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Multimodal spectral imaging of cells using a transmission diffraction grating on a light microscope.

ABSTRACT: A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens.

SUBMITTER: Isailovic D

PROVIDER: S-EPMC3163165 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Multimodal spectral imaging of cells using a transmission diffraction grating on a light microscope.

Isailovic Dragan D Xu Yang Y Copus Tyler T Saraswat Suraj S Nauli Surya M SM

Applied spectroscopy 20110601 6

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originatin ...[more]

PMID: 21639978

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Project description:PurposeTo characterize the first in-human intraoperative imaging using a custom prototype spectral-domain microscope-integrated optical coherence tomography (MIOCT) device during vitreoretinal surgery with instruments in the eye.MethodsUnder institutional review board approval for a prospective intraoperative study, MIOCT images were obtained at surgical pauses with instruments held static in the vitreous cavity and then concurrently with surgical maneuvers. Postoperatively, MIOCT images obtained at surgical pauses were compared with images obtained with a high-resolution handheld spectral-domain OCT (HHOCT) system with objective endpoints, including acquisition of images acceptable for analysis and identification of predefined macular morphologic or pathologic features.ResultsHuman MIOCT images were successfully obtained before incision and during pauses in surgical maneuvers. MIOCT imaging confirmed preoperative diagnoses, such as epiretinal membrane, full-thickness macular hole, and vitreomacular traction and demonstrated successful achievement of surgical goals. MIOCT and HHOCT images obtained at surgical pauses in two cohorts of five patients were comparable with greater than or equal to 80% correlation in 80% of patients. Real-time video-imaging concurrent with surgical manipulations enabled, for the first time using this device, visualization of dynamic instrument-retina interaction with targeted OCT tracking.ConclusionMIOCT is successful for imaging at surgical pauses and for real-time image guidance with implementation of targeted OCT tracking. Even faster acquisition speeds are currently being developed with incorporation of a swept-source MIOCT engine. Further refinements and investigations will be directed toward continued integration for real-time volumetric imaging of surgical maneuvers.Translational relevanceOngoing development of seamless MIOCT systems will likely transform surgical visualization, approaches, and decision-making.

Dataset Information

Multimodal spectral imaging of cells using a transmission diffraction grating on a light microscope.

Publications

Multimodal spectral imaging of cells using a transmission diffraction grating on a light microscope.

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