Abstract
For decades, the confocal microscope has represented one of the dominant imaging systems in biomedical imaging at sub-cellular lengthscales. Recently, however, it has increasingly been replaced by a related, but more powerful successor technique termed image scanning microscopy (ISM). In this article, we present ISM capable of measuring spectroscopic information such as that contained in fluorescence or Raman images. Compared to established confocal spectroscopic imaging systems, our implementation offers similar spectral resolution, but higher spatial resolution and detection efficiency. Color sensitivity is achieved by a grating placed in the detection path in conjunction with a camera collecting both spatial and spectral information. The multidimensional data is processed using multi-view maximum likelihood image reconstruction. Our findings are supported by numerical simulations and experiments on micro beads and double-stained HeLa cells.
Highlights
There has been an increasing need to acquire spectral information in conjunction with high resolution images
confocal spectral imaging (CSI) microscopes are usually designed in a way that the image of the detection pinhole on the multi-anode photo multiplier tubes (PMT) is smaller than the size of an individual detector, because this decouples spectral resolution from the chosen pinhole size [2]
image scanning microscopy (ISM) is an imaging technique that enables the spatial resolution of a confocal microscope with almost fully closed pinhole while essentially all the light reaching the image plane is collected with a detector array, providing bright high-resolution images
Summary
There has been an increasing need to acquire spectral information in conjunction with high resolution images This capability is important to distinguish multiple fluorescent markers simultaneously, even when they present similar emission spectra, such as EYFP and EGFP [1]. One possible technique is confocal spectral imaging (CSI), which combines confocal microscopy and spectroscopy in order to collect high resolution optical sections with speficic color information. CSI microscopes are usually designed in a way that the image of the detection pinhole on the multi-anode PMT is smaller than the size of an individual detector, because this decouples spectral resolution from the chosen pinhole size [2]. We treat the imaging problem under the more general framework of engineered Image Scanning Microscopy (eISM), which we recently defined as ISM using designed excitation and/or detection pupils in conjunction with matched data processing [16]. We treat the imaging problem under the more general framework of engineered Image Scanning Microscopy (eISM), which we recently defined as ISM using designed excitation and/or detection pupils in conjunction with matched data processing [16]. eISM represents an example for an integrated optical design [17], where PSF engineering and data processing work hand in hand to obtain optimal imaging
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