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).

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#174;)</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>

Watch this Scientific Journal Video about Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Biology

3.1K Views.  VIB (BE). 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. 

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

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 ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...