This study was supported by the Netherlands Organization of Scientific Research NWO (Vici grant 09150182110004 to J.v.R. and Veni grant 09150161910151 to H.A.M), the OCENW.GROOT.2019.085 (to J.v.R), and an EMBO postdoctoral fellowship (grant ALTF 452-2019 to H.A.M).

All animal experiments described in this study were carried out in accordance with the guidelines and approval of the Animal Welfare Body (AWB) of the Netherlands Cancer Institute (NKI).
NOTE: Consult the animal ethics committee of the institute before performing any animal experiments.
1. Preparation for surgery and intravital imaging
<ol>
	<li>Pre-surgical treatments
	<ol>
		<li>Induce 8-12-week-old K19-CreERT237: Rosa26-mTmG38 and Lgr5eGFP-IRES-CreERT218: Rosa26-Confetti39 mice by injecting tamoxifen dissolved in sunflower oil intraperitoneally 4-6 weeks prior to surgery (Figure 1).</li>
		<li>Apply analgesia. Inject 200 &#181;L of buprenorphine (0.1 mg/kg body weight) diluted in NaCl 0.9% per 30 g mouse subcutaneously 30 min before surgery. Administer carprofen (0.067 mg/mL) in a bottle containing 125 mL of non-acidified autoclaved drinking water 24 h before surgery. Alternatively, administer a carprofen injection subcutaneously (5 mg/kg) 30 min before the start of surgery.</li>
	</ol>
	</li>
	<li>Preparation for surgery
	<ol>
		<li>Introduce autoclaved sterile surgical tools in the biohazard cabinet.</li>
		<li>Turn on the heating pad and set the temperature to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation for intravital imaging
	<ol>
		<li>Turn on the temperature-controlled chamber of the microscope at least 4 h before imaging to allow sufficient time for temperature stabilization. 
		NOTE: The time needed can vary depending on the machine used.</li>
		<li>Keep the temperature inside the climate chamber stable at 37 &#176;C.</li>
		<li>Turn on the two-photon microscope, scanner, and laser.</li>
		<li>Launch the imaging software.</li>
		<li>Tune the wavelength of the laser to 960 nm and open the shutter.</li>
	</ol>
	</li>
</ol>
2. Surgery and intravital imaging
<ol>
	<li>Surgical exposure of the intestine
	<ol>
		<li>Anesthetize the mouse using 2%-3% isoflurane and place it on a heating pad, covered with a sterile cloth. Check the depth of anesthesia by assessing the frequency and quality of breathing (one breath/second) and by checking the reflex of the mouse.</li>
		<li>Cover the eyes of the mouse with eye ointment.</li>
		<li>Inject 200 &#181;L of pre-warmed sterile saline subcutaneously.</li>
		<li>Shave the abdomen and remove the hair. Change the sterile cloth in the surgical area.</li>
		<li>Insert a rectal probe to monitor the temperature of the mouse. 
		NOTE: The temperature should be approximately 37 &#176;C.</li>
		<li>Put on a new pair of sterile gloves.</li>
		<li>Clean the surgical area in a circular way with alternating scrubs of antiseptic solution followed by 80% ethanol three times. Remove excess ethanol with a sterile cotton swab. 
		NOTE: It is critical to monitor the mouse for hypothermia at this point.</li>
		<li>Cover the mouse with a sterile surgical drape.</li>
		<li>Check the reflex of the mouse.</li>
		<li>Make a ~10 mm vertical midline incision through the skin using a sterile scalpel.</li>
		<li>Incise the linea alba using scissors to separate the rectus abdominis muscles and open the abdomen.</li>
		<li>Find the cecum of the mouse using sterile cotton swabs drenched in sterile pre-warmed saline to use it as a reference point.</li>
		<li>Make a small cut in a piece of sterile gauze, wet it in preheated sterile saline, and place it above the incision.</li>
		<li>Take out the intestine with sterile cotton swabs drenched in pre-warmed sterile saline. Keep the intestine hydrated by adding sterile pre-warmed saline.</li>
		<li>Place a sterile custom-made imaging chamber next to the mouse.</li>
		<li>Transfer the mouse to the preheated sterile imaging box.</li>
		<li>Place the intestine on the sterile glass of the imaging box. Place the head of the mouse inside the inhalation tube of the imaging box, as shown in Figure 1.</li>
		<li>If necessary, secure the mouse with sterile flexible film and tape.</li>
		<li>Place the imaging box containing the mouse in the microscope chamber.</li>
	</ol>
	</li>
	<li>Intravital imaging
	<ol>
		<li>Monitor the mouse during imaging by checking the frequency and depth of breathing and temperature via a rectal probe every 15 min. The percentage of isoflurane should be kept between 1%-2%. Adjust if needed.</li>
		<li>Find a region in the intestine using the eyepiece of the microscope.</li>
		<li>Obtain a wide-field view of the region of interest using the microscope&#39;s internal camera, as seen in Figure 2A.</li>
		<li>Image the region of interest using the 960 nm laser of the multiphoton microscope. Adjust the laser power and wavelength according to the fluorophores used in the experiment.</li>
		<li>Acquire a 10-20 step z-stack of 3 &#181;m of the region of interest.</li>
	</ol>
	</li>
	<li>Laser ablation
	<ol>
		<li>Pick a single position or multiple positions from the tile imaged previously.</li>
		<li>Use the bleach point calibration function in the imaging software at a zoom of 32 and 124 x 124 pixel resolution with a scan speed 400 Hz, using the bidirectional scanning property for 3-10 s, depending on the size of damage aimed for. The initiation of damage in the crypt area can be recognized by an increase in autofluorescence in both the green and red channels.</li>
		<li>Repeat the previous two steps for multiple regions in the same mouse.</li>
		<li>After ablation, acquire z-stacks of the damaged regions to confirm the location and extent of the damage.</li>
	</ol>
	</li>
	<li>Closure of the surgical site
	<ol>
		<li>Place the mouse, while still under anesthesia, in a sterile suturing area.</li>
		<li>Insert the exposed intestine back into the abdomen using sterile cotton swabs drenched in preheated sterile saline.</li>
		<li>Suture the linea alba by performing a simple continuous suture using an absorbable 5-0 suture. Close the extremities of the suture with surgical knots.</li>
		<li>Repeat the same step with the skin layer.</li>
		<li>Switch off the isoflurane station and clean and sterilize the imaging box and inlay.</li>
		<li>Clean the surgery tools with the surgical instrument cleaner, enzymatic cleaner, and surgical instrument lubricant. When dried, sterilize the surgery tools together with the surgery box in the autoclave.</li>
	</ol>
	</li>
	<li>Post-surgery care
	<ol>
		<li>Let the mouse recover from surgery while the cage is placed on the heating pad for ~1 h. 
		NOTE: Place the mouse with other cage mates if possible. Solitary housing is not necessary for recovery.</li>
		<li>Check the temperature of the mouse every 15 min during recovery.</li>
		<li>Inject 200 &#181;L&#160;of buprenorphine (0.1 mg/kg body weight) diluted in NaCl 0.9% per 30 g mouse subcutaneously 6-12 h post-surgery.</li>
		<li>Administer carprofen (0.067 mg/mL) in a bottle containing 125 mL of non-acidified autoclaved drinking water for 72 h post-surgery.</li>
		<li>Weigh the mouse and monitor the welfare every day for 1 week post-surgery.</li>
	</ol>
	</li>
</ol>
3. Repetitive surgery and intravital imaging
<ol>
	<li>Surgery
	<ol>
		<li>On the second time point (at least 1 week after the first imaging session), repeat steps 1.1.2, 1.2, 1.3, and 2.1.</li>
	</ol>
	</li>
	<li>Intravital imaging
	<ol>
		<li>Repeat steps 2.2.1-2.2.3.</li>
		<li>Use the pattern of blood vessels to find the same regions of interest imaged on the first time point.</li>
		<li>Repeat steps 2.2.4 and 2.2.5.</li>
	</ol>
	</li>
	<li>Closure of the surgical site
	<ol>
		<li>Repeat step 2.4.1.</li>
		<li>If the mouse is not meant to be imaged on another time point, sacrifice the mouse by performing cervical dislocation under terminal anesthesia. Otherwise, continue with the next step. 
		NOTE: According to the guidelines of EU Directive 2010/63/EU, appendix IV, cervical dislocation is an acceptable method.</li>
		<li>Repeat steps 2.4.2-2.4.6.</li>
	</ol>
	</li>
	<li>Post-surgery care
	<ol>
		<li>Repeat steps 2.5.1-2.5.5.</li>
	</ol>
	</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intravital+microscopy+toolbox+to+study+mammary+gland+dynamics+from+cellular+level+to+organ+scale.">An intravital microscopy toolbox to study mammary gland dynamics from cellular level to organ scale.</a> Journal of Mammary Gland Biology and Neoplasia. 26 (1), 9-27 (2021).</li></ol>

The authors have nothing to disclose.

This protocol combines microscopic laser ablation and longitudinal intravital microscopy to follow intestinal regeneration from early damage response to long-term tissue remodeling. The technique has been established in strict adherence to ethical considerations to induce and image microscopic laser ablation, and when followed precisely will maintain animal wellbeing. During surgery, it is important to make sure that the integrity of the intestine is well preserved. This can be achieved by gently handling the tissue with sterile wet cotton swabs, which prevents bleeding or drying out of the tissue. The extent of laser-induced microscopic damage should also be carefully assessed by imaging the different intestinal layers of the area after laser ablation. If the researcher wishes to adapt the frequency of the experimental steps described in this protocol, the animal ethics committee of the institute should be consulted before the experiment to establish the consequence on the welfare.
Repetitive intravital microscopy allows to monitor tissue recovery in the same mouse over time. Repeated surgical exposure of the intestine readily grants optical access to the entire intestinal tract. Tissue inherent features, such as the vasculature, serve as landmarks to identify the same intestinal regions in each imaging session. Thus, the same tissue region can be imaged over several weeks, which enables one to quantify long-term tissue regeneration in the same intestinal area in the same mouse. The spatiotemporal control offered by the combined surgery and imaging method brings the advantage that the same organ can be imaged under both homeostatic and regenerating conditions in the same mouse, which stands in contrast to previous whole-organ damage models where controls and regenerating samples originated from different mice11,12,18,19,20. Hence, our experimental setting minimizes the required number of animals needed for the experiment and reduces intra-animal variation.
Protocol troubleshooting should start with a review of the mouse and intestine handling technique, and a control of microscopy equipment and settings. There are many critical steps in this protocol that require extra attention. First, in order to ensure animal wellbeing and continuous high data quality, all work needs to be carried out in a clean sterile environment using aseptic technique, and mouse temperature should be maintained during surgery and each imaging session. Keeping the tissue hydrated with sterile, pre-warmed saline during imaging is essential and prevents tissue fibrosis.
To ensure that the experiment is carried out in a reproducible manner, it is important to align the lasers before use for optimal acquisition and to measure the power output of the multiphoton laser at the beginning of each session. Parameters such as the type of the objective, magnification, dwelling time, laser power, and wavelength have influences on the extent of microscopic laser ablation and should be considered. In this study, both laser ablation and imaging are conducted with a laser power of 1.2 W (out of the lens) at a 960 nm wavelength through a Fluotar VISIR 25x/0.95 WATER objective. Changing the wavelength or optical scanning properties affects the extent of microscopic damage. A lower wavelength (such as 840 nm) translates into higher energy photons and often in a higher output of the laser, and may enhance microscopic damage. A higher zoom results in more energy per region, and therefore less time to ablate crypts, and vice versa. The pixel dwelling time can also be increased or decreased to change the speed of ablation and the extent of damage. When the imaged tissue is not stable (e.g., because of peristaltic movements), ablation needs to be performed rapidly. For this purpose, the speed of ablation should be optimized by, for example, increasing the zoom and/or laser output.
Finding the same intestinal region over multiple imaging sessions is another critical step that needs to be executed properly to ensure the success of the experiment. To do so, the intestine needs to be positioned in the exact same manner at all time points. We recommend to always use the cecum as a reference point to find the same regions in the small and large intestine. Gently stretching the tissue of interest with cotton swabs ensures that the region of interest is in range of the objective working distance and maximizes the number of regions that can be traced back. In addition, we recommend to always ablate and image multiple microscopic positions in each mouse to account for regions that may not be localized back in a later imaging session. If the regions cannot be found, even though the positioning of the intestine is correct, it can help to reposition the mouse and change the orientation of the exposed area. Tracking crypts over time can be cumbersome for experiments where larger intestinal fields of several adjacent crypts are ablated. Such damaging insults can evoke tissue remodeling beyond the epithelial monolayer, which can culminate in modification of the tissue landmarks used for tracking of the region over time. Choosing landmarks at a sufficient distance to the damaged site and capturing larger fields of view that surpass the damaged area by several hundred micrometers increases the chance for successful long-term experiments. In addition to incorrect positioning of the intestine on the microscope stage, peristaltic movements of the gastrointestinal tract may interfere with imaging. This problem may be ameliorated in two ways. If the frequency of movements is not too high, the process may be repeated in the same region with an increased exposure time.&#160;Alternatively, higher amounts of anesthesia can be used to decrease peristalsis. We recommend limiting higher doses of isoflurane to short adjustments. Altogether, the imaging sessions should be kept as short as possible, optimally below 3 h, to ensure fast recovery.
The combined laser ablation and longitudinal intravital microscopy approach has several advantages when compared with other damage models. Previous (chemical) damage models lacked the ability to locally confine the damaging insult6,11,12,19,20. Laser ablation overcomes this shortcoming by restricting the damage to a defined region of interest. This enables researchers to control the location of the injury, as well as the damage extent. Damage severity can be modulated to ablate crypts or entire microscopic intestinal fields to inform about regenerative responses at the crypt scale. In addition to spatial control, laser ablation also allows to precisely time the onset of damage, thereby surpassing the precision of previous drug, chemical, and infection models9,10,11,12,19,20. Our protocol expands on previous studies that used laser-induced thermal ablation as a method to induce localized damage in the intestine21,23. Previous laser-induced damage models imaged local areas in the small intestine21 or the luminal surface of the distal colon23. The combined surgery and laser ablation approach makes it possible to visualize the intestinal epithelium (crypts in particular) at a high resolution, and to perform laser ablation and follow-up imaging of tissue recovery in any position of the small intestine, cecum, and proximal colon. It captures the recovery of the same intestinal regions over time, allowing to visualize different layers of the intestine (mucosa, submucosa, muscularis, and serosa) as per the experimental setup. Our technique is mainly tailored for long-term repeated imaging for a period of multiple weeks/months. To study the short-term recovery dynamics of crypts (e.g., for several consecutive days following damage), the laser ablation approach described here can be combined with intravital imaging windows27,28,40.
This protocol can be utilized for a multitude of research applications from diverse scientific areas that span regeneration, immunology, and cancer research. Longitudinal imaging of intestinal regeneration sheds light on the cellular dynamics that preserve epithelial integrity and barrier function, enable host defense against pathogens in the intestinal lumen, and that underlie the clearance and spread of oncogenic mutations. Each scientific question will cast unique demands on the extent of laser-induced damage and the duration of imaging. Fluorescent reporter mice and injected dyes can drastically expand and refine the data that can be acquired by allowing the visualization of any cell and structure of interest. For example, an Lgr5-CreERt2:Rosa26-Confetti mouse can be used to visualize stem cell progenies, whilst the Rosa26-mTmG reporter informs on tissue architecture. Together, these recent technological advances make intravital intestinal experimentation a lucrative tool to advance our understanding of intestinal biology and disease.

The epithelial lining of the intestine is constantly challenged by gastric acids, toxins, and microbiota that can cause disruption of the epithelial barrier. Intestinal structure and tissue organization are specialized to constantly self-renew and repair damages. The single-layered epithelium of the small intestine is organized into crypt-villus units1. In homeostasis, self-renewing Lgr5+ intestinal stem cells that reside at the base of the crypt give rise to differentiated progeny. The differentiated daughter cells travel to the tip of the villus axis in a conveyer belt fashion, where they are shed so that the intestinal lining is replenished in 3-5 days2,3.In the long term, not all Lgr5+ cells contribute equally to tissue renewal, as this also depends on the ability of cells to move against the conveyer belt toward the base of the crypt (i.e., retrograde movement)4,5. Indeed, upon ablation of Lgr5+ cells by, for example, radiation, progenitor cells outside of the base of the crypt move into the base to dedifferentiate and replenish the stem cell pool6,7,8.
Acute inflammation can cause the loss of Lgr5+ stem cells9,10. In addition to stem cell loss, many external factors can cause acute damage to the epithelium at the crypt scale. Radiation, chemical treatments, and antibiotics have been shown to injure intestinal crypts and villi11. Larger fields of crypts and villi can be affected by bacterial, viral, and parasitic infections12. The intestine possesses a remarkable ability to recover from internal and external damage at the crypt scale by crypt fission (a division of one crypt into two)13. Upon wounding, crypts in the area adjacent to the damage undergo fission to replenish the crypt numbers. This phenomenon also occurs, although to a lesser extent, during homeostasis14,15. To counterbalance a potential increase in crypt numbers during homeostasis, crypts can also fuse (merging two crypts into one)16,17. Whether crypt fusion also plays a role in reestablishing crypt numbers after wounding is unknown. Moreover, the dynamics and regulatory factors of this process remain to be elucidated.
Injury models are indispensable to study&#160;tissue regeneration in vivo. Various injury models have been utilized to study intestinal tissue regeneration. Previous experimental strategies employed high-dose radiation to deplete stem cell pools18 or treatment with dextran sulfate sodium (DSS) to induce chronic and acute colitis and crypt loss in mice19,20. Single-cell ablation by genetic or optic means has been used to refine tissue damage and considered an attractive tool to disentangle the role of stem and progenitor cells21,22 and study vascular regeneration23. Additionally, a biopsy-injury system has been developed to induce damage in larger fields of several crypts and villi24. Importantly, the response to the damaging insult may vary along the proximal-distal axis of the intestine, as reported for radiation, which caused more damage in the small intestine than in the colon25. This highlights the need for targeted methods that control both the extent of the damaging insult and its localization in the intestinal tract.
Damage extent and recovery have conventionally been assessed by static means, which provide limited information about the dynamics of tissue recovery. Intravital microscopy (IVM) has opened unique opportunities to quantify stem cell behavior, epithelial remodeling, and regeneration in many organs26,27,28,29,30, and has provided impactful insights into intestinal biology4,5,21,31,32,33,34,35,36.
Here, we describe a method to cause spatiotemporal-defined intestinal damage and capture the recovery of the intestinal epithelial lining. We utilize two photon-based laser ablation to damage intestinal crypts and follow the immediate wound response and long-term recovery by repetitive intravital microscopy. Our protocol allows one to map the regenerative remodeling of the intestinal tissue architecture in response to local tissue damage. Crypt dynamics, including fission and fusion events, can be readily quantified and tracked over time. The application of laser ablation and repetitive intravital imaging can be utilized as a platform to investigate the tissue-scale dynamics of intestinal architecture during homeostasis and pathophysiology, such as tumor initiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia induction box</td>
			<td>Veterinary Technics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Certoclav</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Mylan</td>
			<td>202809</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaper (underpad)</td>
			<td>Absorin comfort</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11255-20 or 11272-40</td>
			<td>Inox, style #55, used to hold the peritoneum</td>
		</tr>
		<tr>
			<td>Enzymatic instrument cleaner</td>
			<td>Roboz</td>
			<td>EC-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 80%</td>
			<td>homemade</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Duratears Z (Alcon)</td>
			<td>288/28282-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors Straight 9 cm</td>
			<td>Fine Scientific Tools</td>
			<td>14060-09</td>
			<td>Used to cut skin and peritoneum of the mouse</td>
		</tr>
		<tr>
			<td>Gauze 5 cmX5 cm</td>
			<td>Cutisoft (Bsn medical)</td>
			<td>45847-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps Curved Serrated</td>
			<td>Fine Scientific Tools</td>
			<td>11051-10</td>
			<td>Used to hold the skin</td>
		</tr>
		<tr>
			<td>Hartman Hemostat Straight</td>
			<td>Fine Scientific Tools</td>
			<td>13002-10</td>
			<td>Used for suturing</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Comfort</td>
			<td>T5-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging box</td>
			<td>Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incision film</td>
			<td>Nobafilm</td>
			<td>172215</td>
			<td></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 (vetflurane)</td>
			<td>Pharmachemie BV, Haarlem, Netherlands</td>
			<td>305788</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Penlon sigma delta</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore paper tape</td>
			<td>Micropore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Braun</td>
			<td></td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Needle 25G</td>
			<td>BD</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper tape tesa</td>
			<td>Tesa</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-994</td>
			<td>semi-transparent tape</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Supermax stainless steel</td>
			<td></td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Kent Scientific</td>
			<td>20250-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Rimadyl Cattle (carprofen)</td>
			<td>Zoetis B.V</td>
			<td>Registration# REG NL 10130</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Scissors Straight 11.5 cm</td>
			<td>Fine Scientific Tools</td>
			<td>91460-11</td>
			<td>Used to cut gauze</td>
		</tr>
		<tr>
			<td>Surgical instrument cleaner</td>
			<td>Roboz</td>
			<td>IC-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument lubricant</td>
			<td>Roboz</td>
			<td>IL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1 ml)</td>
			<td>BD</td>
			<td>303172</td>
			<td>Other brands available</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Temgesic (Buprenorphine hydrochloride)</td>
			<td>Indivior UK Limited/Reckitt Benckiser Healthcare</td>
			<td>Registration# RVG 08725</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl polyglactin suture 5-0 FS-2 needle</td>
			<td>Ethicon</td>
			<td>V292ZH</td>
			<td></td>
		</tr>
		<tr>
			<td>VirkonS</td>
			<td>Bio-services</td>
			<td></td>
			<td>antiseptic solution</td>
		</tr>
		<tr>
			<td>Wooden cotton swab (sterile)</td>
			<td>Klinion</td>
			<td>531530</td>
			<td>Other brands available</td>
		</tr>
	</tbody>
</table>

laser ablation and intravital microscopy to study intestinal remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

To demonstrate the type of results that can be obtained from the laser ablation method combined with intravital imaging, we performed an experiment as shown in Figure 1. First, we injected K19-CreERT2;mTmG (K19cre-mTmG) and Lgr5eGFP-IRES-CreERT2;Rosa26-Confetti (Lgr5cre-Confetti) mice with tamoxifen to stochastically label cells with one of the Confetti colors (or green fluorescent protein [GFP] in the case of mTmG mice). K19-cre induces lineage tracing from all epithelial cells, while Lgr5-cre induces tracing from stem cells only8,18,37. The tamoxifen injection was administered&#160;4-6 weeks before surgery and the first imaging session in order to obtain crypts in which all the cells are monoclonal for a certain color. Robust identification of the same tissue regions over several weeks is essential to track the fate of the same crypts over time. Inherent tissue architectural features, such as the vasculature, can be used as reliable tissue landmarks, and were here recorded with the internal camera of the microscope. In addition, labeling a small fraction of crypts with (at least two) different colors helped recognize the same crypts in each imaging session and following their behavior during the regenerative process after laser damage. Upon surgically exposing the intestine of the mouse and acquiring both camera and fluorescent images of the region of interest (Figure 2A), bleach point settings of the multiphoton laser can be used to ablate one or more crypts (Figure 2B,C). The initiation of damage could be recognized by an increase in autofluorescence in both the green and red channels. To study the dynamics of crypt recovery, the surgery and imaging procedure was repeated weeks/months after the first imaging session and tissue inherent structures (vasculature and patterns of clonally labelled crypts) were used as landmarks to find the exact same location of the injury (Figure 2). When this method was applied in Lgr5Cre-Confetti mice, where Lgr5-eGFP is expressed in a patchy manner, changes in crypt numbers and shapes following the laser-induced damage could be captured (Figure 3). While some regions showed no changes in the number and structure of crypts (Figure 3B), other regions showed extensive crypt remodeling, as seen in the number of crypt fission and fusion events (Figure 3C) and the loss of crypts 2 weeks after the laser-induced injury (Figure 3D). By introducing different sizes of damage and quantifying the fission, fusion, and disappearance events following ablation, as in the example shown in (Figure 3E), the dynamics of injury-induced regeneration can be mapped in different mouse models.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64756/64756fig01.jpg" /> 
Figure 1: Schematic representation of the experimental setup. In vivo labeling of intestinal cells is achieved by injecting tamoxifen intraperitoneally in genetically modified mouse models to induce Cre-mediated recombination in intestinal crypts. After days or weeks (when the crypts are monoclonal for a certain color), the intestine is surgically exposed and subjected to laser ablation using a multiphoton microscope with a temperature-controlled chamber. The extent of crypt damage is characterized immediately after ablation and the mouse is allowed to recover from the procedure. Repeated surgical exposure and IVM are used to monitor intestinal recovery over time. The pattern and distribution of the blood vasculature and differently colored crypts are used to find back the same regions weeks or months after ablation. Crypt remodeling (e.g., fission or fusion) can be observed at the site of damage weeks after ablation. <a href="https://www.jove.com/files/ftp_upload/64756/64756fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64756/64756fig02v6.jpg" /> 
Figure 2: Longitudinal imaging of tissue regeneration upon laser-induced damage in the intestine. When the intestine is positioned in the exact same manner, blood vessels of the intestine can be used as a map to find and image the exact same regions. (A) Camera overview images of the intestine. Dashed white lines represent the pattern of the vasculature neighboring the site of damage (white box), used as a landmark to find the same laser-ablated region on another time point. The lower two images show the zoomed-in view of the white box. The arrow depicts the exact site of laser ablation at day 1 and 1 month later. (B) Fluorescent images of a single crypt damage captured by multiphoton microscope. The arrow in the blue box shows the site of the damage at day 1 and 1 month later. (C) Fluorescent images of a damaged field of crypts. The arrows in the blue box mark the region of the damage at day 1 and 1 month later. Scale bars: 1 mm (A, top); 0.25 mm (A, bottom); 200 &#181;m (big overview B and C); and 50 &#181;m (zoomed images B and C). <a href="https://www.jove.com/files/ftp_upload/64756/64756fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64756/64756fig03v2.jpg" /> 
Figure 3: Intravital imaging of crypt fates following laser ablation. (A) Using the Confetti model, crypts labeled with different colors can be followed over time to map the dynamics of recovery. The white arrow depicts the site of laser ablation. The yellow box represents a neighboring region (&#60;100 &#181;m from the site of damage), while the blue and purple boxes represent regions far (&#62;100 &#181;m) from the laser-ablated region. Different modes of regeneration are observed. (B) Some regions remain unchanged as in the purple box. (C) Other regions exhibit crypt remodeling in the form of crypt fission or fusion; the dashed white lines and arrows depict crypt fission events and the orange dashed lines and arrows depict crypt fusion events. (D) In some regions, crypt disappearance is observed, as in the yellow box where the crypt highlighted with an orange dashed line disappeared. (E) An example quantification showing the number of crypt remodeling events observed in the areas neighboring (i.e., less than 100 &#181;m) and far from (i.e., more than 100 &#181;m) the damage site. Scale bars: 200 &#181;m (A); 50 &#181;m (B-D). <a href="https://www.jove.com/files/ftp_upload/64756/64756fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling at JoVE.com

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...

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1.3K Views.  The Netherlands Cancer Institute. Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Video: Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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, ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

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.

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 ...