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

Summary

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.

Abstract

The incidence of inflammatory bowel disease, i.e., Crohn'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.

Introduction

Inflammatory bowel disease (IBD), i.e., Ulcerative Colitis (UC) and Crohn'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 attention^2,3. 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 home....

Protocol

All animal experiments were approved by the regional ethics committee (the Landesamt für Natur, Umwelt und Verbraucherschutz, LANUV, Germany) according to German Animal Protection Law. The local ethics committee of the University of Münster approved the use of human tissues for histological and microscope analysis.

1. Animals and Materials

1. 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.
2. Induce acute DSS colitis by administering 3% w/v d....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Azoxymethane (AOM)	Sigma - Aldrich, Deisenhofen, Germany	A5486
Cell Culture Flask	Greiner Bio-One, Frickenhausen, Germany	658170
Costar Stripette	Corning Inc., New York, USA	4488
Dextran sulphate sodium (DSS)	TdB Consulatancy, Uppsala, Sweden	DB001
DMEM/Ham's F12	PAA Laboratories - Pasching - Austria	E15-813
EGF	Sigma - Aldrich, Deisenhofen, Germany	SPR3196
Ethylenediaminetetraacetic acid (EDTA)	Sigma - Aldrich, Deisenhofen, Germany	E 9884
Falcon Tube 50 ml	BD Biosciences, Erembodegem, Belgium	352070
Isopentane (2-Methylbutane)	Sigma - Aldrich, Deisenhofen, Germany	M32631-1L
Methylene blue	Merck, Darmstadt, Germany	1159430025
Mitomycin C	Sigma - Aldrich, Deisenhofen, Germany	M4287
Microscope Slides	G. Menzel, Braunschweig, Germany	J1800AMNZ
O.C.T. Tissue Tek compound	Sakura, Zoeterwonde, Netherlands	4583
Pen/Strep/Amphotericin B	Lonza, Verviers, Belgium	1558
Phosphate buffered saline, PBS	Lonza, Verviers, Belgium	4629
RPMI 1640	Lonza, Verviers, Belgium	3626
Sodium Chloride 0.9%	Braun, Melsungen, Germany	5/12211095/0411
Standard diet	Altromin, Lage, Germany	1320
Tissue-Tek Cryomold	Sakura, Leiden, Netherlands	4566
Trypsin EDTA	Lonza, Verviers, Belgium	7815
Vitro – Clud	R. Langenbrinck, Teningen, Germany	04-0002
µ-Dish 35 mm with Culture-Insert, high	ibidi GmbH, Munich, Germany	81176
DIC Lid for µ-Dishes, with a glass insert	ibidi GmbH, Munich, Germany	80050
Equipment
MICROM HM550	Thermo Fisher Scientific, Inc., Waltham, USA	46320
Digital holographic microscope
Component	Model	Company
Inverted Microscope	iMIC	Till Photonics, Graefelfing, Germany
Laser	Compass 315M	Coherent GmbH, Luebeck, Germany
Microscope lens	Zeiss EC Plan Neofluar 10x/0.3	Zeiss, Goettingen, Germany
CCD camera	DMK 41BF02	The Imaging Source, Bremen, Germany