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.

Drs. Leavesley and Rich disclose financial interest in a start-up company, SpectraCyte LLC, founded to commercialize spectral imaging technology.

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><tbody><tr><td>Airway Smooth Muscle Cells</td><td>National Disease Research Interchange (NDRI)</td><td>Isolated from human lung tissues obtained from NDRI</td><td>Highly autofluorescent, calcium sensitive cells</td></tr><tr><td>Automated Shutter</td><td>Thorlabs Inc.</td><td>SHB1</td><td>Remote-controllable shutter to minimize photobleaching</td></tr><tr><td>Automated Stage</td><td>Prior Scientific</td><td>H177P1T4</td><td>Remote-controllable stage for automated multiple field of view or stitched image collection.</td></tr><tr><td>Automated Stage Controller (XY)</td><td>Prior Scientific</td><td>Proscan III (H31XYZE-US)</td><td>For interfacing automated stage with computer and joystick</td></tr><tr><td>Buffer</td><td>Made in-house</td><td>Made in-house</td><td>145 mM NaCl, 4 mM KCl, 20 mM HEPES, 10 mM D-glucose, 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, and 1mM CaCl&lt;sub&gt;2&lt;/sub&gt;, at pH 7.3</td></tr><tr><td>Cell Chamber</td><td>ThermoFisher Scientific</td><td>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><td>Excitation Filters</td><td>Semrock Inc.</td><td>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><td></td><td>Semrock Inc.</td><td>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><td></td><td>Semrock Inc.</td><td>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><td></td><td>Semrock Inc.</td><td>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><td></td><td>Semrock Inc.</td><td>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><td>Fluorescence Filter Cube Dichroic Beamsplitter</td><td>Semrock Inc.</td><td>FF495-Di03</td><td>Separates excitation and emission light at 495 nm (&gt;98% reflection between 350-488 nm, &gt;93% transmission between 502-950 nm), Filter effective index (1.78)</td></tr><tr><td>Fluorescence Filter Cube Longpass Filter</td><td>Semrock Inc.</td><td>FF01 496/LP-25</td><td>Allows passage of light longer than 496 nm ( &gt;93% average transmission between 503.2-1100 nm), Refractive index (1.86)</td></tr><tr><td>GCaMP Probe</td><td>Addgene</td><td>G-CaMP3; Plasmid #22692</td><td>A single-wavelength GCaMP2-based genetically encoded calcium indicator</td></tr><tr><td>Integrating Sphere</td><td>Ocean Optics</td><td>FOIS-1</td><td>Used for accurate measurement of wide-angle illumination</td></tr><tr><td>Inverted Fluorescence Microscope</td><td>Nikon Instruments</td><td>TE2000</td><td>Inverted microscopes allow direct excitation of sample without the need to penetrate layers of media and/or tissue.</td></tr><tr><td>Mitotracker Green FM</td><td>ThermoFisher Scientific</td><td>M7514</td><td>Labels mitochondria</td></tr><tr><td>NIST-Traceable Calibration Lamp</td><td>Ocean Optics</td><td>LS-1-CAL-INT</td><td>A lamp with a known spectrum for use as a standard</td></tr><tr><td>NIST-Traceable Fluorescein</td><td>ThermoFisher Scientific</td><td>F36915</td><td>For verifying appropriate spectral response of the system</td></tr><tr><td>NucBlue</td><td>ThermoFisher Scientific</td><td>R37605</td><td>Labels cell nuclei</td></tr><tr><td>Objective (10X)</td><td>Nikon Instruments</td><td>Plan Apo &amp;lambda; 10X/0.45 &amp;infin;/0.17 MRD00105</td><td>Useful for large fields of view</td></tr><tr><td>Objective (20X)</td><td>Nikon Instruments</td><td>Plan Apo &amp;lambda; 20X/0.75 &amp;infin;/0.17 MRD00205</td><td>Most often used for tissue samples</td></tr><tr><td>Objective (60X)</td><td>Nikon Instruments</td><td>Plan Apo VC 60X/1.2 WI &amp;infin;/0.15-0.18 WD 0.27</td><td>Most often used for cell samples</td></tr><tr><td>sCMOS Camera</td><td>Photometrics</td><td>Prime 95B (Rev A8-062802018)</td><td>For acquiring high-sensitivity digital images</td></tr><tr><td>Spectrometer</td><td>Ocean Optics</td><td>QE65000</td><td>Used to measure spectral output of excitation-scanning spectral system</td></tr><tr><td>Tunable Filter Changer</td><td>Sutter Instrument</td><td>Lambda VF-5</td><td>Motorized unit for automated excitation filter tuning/switching</td></tr><tr><td>Xenon Arc Lamp</td><td>Sunoptic Technologies</td><td>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...

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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 ...

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Research

JoVE Journal

Engineering

8.0K Views.  University of South Alabama. Dit protocol is belangrijk omdat het een methode beschrijft die kan worden gebruikt om verschillende fluorescerende signalen in één monster te scheiden. In vergelijking met traditionele methoden kan excitatiescannen de snelheid van het experiment verhogen en de gevoeligheid van de gegevens verbeteren. Door het gebruik van verschillende verlichtingsbronnen, zoals een supercontinuumlaser, kan deze methode van toepassing zijn op draaiende schijf- of laserscannende confocale microscoopsystemen....

Video: Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Biochemistry

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Bioengineering

Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based molecular single crystals. As representative example, we chose a single crystal of the heterodinuclear Ln-based complex [TbEu(bpm)(tfaa)6] (bpm=2,2&#8217;-bipyrimidine, tfaa&#8211; =1,1,1-trifluoroacetylacetonate) exhibiting bright visible emission under UV excitation. HSI is an emerging technique that combines 2-dimensional spatial imaging of a luminescent structure with spectral information from each pixel of the obtained image. Specifically, HSI on single crystals of the [Tb-Eu] complex provided local spectral information unveiling variation of the luminescence intensity at different points along the studied crystals. These changes were attributed to the optical anisotropy present in the crystal, which results from the different molecular packing of Ln3+ ions in each one of the directions of the crystal structure. The HSI herein described is an example of the suitability of such technique for spectro-spatial investigations of molecular materials. Yet, importantly, this protocol can be easily extended for other types of luminescent materials (such as micron-sized molecular crystals, inorganic microparticles, nanoparticles in biological tissues, or labelled cells, among others), opening many possibilities for deeper investigation of structure-property relationships. Ultimately, such investigations will provide knowledge to be leveraged into the engineering of advanced materials for a wide range of applications from bioimaging to technological applications, such as waveguides or optoelectronic devices.

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based ...

Chemistry

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

Immunology and Infection

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals

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