Multi-Spectral Imaging and Linear Unmixing Add a Whole New Dimension to Laser Scanning Fluorescence Microscopy

Spectral imaging is a natural extension of the capabilities of confocal microscopes. The first confocal spectral imaging (CSI) instruments were able to acquire spectral data that allowed the emissions of overlapping fluorescent probes to be assigned to data channels representing a spectrum rather than a range of emission wavelengths. This marked a significant improvement over what could be done by channel series with standard confocal microscopes. However the performance of these earlier designs can fall short in one or more of the following areas; sensitivity, spectral resolution and reproducibility, acquisition speed, and unmixing accuracy. Nikon has recently introduced a new CSI instrument, C1si, that overcomes some of the more serious performance deficiencies of earlier designs through unique optical, electronic, and data handling advances. C1si uses a multianode photomultiplier tube (PMT) as the detector and typically acquires spectral data in a single scan. Sensitivity is enhanced over designs diffracting randomly polarized fluorescence by rotating the polarization of all emission photons to the S-plane, the plane for which the diffraction grating is most efficient. Three diffraction gratings are provided supporting wavelength sampling increments of 2.5, 5, and 10 nm. Improvements have been made in the digitization process to increase detection efficiency as well. C1si is calibrated to a high enough standard that it is possible to share and reproduce data between instruments. The algorithm implemented in the EZ-C1 software is able to accurately and repeatedly unmix fluorescent probes with emission peaks separated by as little as 5 nm. It is possible to unmix probes with emission peaks separated by 20 nm with a 10-1 brightness difference. Three probes can be unmixed with emission peaks contained within a 20 nm range. Acquisition is fast enough and the sensitivity is sufficient for C1si to acquire more than 100 frames of spectral time series data without serious photobleaching. q 2006 International Society for Analytical Cytology Key terms: confocal microscopy; spectral imaging; spectral unmixing; fluorescence; Nikon; calibration Confocal fluorescence microscopes were originally developed as a means to produce 3D images with greater information content, by minimizing the reduction in signal to noise caused by out of focus fluorescence common in widefield images. This is accomplished by illuminating and acquiring images through confocal or conjugate pinholes, thereby restricting the in-focus optical section thickness. Stacks of images could then be combined into volume renderings of exquisite clarity. The first confocal microscopes were developed by scientists in their own laboratories seeking solutions to imaging problems encountered in their research (1,2). Commercialization of the technology has expanded the capabilities of confocal microscopes to generate data in multiple fluorescence channels, in time series, and in series of temporally resolved stacks of images acquired at multiple stage coordinates, no...

Section: Limitations Of Previous Csi Systemsmentioning

confidence: 99%

The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed

Larson

2006

“…The results illustrated in Figure 5 and Table 1 show that, while each spectral mapping algorithm gave slightly different results, results were spatially and statistically similar, suggesting that spectral identification algorithms other than the previously used supervised classification (8,9) and LSU (3,4,(8)(9)(10)(11) will have utility for spectral microscopy. Moreover, these results showed that a full range of algorithms could be used to derive similar results, providing the analyst with a host of options for solving any particular spectral analysis problem.…”

Section: Discussionmentioning

confidence: 86%

“…The most widely used methods include both supervised classification techniques (8,9) and linear spectral unmixing (LSU) (3,4,(8)(9)(10)(11). All of these studies demonstrated success utilizing these techniques with spectral microscopy data.…”

Section: Society For Analytical Cytologymentioning

confidence: 99%

Spectral mapping tools from the earth sciences applied to spectral microscopy data

Harris¹

2006

Background:Spectral imaging, originating from the field of earth remote sensing, is a powerful tool that is being increasingly used in a wide variety of applications for material identification. Several workers have used techniques like linear spectral unmixing (LSU) to discriminate materials in images derived from spectral microscopy. However, many spectral analysis algorithms rely on assumptions that are often violated in microscopy applications. This study explores algorithms originally developed as improvements on early earth imaging techniques that can be easily translated for use with spectral microscopy.Methods:To best demonstrate the application of earth remote sensing spectral analysis tools to spectral microscopy data, earth imaging software was used to analyze data acquired with a Leica confocal microscope with mechanical spectral scanning. For this study, spectral training signatures (often referred to as endmembers) were selected with the ENVI (ITT Visual Information Solutions, Boulder, CO) “spectral hourglass” processing flow, a series of tools that use the spectrally over‐determined nature of hyperspectral data to find the most spectrally pure (or spectrally unique) pixels within the data set. This set of endmember signatures was then used in the full range of mapping algorithms available in ENVI to determine locations, and in some cases subpixel abundances of endmembers.Results:Mapping and abundance images showed a broad agreement between the spectral analysis algorithms, supported through visual assessment of output classification images and through statistical analysis of the distribution of pixels within each endmember class.Conclusions:The powerful spectral analysis algorithms available in COTS software, the result of decades of research in earth imaging, are easily translated to new sources of spectral data. Although the scale between earth imagery and spectral microscopy is radically different, the problem is the same: mapping material locations and abundances based on unique spectral signatures. © 2006 International Society for Analytical Cytology

“…In this procedure, using a Zeiss 510 META, a fluorescence spectrum comprising two scans of eight simultaneous channels (10.7 nm each) was made from 555 to 726 nm (43). The Zeiss 510 META system contained a 543 nm laser that was used for excitation of the LT dye and a 488 nm laser that was used for the excitation of the AIF in the tissue.…”

Section: Resultsmentioning

confidence: 99%

“…Spectra were usually recorded in two scans, each consisting of 8 bins of 10.7 nm regions. Spectra were recorded using Zeiss META software from a very small region of interest (ROI) that was made from an individual unaffected granulosa cell, a nucleolus, or an apoptotic granulosa cell (43). An 80/20 reflector was used for spectral analysis and a TD was used for morphology.…”

Section: Zeiss 510 Meta Systemmentioning

confidence: 99%

Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

Zucker

Jeffay

2006

Background:Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy.Methods:Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths.Results:Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image.Conclusions:Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut‐PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut‐PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality. © 2006 International Society for Analytical Cytology