https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2803%2900521-0

Spectral imaging and its applications in live cell microscopy

Background: Spectral imaging extends the capabilities of biological and clinical studies to simultaneously study multiple features such as organelles and proteins qualitatively and quantitatively. Spectral imaging combines two well-known scientific methodologies, namely spectroscopy and imaging, to provide a new advantageous tool. The need to measure the spectrum at each point of the image requires combining dispersive optics with the more common imaging equipment, and introduces constrains as well. Methods and Results: The principles of spectral imaging and a few representative applications are described. Spectral imaging analysis is necessary because the complex data structure cannot be analyzed visually. A few of the algorithms are discussed with emphasis on the usage for different experimental modes (fluorescence and bright field). Finally, spectral imaging, like any method, should be evaluated in light of its advantages to specific applications, a selection of which is described. Conclusions: Spectral imaging is a relatively new technique and its full potential is yet to be exploited. Nevertheless, several applications have already shown its potential. q 2006 International Society for Analytical Cytology

http://www.cb.uu.se/~milan/histo/before2011august/GariniYoung06.pdf

Spectral imaging: Principles and applications

Multicolor fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles, or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using fluorescence resonance energy transfer (FRET). These techniques have become invaluable to all biologists from cytogeneticists to zoologists. Resolving multiple, co-localized chromosome probes and individual fluorescent species in corals share the common problem of spectral overlap. Even in simple systems where two or three fluorochromes are used, spectral overlap or cross-talk can be difficult to eliminate, limiting the ability to distinguish one signal from another with any confidence. However, this problem is not unique to the biologist; geologists who use remote sensing techniques have been struggling with the problem for a long time. In fact, their problems are actually worse because many geological spectra are very similar and the number of possible spectral classes is much larger than the number of fluorescent probes in a biology experiment. Nonetheless, these issues have been tackled successfully, and biologists have recently begun to employ these methods to decipher multiple spectral signatures within cells and organisms, a process known as emission fingerprinting. Here, we review some approaches that have been used to enhance spectral resolution in fluorescence microscopy.

/pdf/multi-spectral-imaging-and-linear-unmixing-add-a-whole-new-1eblcgxq0y.pdf

Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy.

Spectral imaging is a technology that integrates conventional imaging and spectroscopy to get both spatial and spectral information from an object. Although this technology was originally developed for remote sensing, it has been extended to the biomedical engineering field as a powerful analytical tool for biological and biomedical research. This review introduces the basics of spectral imaging, imaging methods, current equipment, and recent advances in biomedical applications. The performance and analytical capabilities of spectral imaging systems for biological and biomedical imaging are discussed. In particular, the current achievements and limitations of this technology in biomedical engineering are presented. The benefits and development trends of biomedical spectral imaging are highlighted to provide the reader with an insight into the current technological advances and its potential for biomedical research.

https://www.spiedigitallibrary.org/journals/journal-of-biomedical-optics/volume-18/issue-10/100901/Review-of-spectral-imaging-technology-in-biomedical-engineering--achievements/10.1117/1.JBO.18.10.100901.pdf

Review of spectral imaging technology in biomedical engineering: achievements and challenges

Cytogenetic analysis provides critical information of diagnostic and prognostic importance for haematological malignancies1,2. In fact, the identification of recurring chromosomal breakpoints in leukaemias and lymphomas has expedited the cloning of genes whose translocation-induced deregulation causes malignant transformation3,4. The pillar of karyotype analysis rests on chromosome banding techniques that have the distinct advantage that the entire genome can be analysed in a single experiment. However, poorly spread or contracted metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, common in tumour cell preparations, are often difficult to interpret unambiguously and subtle chromosomal aberrations, in particular the exchange of telomeric chromatin or small insertions remain elusive. Fluorescence in situ hybridization (FISH) overcomes some of these limitations, but is mainly utilized to confirm the presence of previously characterized or suspected aberrations5. We have developed a novel approach, termed spectral karyotyping or SKY6,7 based on the hybridization of 24 fluorescently labelled chromosome painting probes that allows the simultaneous and differential colour display of all human chromosomes. We have used SKY to complement conventional banding techniques in haematological malignancies by analysing 15 cases with unidentified chromosome aberrations. In all instances SKY provided additional cytogenetic information, including the identification of marker chromosomes, the detection of subtle chromosomal translocations and the clarification of complex chromosomal rearrangements. Thus, SKY in combination with standard chromosome banding allows the characterization of chromosomal aberrations in leukaemia with unprecedented accuracy.

Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104 pixels in a single cell. The net result was I(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food-digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.

Fourier transform multipixel spectroscopy for quantitative cytology

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Fourier transform multipixel spectroscopy for quantitative cytology