JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: September 13th, 2016

DOI:

10.3791/54460

1Department of Medicine B, University Hospital Münster, 2Institute of Palliative Care, University Hospital Münster, 3Biomedical Technology Center, University of Münster, 4Department of Gastroenterology, Klinikum Bielefeld
* These authors contributed equally

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'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.

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 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 home....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 legisl.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 50ml 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

  1. Baumgart, D. C., Sandborn, W. J. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet. 369 (9573), 1641-1657 (2007).
  2. Dieleman, L. A., et al. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 114 (3), 385-391 (1998).
  3. Florholmen, J. Mucosal healing in the era of biologic agents in treatment of inflammatory bowel disease. Scand J Gastroenterol. 50 (1), 43-52 (2015).
  4. Merga, Y., Campbell, B. J., Rhodes, J. M. Mucosal barrier, bacteria and inflammatory bowel disease: possibilities for therapy. Dig Dis. 32 (4), 475-483 (2014).
  5. Young, V. B., Kahn, S. A., Schmidt, T. M., Chang, E. B. Studying the Enteric Microbiome in Inflammatory Bowel Diseases: Getting through the Growing Pains and Moving Forward. Front Microbiol. 2, 144 (2011).
  6. Atreya, R., Neurath, M. F. IBD pathogenesis in 2014: Molecular pathways controlling barrier function in IBD. Nat Rev Gastroenterol Hepatol. 12 (2), 67-68 (2015).
  7. Jones, M. K., Tomikawa, M., Mohajer, B., Tarnawski, A. S. Gastrointestinal mucosal regeneration: role of growth factors. Front Biosci. 4, 303-309 (1999).
  8. Burk, R. R. A factor from a transformed cell line that affects cell migration. Proc Natl Acad Sci U S A. 70 (2), 369-372 (1973).
  9. Singh, A., Nascimento, J. M., Kowar, S., Busch, H., Boerries, M. Boolean approach to signalling pathway modelling in HGF-induced keratinocyte migration. Bioinformatics. 28 (18), 495-501 (2012).
  10. Sakalar, C., et al. Pronounced transcriptional regulation of apoptotic and TNF-NF-kappa-B signaling genes during the course of thymoquinone mediated apoptosis in HeLa cells. Mol Cell Biochem. 383 (1-2), 243-251 (2013).
  11. Serada, S., et al. Serum leucine-rich alpha-2 glycoprotein is a disease activity biomarker in ulcerative colitis. Inflamm Bowel Dis. 18 (11), 2169-2179 (2012).
  12. Turovskaya, O., et al. RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis. Carcinogenesis. 29 (10), 2035-2043 (2008).
  13. Perse, M., Cerar, A. Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol. 2012, 718617 (2012).
  14. Lee, K., et al. Quantitative phase imaging techniques for the study of cell pathophysiology: from principles to applications. Sensors (Basel). 13 (4), 4170-4191 (2013).
  15. Bettenworth, D., et al. Quantitative stain-free and continuous multimodal monitoring of wound healing in vitro with digital holographic microscopy. PLoS One. 9 (9), 107317 (2014).
  16. Sridharan, S., Macias, V., Tangella, K., Kajdacsy-Balla, A., Popescu, G. Prediction of prostate cancer recurrence using quantitative phase imaging. Sci Rep. 5, 9976 (2015).
  17. Wang, Z., Tangella, K., Balla, A., Popescu, G. Tissue refractive index as marker of disease. J Biomed Opt. 16 (11), 116017 (2011).
  18. Majeed, H., et al. Breast cancer diagnosis using spatial light interference microscopy. J Biomed Opt. 20 (11), 111210 (2015).
  19. Lenz, P., et al. Digital holographic microscopy quantifies the degree of inflammation in experimental colitis. Integr Biol (Camb). 5 (3), 624-630 (2013).
  20. Popescu, G., et al. Optical imaging of cell mass and growth dynamics. Am J Physiol Cell Physiol. 295 (2), 538-544 (2008).
  21. Klokkers, J., et al. Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells. Am J Physiol Renal Physiol. 297 (3), 693-703 (2009).
  22. Jourdain, P., et al. Determination of transmembrane water fluxes in neurons elicited by glutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic microscopy study. J Neurosci. 31 (33), 11846-11854 (2011).
  23. Wirtz, S., Neufert, C., Weigmann, B., Neurath, M. F. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2 (3), 541-546 (2007).
  24. Bettenworth, D., et al. The tripeptide KdPT protects from intestinal inflammation and maintains intestinal barrier function. Am J Pathol. 179 (3), 1230-1242 (2011).
  25. Kemper, B., et al. Modular digital holographic microscopy system for marker free quantitative phase contrast imaging of living cells. Proc. SPIE. 6191, (2006).
  26. Kemper, B., von Bally, G. Digital holographic microscopy for live cell applications and technical inspection. Appl Opt. 47 (4), 52-61 (2008).
  27. Langehanenberg, P., von Bally, G., Kemper, B. Autofocusing in digital holographic microscopy. 3D Research. 2 (1), 1-11 (2011).
  28. Daimon, M., Masumura, A. Measurement of the refractive index of distilled water from the near-infrared region to the ultraviolet region. Applied optics. 46 (18), 3811-3820 (2007).
  29. Przibilla, S., et al. Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes. J Biomed Opt. 17 (9), 0970011-0970019 (2012).
  30. Kemper, B., et al. Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy. J Biomed Opt. 12 (5), 054009 (2007).
  31. Carpenter, A. E., et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7 (10), 100 (2006).
  32. Marquet, P., et al. Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy. Opt Lett. 30 (5), 468-470 (2005).
  33. Langehanenberg, P., Kemper, B., Dirksen, D., von Bally, G. Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging. Appl Opt. 47 (19), 176-182 (2008).
  34. Hanauer, S. B., et al. Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial. Lancet. 359 (9317), 1541-1549 (2002).
  35. Colombel, J. F., et al. Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial. Gastroenterology. 132 (1), 52-65 (2007).
  36. Feagan, B. G., et al. Vedolizumab as induction and maintenance therapy for ulcerative colitis. N Engl J Med. 369 (8), 699-710 (2013).
  37. Sandborn, W. J., et al. Vedolizumab as induction and maintenance therapy for Crohn's disease. N Engl J Med. 369 (8), 711-721 (2013).
  38. Monteleone, G., et al. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease. N Engl J Med. 372 (12), 1104-1113 (2015).
  39. Vermeire, S., et al. Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial. Lancet. 384 (9940), 309-318 (2014).
  40. Sandborn, W. J., et al. Ustekinumab induction and maintenance therapy in refractory Crohn's disease. N Engl J Med. 367 (16), 1519-1528 (2012).
  41. Natividad, J. M., et al. Commensal and probiotic bacteria influence intestinal barrier function and susceptibility to colitis in Nod1-/-; Nod2-/- mice. Inflamm Bowel Dis. 18 (8), 1434-1446 (2012).
  42. Melgar, S., et al. Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease. Int Immunopharmacol. 8 (6), 836-844 (2008).
  43. Erben, U., et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 7 (8), 4557-4576 (2014).
  44. Bruckner, M., et al. Murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation. J Vis Exp. (90), (2014).
  45. Zhao, K., Wang, W., Guan, C., Cai, J., Wang, P. Inhibition of gap junction channel attenuates the migration of breast cancer cells. Mol Biol Rep. 39 (3), 2607-2613 (2012).
  46. Pavillon, N., et al. Early cell death detection with digital holographic microscopy. PLoS One. 7 (1), 30912 (2012).
  47. Hindryckx, P., et al. Clinical trials in ulcerative colitis: a historical perspective. J Crohns Colitis. 9 (7), 580-588 (2015).
  48. Neurath, M. F., Travis, S. P. Mucosal healing in inflammatory bowel diseases: a systematic review. Gut. 61 (11), 1619-1635 (2012).
  49. Bryant, R. V., Winer, S., Travis, S. P., Riddell, R. H. Systematic review: histological remission in inflammatory bowel disease. Is 'complete' remission the new treatment paradigm? An IOIBD initiative. J Crohns Colitis. 8 (12), 1582-1597 (2014).
  50. Marchal Bressenot, A., et al. Review article: the histological assessment of disease activity in ulcerative colitis. Aliment Pharmacol Ther. 42 (8), 957-967 (2015).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved