Imaging spectrometer fundamentals for researchers in the biosciences—A tutorial

Abstract

Over the last 2 years there has been a dramatic increase in the number of bioscience laboratories using wavelength dispersive spectroscopy to study in vivo, in situ fluorescence. Transforming spectral information into an image provides a graphic means of mapping localized ionic, molecular, and protein-protein interactions. Spectroscopy also enables fluorophores with overlapping spectral features to be delineation. In this study, we provide the tools that a researcher needs to put into perspective instrumental contributions to a reported spectrum in order to gain greater understanding of the natural emission of the sample. We also show how to deduce the basic capabilities of a spectral confocal system. Finally, we show how to determine the true spectral bandwidth of an object, the illuminated area of a laser-excited object, and what is needed to optimize light throughput. Key terms: spectral imaging; spectrometer; spectrograph; PARISS; hyperspectral imaging; confocal; spectrometer design; wavelength calibration; spectral calibration The use of spectroscopy has greatly simplified the task of characterizing and delineating autofluorescence, natural fluorophores, and multiple man-made fluorophores, many with overlapping spectral profiles, in the same sample. Consequently, spectroscopy is one of the fastest growing techniques to be found in a bioscience laboratory (1,2). It is also one of the least understood, especially when both spectral and spatial information is required. In this study, we focus on wavelength dispersive devices rather than those that acquire spectra sequentially by changing bandpass filters. The transformation of wavelength information into an image is often called hyperspectral or multispectral imaging, but these terms are so blurred that, given the current state of technology, using the simple term ''spectral imaging'' is appropriate.This study provides the researcher with the tools to understand how spectrometers work, and how the limits of instrument performance can affect the accuracy, quality, validity, and interchangeability of acquired data. Spectrometers operate with multiple variables that have a significant influence on bandpass, wavelength dispersion, aberrations, and light throughput. To complicate matters further not all spectrometers work well with linear arrays or charge coupled devices (CCD) as a wavelength detectors. We try to put all these factors into perspective.To provide background we also describe how readily available commercial, plane grating, concave holographic grating, and advanced prism-based spectrometers work, and discuss their inherent limitations and advantages.The goal is to help a researcher optimize light throughput, check accuracy, and understand the real consequences of changing aperture sizes (such as a pinhole in a confocal system) on spectroscopic performance. When comparing spectroscopic results with those of others, it is important to understand that in some spectrometers spectral resolution degrades with an increase in the ratio of magnification to num...

High sensitivity non‐invasive detection of calcifications deep inside biological tissue using Transmission Raman Spectroscopy

“…We have adopted a conceptual design exhibiting exceptionally high detection efficiency. Typically the major bottleneck in collection efficiency in these applications is the etendue of spectrometer [46]. In our design we have increased the etendue of our spectrograph by increasing its slit width (by approximately an order of magnitude) in conjunction with increasing its dispersion (x3.3) to counteract the degradation of spectral resolution.…”

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”

“…The lamp is simply positioned on the microscope stage above the objective lens in an inverted microscope or below the objective lens in an upright microscope. The characteristics of an acquired spectrum enable us to measure wavelength accuracy, spectral sensitivity, contrast, wavelength ratios, and spectral resolution (1,2,5,6,18). The lamp is used to compare the performance of one instrument over time or against another similar instrument at a different location.…”

Advanced Optical Imaging of Endocytosis

“…Lerner provides a comprehensive tutorial covering the general principles of imaging spectrometers applicable to both fluorescence and Raman modalities and the reader is referred there for additional information (Lerner, 2006).…”