes during different developmental phases of the mammary gland in diverse fluorescent reporter mouse models. Taken together, the R.MIW facilitates high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

<table><tbody><tr><td>0.9% NaCl</td><td>BD</td><td>306573</td><td>Other brands available</td></tr><tr><td>10 mm round coverglass</td><td>Fisher scientific</td><td>10696365</td><td>Other brands available</td></tr><tr><td>5-0 braided silk suturs</td><td>Ethicon</td><td>W580</td><td></td></tr><tr><td>80% Ethanol</td><td>homemade</td><td>NA</td><td></td></tr><tr><td>Anesthesia induction box</td><td>Kentscientific</td><td>VetFlo-MSEKIT</td><td></td></tr><tr><td>Buprenorphine (Vetergesic&amp;reg;)</td><td>Ecuphar</td><td>NA</td><td></td></tr><tr><td>Cotton tips (sterile)</td><td>Fisher scientific</td><td>13113743</td><td>Other brands available</td></tr><tr><td>Cyanoacrylate adhesive</td><td>Loctite</td><td>NA</td><td>Other brands available</td></tr><tr><td>Eye ointment</td><td>Duratears</td><td>NA</td><td></td></tr><tr><td>Flexible butterfly needle</td><td>Greiner Bio-One</td><td>450120</td><td>Other brands available</td></tr><tr><td>Graefe forceps (blunt)</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Heating pad</td><td>Kentscientific</td><td>RightTemp Jr.</td><td>Other brands available</td></tr><tr><td>Imaging box</td><td>Custom made</td><td>NA</td><td></td></tr><tr><td>Infuse nutrient mixture</td><td>Braun</td><td>Nutriflex special 70/240</td><td></td></tr><tr><td>Insulin syringes</td><td>BD</td><td>324911</td><td>Other brands available</td></tr><tr><td>Inverted multi-photon microscope with automated stage</td><td>Leica Microsystems</td><td>NA</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>Kentscientific</td><td>VetFlo-1231K</td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12510-14</td><td></td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P7793</td><td>semi-transparent tape</td></tr><tr><td>Paper tape tesa</td><td>Tesa</td><td>NA</td><td></td></tr><tr><td>Petroleum Jelly</td><td>Vaseline</td><td>NA</td><td></td></tr><tr><td>Razor blades</td><td>Fisher scientific</td><td>11904325</td><td>Other brands available</td></tr><tr><td>Silicon tubing</td><td>Solutions Elastomeres</td><td>NA</td><td>Any houseware</td></tr><tr><td>Spring scissors (small)</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>Sterile PBS</td><td>ThermoFisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Syringe (10 ml)</td><td>BD</td><td>305482</td><td>Other brands available</td></tr><tr><td>Thin forceps</td><td>Fine Science Tools</td><td>11413-11</td><td></td></tr><tr><td>Titanium lid for mammary imaging window</td><td>Custom made</td><td>NA</td><td></td></tr><tr><td>Titanium mammary imaging window</td><td>Custom made</td><td>NA</td><td></td></tr><tr><td>Wooden sticks toothpicks</td><td>Fisher scientific</td><td>NC1678836</td><td>Other brands available</td></tr></tbody></table>

longitudinal intravital microscopy using a mammary imaging window with replaceable lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

To study the proliferative dynamics of mammary epithelial cells during the different developmental stages of the mammary gland the described protocol was performed. The design of the R.MIW is depicted in Figure 1, and the procedure to implant an R.MIW is summarized in Figure 2. The R.MIW is surgically implanted between the mammary gland and the skin. A suture is used to hold within the window ring and prevent the mice from pulling on the sutures (Figure 2). The sutures holding the R.MIW are made of non-absorbable silk, which has hypoallergenic properties, a soft texture, and is easy to handle. The R.MIW ring is made of titanium, one of the most biocompatible materials that do not promote inflammation or necrosis after implantation21. If the aseptic conditions are followed and the sutures are well executed, the R.MIW mammary gland implantation poses a low risk of postoperative complications. Moreover, unlike abdominal imaging window implantation22, R.MIW implantation is not a limiting factor for mouse survival because the mammary gland is not a vital organ and allows daily imaging up to the limit specified in the ethical protocol. The only factor that limits the duration of window implantation is the homeostatic turnover of the skin, which will eventually lead to the sutures falling out after 4 - 6 weeks. Therefore, it is important to regularly inspect the stability of the sutures. If desired, the sutures can be removed and replaced by a new purse-string suture under aseptic conditions (as described in steps 3.5 - 3.8) to reassure window stability.
A major challenge when using the multiday imaging approach is retracing an ROI on consecutive days. To this end, a quick overview scan can be included before selecting the ROI(s) (Figure 3B). Multiple tissue landmarks can be used to retrace the same region of the tissue, including the collagen network signal (visualized by second harmonic&#160;generation), tissue structure, as well as local patterns of cells differentially or stochastically labeled with dyes or fluorescent proteins (Figure 3B). In this representative example, an R.MIW was implanted onto the 4th&#160;mammary gland of&#160;an MMTV-PyMT;R26-CreERT2het;R26-Confettihet female mouse at the onset of palpable tumor formation.&#160;Subsequently, stochastic recombination was induced by injection of 1.5 mg tamoxifen, resulting in recombination of the Confetti construct in some cells (Figure 3C). The recombined tumor cells were followed for a period of 20 days (Figure 3B,C). If imaging of the same region of the mammary gland over consecutive days is precluded, the window can be opened in an aseptic environment (as described in step 5.1) to further clear up, and reposition the tissue to improve visibility and image acquisition (Figure 3A).
Using this method, the developing mammary gland during puberty was followed at the single-cell level. Repeated IVM through an R.MIW was performed using R26-CreERT2het;R26-mTmGhet&#160;female mice between 4-6 weeks of age, in which all cells are labeled with membrane tdTomato (Figure 4A)23. Mice were injected with a low dose of tamoxifen (0.2 mg/25 g body weight), resulting in sporadic recombination of the mTmG construct, causing a change from red to green in some cells in all tissues, including the mammary gland (Figure 4B). The same ROIs were revisited over multiple days to visualize morphological changes within the mammary gland, including ductal elongation and ductal branching (Figure 4C) at a cellular resolution. Importantly, this combination of stochastic cell labeling and multi-day IVM also allows visualizing the dynamic changes of the ductal environment, such as the dynamics of single cells in the stroma surrounding the elongating and branching ducts (Figure 4D) as well as the cellular dynamics within the inguinal lymph node (Figure 4E).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63326/63326fig04.jpg" /> 
Figure 4: Multi-day IVM of pubertal branching morphogenesis. (A) Pubertal R26-CreERT2; R26-mTmG mice in age between 4-6 weeks were injected with a low dose of tamoxifen leading to Cre-mediated recombination of the R26-mTmG allele and were imaged on days 1, 3, and 5 to follow the dynamic changes in developing mammary gland. (B) Schematic representation of the R26-mTmG mouse&#160;construct, which in non-recombined conditions results in ubiquitous expression of membrane tdTomato (mT). Upon Cre-mediated recombination, mT is switched for membrane eGFP (mG) expression. (C)&#160;3D rendering of an elongating and branching mammary duct over multiple days imaged through an R.MIW in an R26-CreERT2; R26-mTmG female mouse at 6 weeks of age. (D)&#160;Single Z-plane images of the branching tip (top panels) and the elongating branch (bottom panels). mT is depicted in red, and mG in cyan, scale bars represent 100 &#181;m (A and B). Single cells in the stroma are highlighted by white asterisks. Note that signal intensity was manually increased to highlight the single cells in the stroma, leading to a slightly overexposed appearance of the mammary epithelial cells. (E)&#160;Representative images of the inguinal lymph node of an R26-CreERT2; R26-mTmG female mouse imaged through an R.MIW, showing an overview tile scan (left panel), zoom images (right panels) of a single Z-plane (top), and a 3D rendering (bottom). Second harmonic generation (collagen I) is shown in green, mT in red, and mG in cyan. Scale bars represent 500 &#181;m (overview tilescan) and 100 &#181;m (zoom images). Figure panels C and D are modified from Messal et al. 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63326/63326fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similarly, in the adult mammary gland, the proposed R.MIW approach allows visualization of the same ductal structures over multiple days with uncompromised visibility. Even the use of less bright fluorescent reporter models24, such as the Cdh1-mCFP (Ecadherin-mCFP) mouse model, permits visualization of ductal stability and subtle morphological changes at a cellular resolution (Figure 5). Note that visibility between day 3 and day 5 improved significantly after tissue repositioning (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63326/63326fig05.jpg" /> 
Figure 5: Multi-day imaging of the adult mammary gland. 3D rendered images of a mammary ductal structure of an adult female Cdh1-mCFP (cyan, marking the Ecadherin-positive luminal cells expression) mouse over multiple consecutive days. The collagen I pattern (second harmonic generation, magenta) was used to retrace the same ROI, and the Cdh1-mCFP signal was used to mark the ductal (luminal) cells. Note that visibility after day 3 was improved by the opening of the R.MIW lid and repositioning of the tissue. Scale bars represent 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63326/63326fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess proliferative heterogeneity of the mammary epithelial cells during the hormonal cycle at the single-cell level, the photo-convertible Kikume Green-Red (KikGR) reporter mouse model25,26 was used, which has ubiquitous expression of the KikGR protein. KikGR is a bright fluorophore that undergoes green-to-red conversion upon exposure to violet light and the red/green ratio can be used as a proxy for the proliferative activity of a cell2 (Figure 6A,B). Using the R.MIW approach, we followed the same cells within the ductal tree of the adult mammary gland over several days and found previously unanticipated proliferative heterogeneity throughout the ductal tree (Figure 6C). High and low proliferative cells were equally distributed over the different converted areas (Figure 6C,D). Strikingly, at the local level, neighboring cells showed large differences in their red/green ratio (Figure 6C,D). Quantification of the red/green ratio of different cells (as a proxy for their proliferative activity) 10 days after photo-conversion, revealed highly variable dilution rates of some cells within the same ductal micro-environment (Figure 6E). Together, these data reveal a remarkable local proliferative heterogeneity within the adult mammary gland, and at the same time, a global uniform turnover rate.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63326/63326fig06.jpg" /> 
Figure 6: Longitudinal IVM of proliferative heterogeneity in the adult mammary gland using the KikGR mouse model. (A)&#160;KikGR mice were imaged at day 0 before and after exposure to violet light. The imaging sessions were repeated on days 2, 6, and 10 after conversion. (B) Schematic depiction of the hypothetical outcomes upon photo-conversion of KikGR mammary epithelial cells following proliferation (left panel) and no proliferation (right panel) as a function of time. (C)&#160;The same area of the mammary gland was imaged through an R.MIW over a period of 10 days. Smaller regions were photo-converted on day 0 and show a similar dilution rate of the Kikume red signal over time, indicating an equal turnover rate throughout the epithelium. (D)&#160;Zoom images (single Z-planes) of the indicated regions in panel C show proliferative heterogeneity at the cellular level 6 and 10 days after photo-conversion. Scale bars represent 100 &#181;m (panel C) and 10 &#181;m (panel D). (E)&#160;Quantification of the red/green ratio of randomly selected cells (n = 48 cells) in three photo-converted areas. A high red/green ratio is indicative of a low dilution rate and low-proliferative activity, whereas a low red/green ratio is indicative of a high dilution rate and proliferation. Figure panels C- E have been modified from Messal et al. 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63326/63326fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

longitudinal-intravital-microscopy-using-mammary-imaging-window-with

Research

JoVE Journal

Biology

3.2K Views.  VIB (BE). De borstklier is zeer dynamisch en ondergaat meerdere rondes van remodellering gedurende het hele leven. Ons protocol maakt het mogelijk om de cellulaire dynamiek vast te leggen die weefselherschikkingen in de gezonde en zieke borstklier aandrijft. Ons borstbeeldvormingsvenster met een vervangbaar deksel maakt het mogelijk om longitudinale intravitale beeldvorming van de borstklier uit te voeren gedurende lange perioden, tot enkele weken.Het vervangbare deksel van het borstbeeldvorming...

Video: Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Medicine

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body&#39;s outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

Author Spotlight: Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging 

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...

Pancreatic Intravital Imaging Window Installation in Mouse Model: An Integrated Pancreatic Imaging Window for Time-lapse In Vivo Imaging of Murine Pancreas Microstructure

Source: Park, I. et. al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62538">Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to implant a pancreatic imaging window to separate the pancreas from the intestines for longitudinal time-lapse high-resolution imaging of the pancreas microstructure.

Source: Park, I. et. al. Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

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