We report the use of a thin-film transistor (TFT) twisted nematic liquid crystal spatial light modulator (SLM) for Hadamard transform two-dimensional spectral imaging in a fluorescence microscope. The liquid crystal SLM placed in the primary image plane of the microscope generates a set of spatial encoding masks defined by a cyclic S-matrix. The light passing through the mask is relayed by anamorphic optics to the entrance of an imaging spectrograph and detected with a charge-coupled device (CCD) camera. The SLM allows for the convenient generation of arbitrary masks without moving parts. The Hadamard transform approach transmits up to 50% of the light from the image field for multichannel detection, but is subject to transmission losses in the SLM. The system allows the convenient acquisition of two-dimensional spectral images. We characterized and tested many of the parameters controlling both spatial and spectral resolution, and demonstrated the system in the analysis of both naturally fluorescing and stained biological samples.

Spectral Imaging in a Programmable Array Microscope by Hadamard Transform Fluorescence Spectroscopy

Applications of Hadamard transform-gas chromatography/mass spectrometry to the detection of acetone in healthy human and diabetes mellitus patient breath

Publisher Summary This chapter describes the implications for quantitative microscopy. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its mutants constitute a class of fluorophores that has revolutionized the evaluation of molecular interactions and visualization of biological systems. When fused to proteins of interest and expressed in vivo , fluorescent proteins (FPs) act as versatile indicators of structure and function within cells and can be imaged with the full repertoire of fluorescence microscopy techniques. FPs and their constructs are finding increasing use in fluorescence lifetime imaging microscopy (FLIM) and fluorescence resonance energy transfer (FRET) modes of microspectroscopy for the elucidation of protein-protein interactions, signaling, and trafficking in cellular systems. The simple and the most common, application of FPs is as a passive marker fused to a target protein of interest for visualizing its spatiotemporal distribution. Mutations in and around the chromophore have systematic effects on the spectra. Green and red fluorescent proteins exhibit photophysical properties that are generally more complex than those of traditional fluorophores, for example, fluorescein and rhodamine.

Photophysics of green and red fluorescent proteins: implications for quantitative microscopy.

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.

Three-dimensional spectral imaging by hadamard transform spectroscopy in a programmable array microscope.

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N,N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced ∼10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1...

Zeng Xiaobin

Papers

Instrument for Hadamard transform three-dimensional fluorescence microscope image analysis