We thank Faekah Gohar for proofreading the manuscript. We thank Sonja Dufentester and Elke Weber for expert technical assistance.

All animal experiments were approved by the regional ethics committee (the Landesamt fu&#776;r Natur, Umwelt und Verbraucherschutz, LANUV, Germany) according to German Animal Protection Law. The local ethics committee of the University of M&#252;nster approved the use of human tissues for histological and microscope analysis.
1. Animals and Materials
<ol>
	<li>Use female or male mice of the required DSS-susceptible strain that weigh 20 to 25 g, and house according to local animal care legislation. Provide special chow for rodents and autoclaved drinking water ad libitum.</li>
	<li>Induce acute DSS colitis by administering 3% w/v dextran sulfate sodium (DSS, molecular weight: 36,000-50,000 Da) in autoclaved tap water for 5 days. 
	NOTE: The potency of DSS is highly variable depending on manufacturer and batch. Test your supplier-provided DSS first for induction of disease activity, for which daily body weight is a reliable and objective indicator.</li>
	<li>For histological evaluation of colonic tissue samples, euthanize mice by CO2 insufflation (or as specified by national and institutional guidelines) at the end of the experiment.</li>
</ol>
2. Experimental Setup for DSS-colitis and In-vitro Wound Healing Assays
<ol>
	<li>Cell culture and establishment of wound healing assay.
	<ol>
		<li>Grow Caco-2 cells in a 95% humidity and 5% CO2 environment at 37 &#176;C. Use RPMI medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.</li>
		<li>Seed Caco-2 cells at a density of 4 x 105 cells/cm2 on 35 mm Petri dishes with high culture-insert (see Figure 4A). 
		NOTE: The insert generates two cell covered areas that are separated with a defined cell free space representing the wounded area to be analyzed.</li>
		<li>Change medium two days after seeding. Perform by first aspirating residual medium with cell debris by using an automatic pipette, rinse with 100 &#181;l of phosphate buffered saline (PBS) and add fresh RPMI medium or serum-deprived medium.</li>
		<li>Culture cells for 24 hr in serum-deprived medium (0.1% FBS) supplemented with 20 ng EGF/ml serum or 2 &#181;g mitomycin c/ml serum to detect alterations in wound healing behavior. Add normal RPMI medium to control cells for 24 hr.</li>
		<li>After 24 hr of culture, remove and discard culture inserts as described in step 3.4 and perform DHM.</li>
	</ol>
	</li>
	<li>Induction of acute DSS colitis
	<ol>
		<li>Dissolve 3 g of DSS in 100 ml autoclaved water to get a 3% (w/v) DSS solution. Provide this solution in place of drinking water to mice ad libitum for 7 days. Calculate 5 ml of DSS-solution per mouse/day. Provide autoclaved water without DSS for control mice ad libitum.</li>
	</ol>
	</li>
	<li>Preparation of cryostatic sections of murine and human colon
	<ol>
		<li>Euthanize mice by CO2 insufflation at the end of the experiment.</li>
		<li>Dissect the animals&#39; abdomen by laparotomy23. Remove the whole colon carefully using tweezers and cut the ileal and rectal end using surgical scissors. Cut the colon with a scissors longitudinally from the cecal to the rectal end and open the colon. Remove all feces from the specimen using tweezers followed by washing with PBS24.</li>
		<li>Prepare Swiss rolls by rolling up the whole colon with a cotton bud longitudinally from the cecal to the rectal end with the mucosa curved inwards. Embed colonic samples in optimal cutting temperature (OCT) compound and keep frozen at -80 &#176;C until further use.</li>
		<li>Embed human colonic tissue from surgical specimen in optimal cutting temperature OCT compound and keep frozen at -80 &#176;C until further use.</li>
		<li>Cut sections of 7 &#181;m thickness of the OCT-compound-embedded specimens with the help of a cryotome just prior to examination. 
		&#8203;NOTE: The optimum sample thickness depends on the persistence and the scattering properties of the tissue type under investigation. For the described experiments using colon tissue, slice thickness &#62; 10 &#181;m cause significant increase in noise due to light scattering in quantitative DHM phase contrast images, while samples of thickness &#60; 5 &#181;m show a higher risk for damage induced artefacts from the cryo cutting process.</li>
		<li>Transfer sections on to a glass object carrier slide.</li>
	</ol>
	</li>
</ol>
3. Technical Equipment, Software and Procedures for Acquisition and Evaluation of Digital Holograms
<ol>
	<li>Digital holographic microscope for quantitative cell and tissue imaging
	<ol>
		<li>Use an off-axis Mach-Zehnder digital holographic microscopy system for live cell imaging25, as shown in Figure 1. Ensure that the microscope is equipped with a 10X microscope lens, a microscope stage with a holder for glass object carrier slides and Petri dishes with a diameter of 35 mm, a heating chamber to preserve physiological temperature at 37 &#176;C and software for quantitative phase imaging25. 
		NOTE: For example, as described in Kemper et al.26 and Langehanenberg et al.27. 
		NOTE: Alternatively, use a similar system that is capable of performing bright field microscopy and quantitative phase imaging of living cells and dissected tissue slides.</li>
		<li>Clean microscope lens and condenser with lens cleaning paper and a cleaning agent (e.g., ethanol) as recommended by the manufacturer of the microscope to remove dust or other contaminations.</li>
		<li>Start the image acquisition software of the DHM microscope, select &#34;bright field&#34; imaging mode and switch &#34;on&#34; white light illumination. Ensure K&#246;hler-illumination of the sample as recommended by the microscope manufacturer while observing the sample in the live imaging window of the image acquisition software (alternatively a standard image acquisition software can be used in this step). 
		NOTE: The image intensity should be distributed homogeneously in the field of view and the sample position should not move during optical refocusing with the focus drive of the microscope.</li>
		<li>Select &#34;DHM&#34; imaging mode, turn &#34;off&#34; white light illumination and switch &#34;on&#34; the laser light. Check that the illumination with laser light is homogeneous (i.e., that light intensity is homogeneously distributed in the live imaging window of the imaging acquisition software of the DHM microscope) and observe that the off-axis carrier interference fringe pattern appears with adequate contrast in the captured images (digital holograms).</li>
	</ol>
	</li>
	<li>Preparation of cryostat sections for imaging with DHM
	<ol>
		<li>Take the sample (cryostat section on a glass object carrier, thickness: 7 &#181;m, as described in 2.3) out of the freezer. Defrost the sample for about 5 min at RT and normal atmosphere.</li>
		<li>Add 50 - 100 &#181;l phosphate buffered saline (PBS) as embedding medium onto the tissue section using a pipette until it is completely covered with buffer. Cover the sample with a clean glass cover slip (glass thickness 170 &#181;m). 
		NOTE: Over-drying can induce significant changes of the refractive index and scattering properties of the sample.</li>
		<li>Ensure that the bottom of the glass carrier and the cover slip are cleaned from dust and other contamination that may induce light scattering. The sample is ready for investigation with bright field microscopy and DHM.</li>
	</ol>
	</li>
	<li>Quantitative phase imaging of tissue sections with DHM
	<ol>
		<li>Switch on the digital holographic microscope, choose the 10X microscope lens for imaging. Start the image acquisition software of the DHM microscope and select bright file imaging mode.</li>
		<li>Place the tissue slide as described in 2) in the microscope slide holder, with the cover slip facing the microscope objective.</li>
		<li>Switch &#34;on&#34; the bright field illumination of the DHM microscope. Position the sample with the microscope stage and ensure that the tissue area of interest is visible in the live monitoring window. Improve the sharpness of the image using the microscope&#39;s focus drive. 
		NOTE: As well as the area of interest, an area of slide without tissue should also be present in the field of view.</li>
		<li>Capture a bright field image of the sharply focused sample using the image acquisition software.</li>
		<li>Select &#34;DHM&#34; imaging mode, turn &#34;off&#34; the white light illumination and turn &#34;on&#34; the laser light illumination. Select the &#34;exposure time&#34; for hologram recording below 3 msec, observe that holographic off-axis interference fringes appear with adequate contrast in the live imaging window of the imaging acquisition software and capture a digital hologram.</li>
		<li>Repeat steps 3.3.3 - 3.3.5 until a sufficient number of bright field images and digital holograms of different sample areas have been recorded. Hologram acquisition is now complete.</li>
	</ol>
	</li>
	<li>Preparation of wound healing assays for DHM imaging
	<ol>
		<li>Switch on the Petri dish heating chamber of the DHM microscope about 1 - 3 hr prior to the start of the experiment to ensure stable temperature conditions during the DHM measurements.</li>
		<li>Prepare workbench with the required equipment (Petri dish for wound healing assay is described in 2.3): pipettes, tweezers, glass lid for Petri dish, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered cell culture medium with physiological temperature (37 &#176;C) for sample preparation in sterile environment. 
		&#8203;NOTE: Dulbecco&#39;s Modified Eagle Medium (DMEM) is composed of 10 % fetal calve serum (FCS), 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 850 mg/L NaHCO3.</li>
		<li>Remove the plastic lid from the Petri dish and remove the cell culture medium with a pipette. Remove the insert from the Petri dish bottom using tweezers.</li>
		<li>Wash the sample 1 - 2 times with 1 ml HEPES buffered cell culture medium in order to remove dead cells and remaining cellular components (e.g., serum) in the wound area. Add 2 ml HEPES buffered cell culture medium and cap the Petri dish with the glass lid.</li>
		<li>Ensure that the glass lid and the Petri dish bottom have been cleaned from dust and other contamination. Sample is ready for time lapse observation with DHM.</li>
	</ol>
	</li>
	<li>Continuous multimodal monitoring of wound healing in vitro with DHM
	<ol>
		<li>Switch on the Petri dish heating chamber of the DHM microscope about 1 - 3 hr prior to the start of the experiment to ensure stable temperature conditions during the DHM measurements.</li>
		<li>Switch &#34;on&#34; the digital holographic microscope, select the 10X microscope lens for imaging. Start the image acquisition software of the DHM microscope and select &#34;bright field&#34; imaging mode. Ensure that the heating chamber for the Petri dish is operating a physiological temperature (37 &#176;C).</li>
		<li>Place the Petri dish with the wound healing assay, prepared as described in 4), in the heating chamber of the DHM microscope.</li>
		<li>Select bright field imaging mode and position the sample with the microscope stage while observing it in the live monitoring window of the image acquisition software of the DHM microscope. Observe that the desired area of the sample appears sharply focused under white light illumination.</li>
		<li>Capture bright field images of different areas of the sample (wound area and surrounding areas with confluent cells) under white light illumination with the image acquisition software and document appearance, cell density and homogeneity.</li>
		<li>Select &#34;bright field&#34; imaging mode of the DHM microscope. Choose a suitable wound area under white light illumination in the live monitoring window with the image acquisition software of the DHM microscope. Ensure the wound area is free from dead cells and no serum remains, and ensure that both sides include a single homogeneous cell layer, preferably with straight borders.</li>
		<li>Capture a white light image of the initial wound area in bright field imaging mode with the image acquisition software of the DHM microscope.</li>
		<li>Turn &#34;off&#34; white light illumination, select &#34;DHM&#34; mode and switch &#34;on&#34; laser illumination. Select an exposure time of below 3 msec for hologram recording (observe that holographic off-axis interference fringes appear with adequate contrast in the live imaging window of the image acquisition software of the DHM microscope) and capture a digital hologram.</li>
		<li>Capture a sample hologram in DHM mode with the image acquisition software and reconstruct a quantitative phase image with the reconstruction software of the DHM microscope in order to check the image quality.</li>
		<li>Select a suitable time delay (e.g., 3 - 5 min) for time-lapse hologram acquisition with the image acquisition software of the DHM microscope.</li>
		<li>Select the time-lapse acquisition mode of the image acquisition software in which the sample is only illuminated with laser light during hologram acquisition.</li>
		<li>Start the time-lapse DHM observation of the wound healing assay.</li>
		<li>Stop the time-lapse acquisition after the intended time, select bright field imaging mode and document the final appearance of the sample under white light imaging.</li>
	</ol>
	</li>
	<li>Reconstruct digital holograms of dissected tissues and determine the average refractive index as a parameter to quantify tissue density
	<ol>
		<li>Reconstruct quantitative phase images from the digital holograms of dissected tissues with the software of the DHM microscope, e.g., as described in Kemper et al.26 and Langehanenberg et al.27.</li>
		<li>Determine the average phase contrast&#160;&#916;&#966; in different tissue layers (epithelium, submucosa, stroma) in appropriately chosen regions of interests (ROIs)19.</li>
		<li>Determine the refractive index of the embedding medium by using a refractometer or alternatively by using an appropriate value from the literature. (refractive index values for typical embedding media: nwater = 1.334 28, nPhosphate buffered saline (PBS) = 1.337 29, ncell culture medium = 1.337-1.339 29,30).</li>
		<li>Calculate the refractive indices of different tissue layers from the average phase contrast values19 
		<img alt="Equation 1" src="/files/ftp_upload/54460/54460eq1.jpg" />&#160;&#160;&#160;&#160; (1) 
		NOTE: In Eq. 1 &#955; is the wavelength of the laser light (here: &#955; = 532 nm), d the thickness of the dissected tissues (here: 7 &#181;m) and nmedium is the refractive index of the embedding medium (here: nmedium = nPBS = 1.337, determined by an Abbe-Refractometer).</li>
	</ol>
	</li>
	<li>Reconstruct and evaluate digital holograms from the time-lapse wound healing series observation
	<ol>
		<li>Reconstruct quantitative phase images from the time lapse hologram series obtained during wound healing observation with the DHM microscope software26,27.</li>
		<li>Normalize each series of quantitative phase images to the image with maximum phase contrast.</li>
		<li>Determine the area Sc that is covered by the cells in the quantitative DHM phase images by image segmentation, which can be performed using the free software cell profiler (www.cellprofiler.org31).</li>
		<li>Calculate the average phase contrast of the cells &#916;&#966;cell in the area Sc.</li>
		<li>Retrieve the cellular dry mass DM from the average phase contrast &#916;&#966;cell in the area Sc15 
		<img alt="Equation 2" src="/files/ftp_upload/54460/54460eq2.jpg" />&#160;&#160;&#160;&#160; (2) 
		NOTE: In Equation 2 DM denotes the cellular dry mass, Sc presents the area that is occupied by the cells and &#945; = 0.002 m2/kg.</li>
		<li>Determine the integral cellular refractive index ncell and the refractive index of the cell culture medium nmedium. Determine ncell separately experimentally from suspended cells as described in 30 and measure nmedium with a refractometer. Alternatively use literature values for ncell30 and nmedium29,30.</li>
		<li>Calculate the average cell thickness dcell from &#955;, &#916;&#966;cell, ncell and nmedium 26,32 
		<img alt="Equation 3" src="/files/ftp_upload/54460/54460eq3.jpg" />&#160;&#160;&#160;&#160; (3) 
		NOTE: In Eq. 3 the parameter dcell is the average cell thickness, &#955; denotes the light wavelength of the laser light, &#916;&#966;cell denotes the average phase contrast, and ncell and nmedium are the integral cellular refractive index and the refractive index of the surrounding medium.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

We demonstrate that DHM provides accurate assessment of histological damage in murine colitis models and human colonic tissue samples ex vivo. Furthermore, we shown DHM can continuously monitor epithelial wound healing whilst simultaneously providing multimodal information about cellular alterations. In DHM, the reconstruction of digitally captured holograms is performed numerically32. Therefore, in comparison to bright field microscopy, Zernike phase contrast and differential interference contrast mi...

Inflammatory bowel disease (IBD), i.e., Ulcerative Colitis (UC) and Crohn&#39;s disease (CD) are idiopathic inflammatory disorders of the gastrointestinal tract1. Research into the underlying pathophysiology of IBD and the evaluation of potential new drugs or novel diagnostic approaches is particularly of importance. In both basic research and the clinical management of IBD patients, the intestinal mucosa has become a focus of attention2,3. The mucosa represents an anatomical boundary, at which the interaction between commensal bacteria, epithelial cells and various cellular components of the intestinal immune system orchestrate gut homeostasis4,5. However, in IBD patients, uncontrolled and persistent intestinal inflammation leads to mucosal damage, detectable as ulcerations or stenosis, which can finally culminate in breakdown of epithelial barrier function, which itself aggravates local inflammation6.
Epithelial wound healing is therefore crucial for epithelial regeneration following inflammation but is also a core requirement for the healing of gastrointestinal ulcers or anastomotic leakage after gastrointestinal surgery7. Epithelial wound healing can be simulated in in vitro wound healing assays and in murine models of intestinal inflammation8,9. Both in vitro and in vivo approaches have drawbacks, which limit the accuracy of experimental assessment. In vitro assays, like classical scratch assays, require protracted staining procedures or transfection with fluorescent chromophores. They are often limited by their discontinuous monitoring of cell proliferation and migration that cannot be automated10. In vivo models, such as dextran sodium sulphate (DSS)-induced colitis, frequently lack robust read-outs, in part due to the significant variation seen in laboratory markers, making such markers inappropriate to evaluate colitis severity11,12. Histological analysis of the inflamed mucosa is currently still the most valid approach to determine colitis severity but this, like in vitro epithelial wound healing assays, requires staining and is dependent on investigator&#39;s expertise13.
Recently digital holographic microscopy (DHM), a variant of quantitative phase microscopy14, was identified as useful tool for the evaluation of epithelial wound healing in vitro and in vivo15. DHM allows assessment of tissue density by measuring optical path length delay (OPD), which prospects novel cancer diagnosis16-18 and quantification of inflammation related tissue alterations19. Additionally, DHM allows monitoring of cell morphology dynamics by determining cell thickness, cell covered surface area and intracellular (protein) content quantity15,20. In in vitro assays, DHM also enables the analysis of physiological processes, e.g., cellular water permeability by evaluating changes in cell volume and thickness21,22. Moreover, DHM measurements can be automated which prevents investigator-associated sample bias.
Here, we demonstrate the use of DHM in a murine model of intestinal inflammation, and also apply DHM to analysis of human tissues samples for quantitative monitoring of wound healing as a label-free in vitro assay. First, we evaluate inflammatory alterations of different colonic wall-layers in colitic mice and tissue sections from humans with IBD. After describing the DHM quantitative phase imaging procedure, we provide detailed instructions for using the microscope components, the preparation of tissue sections and also describe the evaluation of the acquired quantitative phase images.
Next, we show that DHM can be utilized for continuous multimodal monitoring of epithelial wound healing in vitro, and describe the analysis of cellular characteristics like cell layer thickness, dry mass and cellular volume give insight into drug induced and physiologic cell alterations.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Azoxymethane (AOM)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>A5486</td><td></td></tr><tr><td>Cell Culture Flask</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>658170</td><td></td></tr><tr><td>Costar Stripette</td><td>Corning Inc., New York, USA</td><td>4488</td><td></td></tr><tr><td>Dextran sulphate sodium (DSS)</td><td>TdB Consulatancy, Uppsala, Sweden</td><td>DB001</td><td></td></tr><tr><td>DMEM/Ham&#039;s F12</td><td>PAA Laboratories - Pasching - Austria</td><td>E15-813</td><td></td></tr><tr><td>EGF</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>SPR3196</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>E 9884</td><td></td></tr><tr><td>Falcon Tube 50 ml</td><td>BD Biosciences, Erembodegem, Belgium</td><td>352070</td><td></td></tr><tr><td>Isopentane (2-Methylbutane)</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>M32631-1L</td><td></td></tr><tr><td>Methylene blue</td><td>Merck, Darmstadt, Germany</td><td>1159430025</td><td></td></tr><tr><td>Mitomycin C</td><td>Sigma - Aldrich, Deisenhofen, Germany</td><td>M4287</td><td></td></tr><tr><td>Microscope Slides</td><td>G. Menzel, Braunschweig, Germany</td><td>J1800AMNZ</td><td></td></tr><tr><td>O.C.T. Tissue Tek compound&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sakura, Zoeterwonde, Netherlands</td><td>4583</td><td></td></tr><tr><td>Pen/Strep/Amphotericin B</td><td>Lonza, Verviers, Belgium</td><td>1558</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Lonza, Verviers, Belgium</td><td>4629</td><td></td></tr><tr><td>RPMI 1640</td><td>Lonza, Verviers, Belgium</td><td>3626</td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td>Braun, Melsungen, Germany</td><td>5/12211095/0411</td><td></td></tr><tr><td>Standard diet</td><td>Altromin, Lage, Germany</td><td>1320</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, Leiden, Netherlands</td><td>4566</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Lonza, Verviers, Belgium</td><td>7815</td><td></td></tr><tr><td>Vitro &amp;ndash; Clud</td><td>R. Langenbrinck, Teningen, Germany</td><td>04-0002&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;&amp;micro;-Dish 35 mm with Culture-Insert, high</td><td>ibidi GmbH, Munich, Germany</td><td>81176</td><td></td></tr><tr><td>DIC Lid for &amp;micro;-Dishes, with a glass insert</td><td>ibidi GmbH, Munich, Germany</td><td>80050</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MICROM HM550</td><td>Thermo Fisher Scientific, Inc., Waltham, USA</td><td>46320</td><td></td></tr><tr><td>&lt;strong&gt;Digital holographic microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Component&lt;/strong&gt;</td><td>&lt;strong&gt;Model&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td></td></tr><tr><td>Inverted Microscope</td><td>iMIC</td><td>Till Photonics, Graefelfing, Germany</td><td></td></tr><tr><td>Laser</td><td>Compass 315M</td><td>Coherent GmbH, Luebeck, Germany</td><td></td></tr><tr><td>Microscope lens</td><td>Zeiss EC Plan Neofluar 10x/0.3</td><td>Zeiss, Goettingen, Germany</td><td></td></tr><tr><td>CCD camera</td><td>DMK 41BF02</td><td>The Imaging Source, Bremen, Germany</td><td></td></tr></tbody></table>

multimodal quantitative phase imaging with digital holographic microscopy accurately assesses intestinal inflammation and epithelial wound healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Typical Setup for DHM Imaging for Digital Holographic Microscopy (DHM)
To perform bright field imaging and quantitative DHM phase contrast imaging, we applied an inverted microscope as depicted in Figure 1B. The system was modified by attaching a DHM module, as described earlier25. Digital holograms were generated by illumination the sample with the l...

Watch this Scientific Journal Video about Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing at JoVE.com

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

multimodal-quantitative-phase-imaging-with-digital-holographic

Research

JoVE Journal

Medicine

8.3K Views.  University Hospital Münster. Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro. 

Video: Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Bioengineering

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Developmental Biology

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Immunology and Infection

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Biology

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

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