The authors would like to acknowledge support from NSF 1725937, NIH P01HL066299, NIH R01HL058506, NIH S10OD020149, NIH UL1 TR001417, NIH R01HL137030, AHA 18PRE34060163, and the Abraham Mitchell Cancer Research Fund.

1. Device set-up
<ol>
	<li>Light source: select a broad-band spectral light source with high power output and high collimation (a 300 W Xe arc lamp was used for these studies).</li>
	<li>Shutter (optional): add a shutter to the optical path to reduce photobleaching for time-lapse imaging.</li>
	<li>Tunable filter system: incorporate a mechanical tuning assembly and thin-film tunable filter (TFTF) set to enable the desired wavelength-adjustable excitation range (e.g., 360-485 nm).</li>
	<li>Microscope: use an inverted f...

The optimal use of an excitation-scanning hyperspectral imaging set-up begins with construction of the light path. In particular, choice of light source, filters (tunable and dichroic), filter switching method, and camera determine the available spectral range, possible scan speed, detector sensitivity, and spatial sampling. Mercury arc lamps offer many excitation wavelength peaks but do not provide a flat spectral output and will require significant signal reduction at the output peaks to correct the spectral image data...

Spectral imaging may be performed in a variety of ways and is referred to by several terms1,2,3,4. In general, spectral imaging refers to data acquired in at least two spatial dimensions and one spectral dimension. Multispectral and hyperspectral imaging are most often distinguished by the number of wavelength bands or whether the spectral bands are contiguous1. For this application, hyperspectral data is defined as spectral data acquired with contiguous wavelength bands achieved by spacing of center wavelengths no less than half the full width at half maximum (FWHM) of each bandpass filter used for excitation (i.e., 5 nm center wavelength spacing for bandpass filters with 14-20 nm bandwidths). The contiguous nature of the data bands allows for an oversampling of the dataset, ensuring that Nyquist criteria are satisfied when sampling the spectral domain.
Hyperspectral imaging was developed by NASA in the 1970s and 1980s in conjunction with the first Landsat satellite5,6. Collecting data from several contiguous spectral bands allowed the generation of a radiance spectrum of each pixel. Identifying and defining the radiance spectrum of individual components made it possible to not only detect surface materials by their characteristic spectra, but it also allowed for the removal of intervening signals, such as variations in the signal due to atmospheric conditions. The concept of detecting materials using their characteristic spectra was applied to biological systems in 1996 when Schr&#246;ck et al. used combinations of five different fluorophores and their known spectra to distinguish labeled chromosomes in a process termed spectral karyotyping7. This technique was elaborated upon in 2000 by Tsurui et al. for fluorescence imaging of tissue samples, using seven fluorescent dyes and singular value decomposition to achieve spectral separation of each pixel into linear combinations of spectra in the reference library8. Similar to their remote sensing counterparts, the contribution of each known fluorophore can be calculated from the hyperspectral image, given a priori information of the spectrum of each fluorophore.
Hyperspectral imaging has also been used in the areas of agriculture9, astronomy10, biomedicine11, chemical imaging12, environmental applications13, eye care14, food science15, forensic science16,17, medical science18, mineralogy19, and surveillance20. A key limitation of current fluorescence microscope hyperspectral imaging systems is that the standard hyperspectral imaging technology isolates fluorescence signals in narrow bands by 1) first filtering the excitation light to control sample excitation, then 2) further filtering emitted light to separate the fluorescence emission into narrow bands that can later be separated mathematically21. Filtering both the excitation illumination and emitted fluorescence reduces the amount of available signal, which lowers the signal-to-noise ratio and necessitates lengthy acquisition times. The low signal and lengthy acquisition times limit the applicability of hyperspectral imaging as a diagnostic tool.
An imaging modality has been developed that makes use of hyperspectral imaging but boosts the available signal, thereby reducing the necessary acquisition time21,22. This new modality, called excitation-scanning hyperspectral imaging, acquires spectral image data by varying the excitation wavelength and collecting a broad range of emitted light. It has been previously shown that this technique yields orders of magnitude increases in signal-to-noise ratio compared to emission scanning techniques21,22. The increase in signal-to-noise ratio is largely due to the wide bandpass (~600 nm) of emission light detected, while specificity is provided by filtering only the excitation light instead of the fluorescence emission. This allows all emitted light (for every excitation wavelength) to reach the detector21. Additionally, this technique can be used to discriminate autofluorescence from exogenous labels. Furthermore, the ability to reduce acquisition time due to increased detectable signal reduces the danger of photobleaching as well as allows spectral scans at an acquisition rate that is acceptable for spectral video imaging.
The goal of this protocol is to serve as a data acquisition guide for excitation-scanning hyperspectral imaging microscopy. In addition, descriptions are included that help to understand the light path and hardware. Also described is the implementation of open-source software for an excitation-scanning hyperspectral imaging microscope. Finally, descriptions are provided for how to calibrate the system to a NIST-traceable standard, adjust software and hardware settings for accurate results, and unmix the detected signal into contributions from individual components.

<table border="0" cellpadding="0" cellspacing="0" style="width:2119px;" width="2120">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Airway Smooth Muscle Cells</td>
			<td style="width:327px;">National Disease Research Interchange (NDRI)</td>
			<td style="width:360px;">Isolated from human lung tissues obtained from NDRI</td>
			<td style="width:1105px;">Highly autofluorescent, calcium sensitive cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Automated Shutter</td>
			<td style="width:327px;">Thorlabs Inc.</td>
			<td style="width:360px;">SHB1</td>
			<td>Remote-controllable shutter to minimize photobleaching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Automated Stage</td>
			<td style="width:327px;">Prior Scientific</td>
			<td style="width:360px;">H177P1T4</td>
			<td>Remote-controllable stage for automated multiple field of view or stitched image collection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Automated Stage Controller (XY)</td>
			<td style="width:327px;">Prior Scientific</td>
			<td style="width:360px;">Proscan III (H31XYZE-US)</td>
			<td>For interfacing automated stage with computer and joystick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Buffer</td>
			<td style="width:327px;">Made in-house</td>
			<td style="width:360px;">Made in-house</td>
			<td>145 mM NaCl, 4 mM KCl, 20 mM HEPES, 10 mM D-glucose, 1 mM MgCl2, and 1mM CaCl2, at pH 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Cell Chamber</td>
			<td style="width:327px;">ThermoFisher Scientific</td>
			<td style="width:360px;">Attofluor Cell Chamber, A7816</td>
			<td>Coverslip holder composed of surgical stainless steel and a rubber O-ring to seal in media and prevent sample and/or objective contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Excitation Filters</td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">TBP01-378/16</td>
			<td>Center wavelength range (340-378 nm), Bandwidth (Minimum 16 nm, nominal FWHM 20 nm), Refractive index (1.88)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;"></td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">TBP01-402/16</td>
			<td>Center wavelength range (360-400 nm), Bandwidth (Minimum 16 nm, nominal FWHM 20 nm), Refractive index (1.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;"></td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">TBP01-449/15</td>
			<td>Center wavelength range (400-448.8 nm), Bandwidth (Minimum 15 nm, nominal FWHM 20 nm), Refractive index (1.8)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;"></td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">TBP01-501/15</td>
			<td>Center wavelength range (448.8-501.5 nm), Bandwidth (Minimum 15 nm, nominal FWHM 20 nm), Refractive index (1.84)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;"></td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">TBP01-561/14</td>
			<td>Center wavelength range (501.5-561 nm), Bandwidth (Minimum 14 nm, nominal FWHM 20 nm), Refractive index (1.83)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Fluorescence Filter Cube Dichroic Beamsplitter</td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">FF495-Di03</td>
			<td>Separates excitation and emission light at 495 nm (&#62;98% reflection between 350-488 nm, &#62;93% transmission between 502-950 nm), Filter effective index (1.78)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Fluorescence Filter Cube Longpass Filter</td>
			<td style="width:327px;">Semrock Inc.</td>
			<td style="width:360px;">FF01 496/LP-25</td>
			<td>Allows passage of light longer than 496 nm ( &#62;93% average transmission between 503.2-1100 nm), Refractive index (1.86)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">GCaMP Probe</td>
			<td style="width:327px;">Addgene</td>
			<td style="width:360px;">G-CaMP3; Plasmid #22692</td>
			<td>A single-wavelength GCaMP2-based genetically encoded calcium indicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Integrating Sphere</td>
			<td style="width:327px;">Ocean Optics</td>
			<td style="width:360px;">FOIS-1</td>
			<td>Used for accurate measurement of wide-angle illumination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Inverted Fluorescence Microscope</td>
			<td style="width:327px;">Nikon Instruments</td>
			<td style="width:360px;">TE2000</td>
			<td>Inverted microscopes allow direct excitation of sample without the need to penetrate layers of media and/or tissue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Mitotracker Green FM</td>
			<td style="width:327px;">ThermoFisher Scientific</td>
			<td style="width:360px;">M7514</td>
			<td>Labels mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NIST-Traceable Calibration Lamp</td>
			<td style="width:327px;">Ocean Optics</td>
			<td style="width:360px;">LS-1-CAL-INT</td>
			<td>A lamp with a known spectrum for use as a standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NIST-Traceable Fluorescein</td>
			<td style="width:327px;">ThermoFisher Scientific</td>
			<td style="width:360px;">F36915</td>
			<td>For verifying appropriate spectral response of the system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NucBlue</td>
			<td style="width:327px;">ThermoFisher Scientific</td>
			<td style="width:360px;">R37605</td>
			<td>Labels cell nuclei</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Objective (10X)</td>
			<td style="width:327px;">Nikon Instruments</td>
			<td style="width:360px;">Plan Apo &#955; 10X/0.45 &#8734;/0.17 MRD00105</td>
			<td>Useful for large fields of view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Objective (20X)</td>
			<td style="width:327px;">Nikon Instruments</td>
			<td style="width:360px;">Plan Apo &#955; 20X/0.75 &#8734;/0.17 MRD00205</td>
			<td>Most often used for tissue samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Objective (60X)</td>
			<td style="width:327px;">Nikon Instruments</td>
			<td>Plan Apo VC 60X/1.2 WI &#8734;/0.15-0.18 WD 0.27</td>
			<td>Most often used for cell samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">sCMOS Camera</td>
			<td style="width:327px;">Photometrics</td>
			<td style="width:360px;">Prime 95B (Rev A8-062802018)</td>
			<td>For acquiring high-sensitivity digital images</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Spectrometer</td>
			<td style="width:327px;">Ocean Optics</td>
			<td style="width:360px;">QE65000</td>
			<td>Used to measure spectral output of excitation-scanning spectral system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Tunable Filter Changer</td>
			<td style="width:327px;">Sutter Instrument</td>
			<td style="width:360px;">Lambda VF-5</td>
			<td>Motorized unit for automated excitation filter tuning/switching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Xenon Arc Lamp</td>
			<td style="width:327px;">Sunoptic Technologies</td>
			<td style="width:360px;">Titan 300HP Lightsource</td>
			<td>Light source with relatively uniform spectral output</td>
		</tr>
	</tbody>
</table>

excitation-scanning hyperspectral imaging microscopy to efficiently discriminate fluorescence signals

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence signals were proven cumbersome until the advent of hyperspectral imaging, in which fluorescence sources can be separated from each other as well as from background signals and autofluorescence (given knowledge of their spectral signatures). However, traditional, emission-scanning hyperspectral imaging suffers from slow acquisition times and low signal-to-noise ratios due to the necessary filtering of both excitation and emission light. It has been previously shown that excitation-scanning hyperspectral imaging reduces the necessary acquisition time while simultaneously increasing the signal-to-noise ratio of acquired data. Using commercially available equipment, this protocol describes how to assemble, calibrate, and use an excitation-scanning hyperspectral imaging microscopy system for separation of signals from several fluorescence sources in a single sample. While highly applicable to microscopic imaging of cells and tissues, this technique may also be useful for any type of experiment utilizing fluorescence in which it is possible to vary excitation wavelengths, including but not limited to: chemical imaging, environmental applications, eye care, food science, forensic science, medical science, and mineralogy.

Several important steps from this protocol are necessary to ensure the collection of data that is both accurate and devoid of imaging and spectral artifacts. Skipping these steps may result in data that appear significant but cannot be verified or reproduced with any other spectral imaging system, thereby effectively nullifying any conclusions made with said data. Chief among these important steps is proper spectral output correction (section 3). The correction factor compensates for wave...

Watch this Scientific Journal Video about Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals at JoVE.com

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Spectral imaging has become a reliable solution for identification and separation of multiple fluorescence signals in a single sample and can readily distinguish signals of interest from background or autofluorescence. Excitation-scanning hyperspectral imaging improves on this technique by decreasing the necessary image acquisition time while simultaneously increasing the signal-to-noise ratio.

Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence ...

This protocol is significant as it describes a method which can be used to separate several fluorescent signals in a single sample. Compared to traditional methods, excitation-scanning can increase the speed of the experiment and improve the sensitivity of the data. By using different illumination sources such as a supercontinuum laser, this method may be applicable to spinning disk or laser scanning confocal microscope systems.When performing excitation-scannings, spectral imaging microscopy, it is important to check that the excitation peak of each fluorescent label occurs at a different wavelength. To begin, set up an inverted fluorescence microscope and camera as described in the text protocol. Then load the sample on the stage.From the drop-down menu of the micromanager main window, select 475 nanometers for initial sample viewing. Then double-click at the box next to exposure and enter 100 to set the exposure time at 100 milliseconds. Finally, click live to view the sample.Now click on Auto. The automatic intensity viewing range button to bring the minimum and maximum values into meaningful, visual ranges. Next, use the microscope's focus knobs to focus the sample.Once in focus, adjust the position of the sample so that it's edge is in the center of the field of view. Then fine-tune the focus. The sample is in focus when the edge features in the image appear sharp.Now, open the multi-dimensional acquisition tool. In the menu, click the load button and choose a preset setting containing an appropriate wavelength range for spectral acquisition. Once the acquisition settings are confirmed, move to a blank region of the sample and click acquire to collect a spectral image stack containing background and noise to use for subtraction later.Then, move about the sample to find the brightest regions. Samples are likely to have more intense fluorescence away from the edge of the sample. Once the brightest region is located, take a single image stack.Load the acquired image into Image J and use Image J's measure function to confirm that no wavelengths contain overexposed pixels. If a reduced exposure time is necessary due to overexposure, the background image should be retaken with a new exposure time as well. If any of the images were found to contain the upward detectional limit of the camera, adjust the exposure time of the spectral scan to ensure that the maximum signal throughout the spectral range does not exceed the dynamic range of the camera.With an appropriate exposure time now determined, place an unlabeled sample onto the stage. Acquire spectral image data from the unlabeled sample to determine if there is any underlying autofluoresence. Then, place a single-labeled sample on the stage.Acquire spectral image data from each of the single-labeled samples to use as spectral controls to build a spectral library. Perform the scan for each control sample using an identical wavelength range exposure time and camera settings. Next, place a slide containing the experimental sample on the microscope.Select a field of view that has appropriately labeled cells and acquire the spectral image data from the sample using the determined acquisition settings. Correct images to a flat spectral response by subtracting the background spectrum from the sample image and multiplying the image by the correction coefficient. Here, a MATLAB script is used to quickly process the images.Next, generate the spectral library. For best results, normalize each end member to the wavelength band of the greatest intensity by determining which wavelength band has the most intense value and dividing the measurement at each wavelength band by that maximum value. Then save the spectral data as a dat file.When complete, unmix the spectral image data. The unmixing step will generate an abundance image for each fluorescent label, where abundance is the amount of relative fluorescent signal in the image from the prospective label. Next, open each unmixed image to visually inspect the distribution of pure components.Compare the magnitude of the error image to those of the unmixed images. If the magnitude of the error image is similar to the unmixed abundance images, signals in the spectral image data may not be accurately accounted for. Finally, check that there are no unidentified residual structures by comparing the error image to the unmixed images.When performed correctly, the spectral unmixing process allows separation of a spectral image stack into respective contributions from each fluorescence label. Here, an unmixed spectral image set with individual images highlighting airway smooth muscle cell autofluorescence, a GCaMP probe and a mitochondrial label. There is high error associated with the nuclear and perinuclear regions of the cells indicating that the measured spectra of those regions are not well accounted for by the spectra in the spectral library.Pure spectra collected from labeled cells that are also autofluorescent will likely contaminate the spectral profile for the pure component. Here, one can see the difference of unmixing with pure spectra contaminated with autofluorescence, versus the corrected pure spectra after scaled subtraction. Proper data acquisition is essential when imaging sensitive samples as they may only survive a single imaging session unlike the background and controls which may be reimaged.Ensure that the proper hardware and acquisition settings are predetermined as much as possible. As a next step, videos created from time-lapse data acquired using this system can be used to track fluorescent sources in discreet locations over time such as second messenger signal localization. Take care when using powerful light sources to avoid eye exposure.Also, be sure to wear proper gloves when handling biological labels.

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7.9K Views.  University of South Alabama. Questo protocollo è significativo in quanto descrive un metodo che può essere utilizzato per separare diversi segnali fluorescenti in un singolo campione. Rispetto ai metodi tradizionali, l'eccitazione-scansione può aumentare la velocità dell'esperimento e migliorare la sensibilità dei dati. Utilizzando diverse fonti di illuminazione come un laser supercontinuo, questo metodo può essere applicabile ai sistemi di microscopio confocale a scansione laser o disco rotante.Quando si es...