title: Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.
abstract: The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.
authors.affiliation: Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Ortrud
authors.initials: O
authors.lastname: Uckermann
authors.affiliation: Clinical Sensoring and Monitoring, Clinic of Anesthesiology and Intensive Care Therapy, Medical Faculty, TU Dresden, Dresden, Germany.
authors.forename: Roberta
authors.initials: R
authors.lastname: Galli
authors.affiliation: Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Leupold
authors.affiliation: Department of Neuropathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Coras
authors.affiliation: Neuropathology, Institute of Pathology, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meinhardt
authors.affiliation: Neurology, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.|Center for Epilepsy surgery, Dresden, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Hallmeyer-Elgner
authors.affiliation: Center for Epilepsy surgery, Dresden, Germany.|Sächsisches Epilepsiezentrum Radeberg/Kleinwachau, Radeberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mayer
authors.affiliation: Neurology, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Storch
authors.affiliation: Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.|Center for Epilepsy surgery, Dresden, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schackert
authors.affiliation: Clinical Sensoring and Monitoring, Clinic of Anesthesiology and Intensive Care Therapy, Medical Faculty, TU Dresden, Dresden, Germany.|Center for Regenerative Therapies, TU Dresden, Dresden, Germany.
authors.forename: Edmund
authors.initials: E
authors.lastname: Koch
authors.affiliation: Department of Neuropathology, University Hospital Erlangen, Erlangen, Germany.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Blümcke
authors.affiliation: Clinical Sensoring and Monitoring, Clinic of Anesthesiology and Intensive Care Therapy, Medical Faculty, TU Dresden, Dresden, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Steiner
authors.affiliation: Neurosurgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany.|Center for Epilepsy surgery, Dresden, Germany.|Center for Regenerative Therapies, TU Dresden, Dresden, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kirsch
mesh_terms: D000328:Adult; D004833:Epilepsy, Temporal Lobe / Q000150:complications / Q000473:pathology*; D005260:Female; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008297:Male; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D008875:Middle Aged; D009474:Neurons / Q000473:pathology*; D012598:Sclerosis / Q000209:etiology / Q000473:pathology; D013059:Spectrum Analysis, Raman; D055815:Young Adult
keywords: Coherent anti-Stokes Raman scattering; Corpora amylacea; Endogenous fluorescence; Human; Second harmonic generation