Richard Torres1, Sam Vesuna, Michael J Levene. 1. From the Departments of Laboratory Medicine (Dr Torres) and Neurology (Dr Levene), Yale University School of Medicine, New Haven, Connecticut; and the Department of Biomedical Engineering, Yale University School of Engineering and Applied Science, New Haven, Connecticut (Drs Torres and Levene and Mr Vesuna).
Abstract
CONTEXT: Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. OBJECTIVE: To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. DESIGN: Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. RESULTS: Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. CONCLUSIONS: Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.
CONTEXT: Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. OBJECTIVE: To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. DESIGN:Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. RESULTS: Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. CONCLUSIONS: Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.
Authors: Elizabeth A Calle; Sam Vesuna; Sashka Dimitrievska; Kevin Zhou; Angela Huang; Liping Zhao; Laura E Niklason; Michael J Levene Journal: Tissue Eng Part C Methods Date: 2014-01-16 Impact factor: 3.056
Authors: Michael G Monaghan; Sebastian Kroll; Sara Y Brucker; Katja Schenke-Layland Journal: Tissue Eng Part C Methods Date: 2016-04-25 Impact factor: 3.056
Authors: Richard Torres; Heino Velazquez; John J Chang; Michael J Levene; Gilbert Moeckel; Gary V Desir; Robert Safirstein Journal: J Am Soc Nephrol Date: 2015-08-24 Impact factor: 10.121