Spatially varying regularization of deconvolution in 3D microscopy

Confocal microscopy has become an essential tool to explore biospecimens in 3D. Confocal microcopy images are still degraded by out‐of‐focus blur and Poisson noise. Many deconvolution methods including the Richardson–Lucy (RL) method, Tikhonov method and split‐gradient (SG) method have been well received. The RL deconvolution method results in enhanced image quality, especially for Poisson noise. Tikhonov deconvolution method improves the RL method by imposing a prior model of spatial regularization, which encourages adjacent voxels to appear similar. The SG method also contains spatial regularization and is capable of incorporating many edge‐preserving priors resulting in improved image quality. The strength of spatial regularization is fixed regardless of spatial location for the Tikhonov and SG method. The Tikhonov and the SG deconvolution methods are improved upon in this study by allowing the strength of spatial regularization to differ for different spatial locations in a given image. The novel method shows improved image quality. The method was tested on phantom data for which ground truth and the point spread function are known. A Kullback–Leibler (KL) divergence value of 0.097 is obtained with applying spatially variable regularization to the SG method, whereas KL value of 0.409 is obtained with the Tikhonov method. In tests on a real data, for which the ground truth is unknown, the reconstructed data show improved noise characteristics while maintaining the important image features such as edges.     

Multiphoton confocal microscopy using a femtosecond Cr:forsterite laser

With its output wavelength covering the infrared penetrating window of most biological tissues at 1,200-1,250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1,230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or three-photon excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1,230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.     

Multiphoton imaging with a novel compact diode‐pumped Ti:sapphire oscillator

Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal‐footprint Ti:sapphire oscillator, pumped by a frequency‐doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire‐based systems for high‐quality multiphoton imaging at a significantly size and weight compared to current systems will become possible. Microsc. Res. Tech. 78:1154–1158, 2015. © 2015 Wiley Periodicals, Inc.     

Dimensional metrology of lab‐on‐a‐chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in‐house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser‐scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three‐dimensional metrology of critical internal structures in lab‐on‐a‐chip devices because scans can be performed rapidly and noninvasively prior to their use.