Fourier transform multipixel spectroscopy for quantitative cytology

Abstract

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.

Spectral imaging: Principles and applications

“…One of these methods is Fourier spectroscopy (15,16), see Figure 3F. In this method there are no filters and the spectrum is measured by using the interference of light.…”

“…The interferogram is then Fourier-transformed in order to determine the spectrum. Figure 3F shows a Michelson interferometer but other interferometers, such as Sagnac, exist that have stability advantages (16). This method has the advantage that the intensity at each wavelength is collected throughout the whole duration of the measurement.…”

Advanced Optical Imaging of Endocytosis

“…Hyperspectral fluorescence microscopes typically use one of three approaches to generate spectrally-resolved information: (1) a prism or grating to disperse the fluorescence emission onto a linear detector array or a charge-coupled device (CCD) detector in point-scanning (Sinclair, et al, 2006) or line-scanning (Sinclair, et al, 2004) formats, (2) interferometric methods that measure the intensity as a function of optical path length difference and glean spectral information through Fourier analysis (Malik, et al, 1996), (3) sequential, narrow bandpass filter scanning of discrete wavelength regions using acousto-optical or liquid crystal tunable filter (Gat, 2000).…”

Chromatin Condensation in Erythropoiesis Resolved by Multipixel Spectral Imaging: Differentiation Versus Apoptosis

“…The inherent mechanical stability of this interferometer allows the Fourier technique to be successfully applied to the visible spectral region. The measurement is done by recording successive CCD frames in synchronization with the steps of the motor used to rotate the collimated beam, so that the instantaneous OPD is known for every pixel in every recorded frame and can be used in the FFT calculation (Malik et al 1996a). During a measurement (20 sec), each pixel of the CCD (512 ϫ 512) is collecting the interferogram, which is then Fourier transformed to give the spectrum.…”