A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labels can be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcated organelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregated regions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern many fluorescence labels by their unique spectral properties, provided there is significant differentiation of their emission spectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reduction from separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaging have allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores. Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detection using excitation-scanning hyperspectral imaging. Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17 nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated and analyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels, spectral range of scan, spacing of center wavelengths, and acquisition time.
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Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging
In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF- 5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.
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- Award ID(s):
- 1725937
- NSF-PAR ID:
- 10107064
- Date Published:
- Journal Name:
- Proc. SPIE 10883, Three- Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 108831A
- Volume:
- 10883
- Page Range / eLocation ID:
- 45
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2510562
