Acousto-optic tunable filters (AOTF) and liquid crystal tunable filters (LCTF) are evaluated for their suitability as fluorescence microscopy imaging spectrometers. AOTFs are solid-state birefringent crystals that provide an electronically tunable spectral notch passband in response to an applied acoustic field. LCTFs also provide a notch passband that can be controlled by incorporating liquid crystal waveplate retarders within a Lyot birefringent filter. In this paper, spectroscopic performance and imaging quality are contrasted by evaluation of model systems. Studies include transmission imaging of standard resolution targets, multispectral fluorescence emission imaging of tagged polystyrene microspheres, and immunofluorescence imaging of neurotransmitters within rat-brainstem thin sections. In addition, the first use of LCTFs for Raman microscopy is demonstrated. Raman microscopy is a noninvasive spectral imaging technique that can provide chemically significant image contrast complementary to fluorescence microscopy without the use of stains or tags.

The past decade has seen an enormous increase in the number and breadth of imaging techniques developed for analysis in many industries, including pharmaceuticals, food, and especially biomedicine. Rather than accept single-dimensional forms of information, users now demand multidimensional assessment of samples. High specificity and the need for little or no sample preparation make Raman imaging a highly attractive analytical technique and provide motivation for continuing advances in its supporting technology and utilization. This review discusses the current tools employed in Raman imaging, the recent advances, and the major applications in this ever-growing analytical field.

An optical detection method, Raman chemical imaging spectroscopy (RCIS), is reported, which combines Raman spectroscopy, fluorescence spectroscopy, and digital imaging. Using this method, trace levels of biothreat organisms are detected in the presence of complex environmental backgrounds without the use of amplification or enhancement techniques. RCIS is reliant upon the use of Raman signatures and automated recognition algorithms to perform species-level identification. The rationale and steps for constructing a pathogen Raman signature library are described, as well as the first reported Raman spectra from live, priority pathogens, including Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Francisella tularensis, Brucella abortus, and ricin. Results from a government-managed blind trial evaluation of the signature library demonstrated excellent specificity under controlled laboratory conditions.

A Lyot-type liquid crystal tunable filter (LCTF) suitable for high-definition Raman chemical imaging has been developed. The LCTF has been incorporated into an efficient Raman imaging system that provides significant performance advantages relative to any previous approach to Raman microscopy. The LCTF and associated optical path is physically compact, which accommodates integration of the LCTF within an infinity-corrected optical microscope. The LCTF simultaneously provides diffraction-limited spatial resolution and 7.6-cm-1 spectral bandpass across the full free spectral range of the imaging spectrometer. The LCTF Raman microscope successfully integrates, in a facile manner, the utility of optical microscopy and the analytical capabilities of Raman spectroscopy. In this paper the LCTF Raman imaging system is described in detail, as well as results of initial studies of polymer and corrosion product model systems.