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May/June 2009      BioOpticsWorld.com




          Advances in lasers, optics, and
            imaging for the life sciences

Also:
Deep, hi-res




                   Hyperspectral
mesoscopic
imaging in vivo

The right stuff
for Raman-based



                   microscopy
cancer diagnosis

Super-resolution
optical
microscopy

Bioimaging
pioneer
                         more info per view
Sunney Xie

Stimulus funding
for biophotonics

Deeper cancer
detection with
SORS

Unmasking
kidney-stone
development

Quantum OCT
hyperspectral imaging

By James Beach




A richer view of
bio structures
Hyperspectral microscopy combines disparate
methodologies to produce a data‑rich
view of biological structures useful in
research and clinical applications.




T
             he value of hyperspectral
             microscopy for life scientists
             is the ability to acquire the
             optical spectrum of all points
in a microscope image, coupled with spe-
cialized spectral analysis. The approach
produces uniquely rich views of biolog-
ical tissue, yielding revelations for both
research and clinical applications. For
instance, hyperspectral imaging (HSI)
can distinguish normal, precancerous,
and cancerous cervical cells on Pap-test
slides based on the combination of their
morphological and spectral characteris-
tics, as a prelude to development of pre-
screening tests for more efficient cervi-      FIGURE 1. The HSI microscope system includes a smooth spectrum halogen light source, spectral
cal-cancer diagnosis.                          imager (Headwall Photonics), color camera (Dage-MTI, Michigan City, IN) and automated stage (Prior
                                               Scientific). Dual monitors allow the image and spectral data to appear on separate screens.
methodology and instrumentation
Hyperspectral microscopy grew out of           surfaces of distant targets like the Earth’s       liquid-crystal and acousto-optic filters
two unrelated disciplines born in the          surface below an aircraft.                         increased the speed and number of
1970s and 1980s: microspectrophotom-             The disciplines began to merge when              wavelengths, and added programming
etry and spectral remote sensing. The          cell scientists turned to digital cameras          flexibility to spectral sequences. As far
former is well known to cell researchers       and image-analysis software designed               back as the early 1980s, flat-field spectro-
for following photochemical reactions          for image arrays. It became possible,              graphs using holographic gratings could
and revealing properties of components         with technologies like the filter wheel,           faithfully reproduce the spectrum of all
within cells’ interior. The latter was a       to rapidly change wavelengths while                points along the spectrograph slit, cre-
NASA creation for capturing and inter-         taking a series of pictures to create              ating one-dimensional spectral images.
preting spectral information from the          spectral images. Electronically tuned              When the scene is scanned across the
                                                                                                  spectrograph slit, a two-dimensional
James Beach is president of Willis Optics and associate professor at Louisiana State              hyperspectral image (a hypercube with
University Health Sciences Center (New Orleans, LA). He was the lead developer for the            two spatial dimensions and a third spec-
CytoViva hyperspectral microscope. Contact him at eadeae@gmail.com; www.cytoviva.com.             tral dimension) is produced.
                 Reprinted with revisions to format, from the May/June 2009 edition of BioOPTICS WORLD
                                           Copyright 2009 by PennWell Corporation
hyperspectral imaging



   For microscopists, scanning was already in place with motor-               ing researchers
ized stages. The remaining problem was to integrate all of the                a much brighter
hardware and software components into a system for hyper-                     view of nanoscale
spectral microscopy, and aim this at a market that would ben-                 str uctures than
efit from the power of the technology.                                        is available with
                                                                              other methods.
hsi for bio                                                                   It provides annu-
In early 2008, CytoViva (Auburn, AL) saw the opportunity for lar structured illu-
commercial hyperspectral microscopy to serve the growing mination at a low
research in nanomedicine. The company worked with Head- angle of incidence
wall Photonics (Fitchburg, MA) to incorporate its VIS-to-near- onto the sample
                                              infrared (IR) spectro- located just above
                                                 Intensity
                                              graphic camera to enable the condenser. The
                                              sensitivity over the visi- TiO2 spectral profilesfield
                                                2500                          resulting dark
                                              ble and near-IR spectral of illuminat ion
                                                2000
                                              ranges that have been is approximately
                                                1500
                                              reported for nanoparti- 150 times brighter
                                                1000
                                              cle applications. ITT-VIS t han is possi-
                                                  500
                                              (Visual Information Solu- ble with conven-
                                              tions of Boulder, CO), tional dark field,
                                                            500    600    700    800   900 1000
                                              developer of remote sens- and can effectively
                                                                    Wavelength (nm)
 Intensity                                                 ing software, scatter light from
                               TiO2 spectral profiles
                                                           supplied the nanoparticles with
2500                                                       sophist icated enough brightness 1000
2000                                                       spectral-anal- to enable the cap-
1500                                                       ysis features ture of spectral              800

1000                                                       of its ENVI information. Vir-               600
  500                                                      software. And tually all the light
                                                           Bruxton (Seat- that is collected has        400
            500      600    700      800    900 1000 tle, WA) pro- interacted multiple
                      Wavelength (nm)                                                                  200
                                                           vided its SIDX times with sample
FIGURE 2. The presence of TiO nanoparticles                device inter- components and
                                   2                                                                                  500         600          700
becomes clear following intradermal injection              face product to carries the unique                             Wavelength
into mouse skin; bright spots indicate clusters of         add instrument spect r a l       sig na -
                                                                                                     FIGURE 3. Hyperspectral microscopy has proven
the highly reflective particles (top). The spectral        controls, con- tures of the sam-
                                                                                                     helpful for investigating Anthrax spores, (top)
signature of TiO extracted from pixel groups in            verting the file- ple constituents.
                  2,                                                                                 shown at 100X; pixels matching the Anthrax
the HSI image with ENVI software, resembles shark input orienta- The smooth spec- library spectra are pseudo colored in red (center).
fins, and is distinct from that of other particles         tion of ENVI tral output from a
                                                                                                     Anthrax reference spectra, obtained from
(bottom). The spectral peak wavelength of TiO              into an inte- halogen source is
                                                   2                                                 different regions of the spore, can help to identify
nanoparticles appears to depend on the number of gral part of the used to avoid prob- strains and possible sources (bottom).
particles in clusters.                                     product.           lems with spectral
                                                              The group analysis when line structure is present, as it is with commonly
designed the hyperspectral imagery (HSI) microscope sys- used mercury, xenon, and metal halide sources.
tem to work with bright- and dark-field transmission modes,                      Visible and near-infrared wavelengths between 400 and
and with incident light for reflectance and epifluorescence. 1000 nm are resolved with an imaging spectrograph contain-
It includes the spectral detector, a second color camera, an ing an original holographic grating, and recorded at 12-bit
automated stage, a halogen light source and dual monitors, depth. Hyperspectral images are produced by moving the tar-
which are integrated with a research microscope (Fig. 1). An get under the microscope objective to the position sampled by
optional live chamber mounts to the microscope stage so the spectrograph slit, and recording the spectra of all points
researchers can examine living cells in real time at high res- along the line onto the digital camera. The target is then
olution. By November 2008, two CytoViva HSI microscopes moved a very short distance using the automated stage (from
were delivered to government research facilities at the FDA Prior Scientific, Boston) to bring the adjacent region of the
and USDA.                                                                     target to the recording position. The process repeats until the
   The hyperspectral microscope is designed to take full area of interest surrounding the target has been recorded.
advantage of its high-intensity dark-field illuminator, giv- The resulting HSI data are represented as a three-dimen-
hyperspectral imaging



sional structure that holds a stack of      spectral imaging can also serve as a tool   it is possible to quantify particle abun-
conventional two-dimensional images,        for “data mining” to determine optimal      dance and cluster size.
each containing a narrow band of wave-      wavelengths and recording conditions           Similar methods have been demon-
lengths that collectively cover the spec-   for specific applications.                  strated for using captured spectra to
tral range.                                                                             identify strains and possible sources of
   The result is the ability to acquire     sample applications                         Anthrax spores (Fig. 3). Since there is a
spectral information from samples in        Applications for the HSI microscopy         growing need to increase biofuels pro-
a way that allows one to separate the       system include nanotoxicology, drug         duction from nonfood sources such as
unique spectral features of a target mol-   delivery, and biomass analytics. At the     forest products, microbial activity dur-
ecule or other object from background       FDA’s National Center for Toxicological     ing the fermentation process is being
spectra in a sample, or to unmix the        Research, Drs. Neera Gopee and Paul         investigated with HSI methods. Cellu-
spectral information from single pixels.    Howard realized the approach could          losic, hemicellulosic, and lignin com-
Objects such as gold, silver, and TiO       provide benefits as a primary detection     ponents can be identified spectrally in
                                        2
nanoparticles, carbon nanotubes, flu-       tool in research on dermal penetration      thin slices of plant materials.
orescent probes, quantum dots, and          of topically applied formulations includ-      Additional uses now being investi-
many endogenous components of cells         ing nanoparticles. They found HSI help-     gated include targeted drug delivery
and biomass have their own unique           ful for quantifying nanomaterials in tis-   with nanoparticles and quantum dots,
spectral features that can be identi-       sue samples based on their unique spec-     and tumor-cell differentiation. In diag-
fied. The hyperspectral image contains      tral signatures (see Fig. 2) The approach   nostic imaging, HSI is being combined
these signatures within pixels associ-      provided a relatively simple and quick      with colonoscopy, ocular funduscopy,
ated with distinct objects in the image.    quantitative method for screening sam-      and body scanning for detection of can-
With the image viewer, individual pix-      ples prior to using more time-consum-       cer and eye disease, and examination of
els of objects can be selected and the      ing methods such as inductively cou-        skin ulcers. We expect HSI microscopy
spectra of those pixels saved to a data-    pled plasma mass spectrometry or elec-      will begin to play a significant role in
base. The spectra are used with spectral    tron microscopy. By collecting spectra      new contributions to basic and applied
classification methods in the software to   of particles dispersed in liquid media,     research, in a wide range of life-science
determine the number and locations of
similar objects in other samples. Hyper-
                                            and then looking for these spectra in
                                            hyperspectral images of treated tissues,
                                                                                        disciplines.«




                         For More Information: Kelly Marino · 888-737-3130 · kelly.marino@cytoviva.com
                                  300 North Dean Road · Suite 5 – PMB 157 · Auburn, AL 36830
                                                      www.cytoviva.com

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上海必和 Bio optics-5-09超光谱高光谱多光谱

  • 1. May/June 2009 BioOpticsWorld.com Advances in lasers, optics, and imaging for the life sciences Also: Deep, hi-res Hyperspectral mesoscopic imaging in vivo The right stuff for Raman-based microscopy cancer diagnosis Super-resolution optical microscopy Bioimaging pioneer more info per view Sunney Xie Stimulus funding for biophotonics Deeper cancer detection with SORS Unmasking kidney-stone development Quantum OCT
  • 2. hyperspectral imaging By James Beach A richer view of bio structures Hyperspectral microscopy combines disparate methodologies to produce a data‑rich view of biological structures useful in research and clinical applications. T he value of hyperspectral microscopy for life scientists is the ability to acquire the optical spectrum of all points in a microscope image, coupled with spe- cialized spectral analysis. The approach produces uniquely rich views of biolog- ical tissue, yielding revelations for both research and clinical applications. For instance, hyperspectral imaging (HSI) can distinguish normal, precancerous, and cancerous cervical cells on Pap-test slides based on the combination of their morphological and spectral characteris- tics, as a prelude to development of pre- screening tests for more efficient cervi- FIGURE 1. The HSI microscope system includes a smooth spectrum halogen light source, spectral cal-cancer diagnosis. imager (Headwall Photonics), color camera (Dage-MTI, Michigan City, IN) and automated stage (Prior Scientific). Dual monitors allow the image and spectral data to appear on separate screens. methodology and instrumentation Hyperspectral microscopy grew out of surfaces of distant targets like the Earth’s liquid-crystal and acousto-optic filters two unrelated disciplines born in the surface below an aircraft. increased the speed and number of 1970s and 1980s: microspectrophotom- The disciplines began to merge when wavelengths, and added programming etry and spectral remote sensing. The cell scientists turned to digital cameras flexibility to spectral sequences. As far former is well known to cell researchers and image-analysis software designed back as the early 1980s, flat-field spectro- for following photochemical reactions for image arrays. It became possible, graphs using holographic gratings could and revealing properties of components with technologies like the filter wheel, faithfully reproduce the spectrum of all within cells’ interior. The latter was a to rapidly change wavelengths while points along the spectrograph slit, cre- NASA creation for capturing and inter- taking a series of pictures to create ating one-dimensional spectral images. preting spectral information from the spectral images. Electronically tuned When the scene is scanned across the spectrograph slit, a two-dimensional James Beach is president of Willis Optics and associate professor at Louisiana State hyperspectral image (a hypercube with University Health Sciences Center (New Orleans, LA). He was the lead developer for the two spatial dimensions and a third spec- CytoViva hyperspectral microscope. Contact him at eadeae@gmail.com; www.cytoviva.com. tral dimension) is produced. Reprinted with revisions to format, from the May/June 2009 edition of BioOPTICS WORLD Copyright 2009 by PennWell Corporation
  • 3. hyperspectral imaging For microscopists, scanning was already in place with motor- ing researchers ized stages. The remaining problem was to integrate all of the a much brighter hardware and software components into a system for hyper- view of nanoscale spectral microscopy, and aim this at a market that would ben- str uctures than efit from the power of the technology. is available with other methods. hsi for bio It provides annu- In early 2008, CytoViva (Auburn, AL) saw the opportunity for lar structured illu- commercial hyperspectral microscopy to serve the growing mination at a low research in nanomedicine. The company worked with Head- angle of incidence wall Photonics (Fitchburg, MA) to incorporate its VIS-to-near- onto the sample infrared (IR) spectro- located just above Intensity graphic camera to enable the condenser. The sensitivity over the visi- TiO2 spectral profilesfield 2500 resulting dark ble and near-IR spectral of illuminat ion 2000 ranges that have been is approximately 1500 reported for nanoparti- 150 times brighter 1000 cle applications. ITT-VIS t han is possi- 500 (Visual Information Solu- ble with conven- tions of Boulder, CO), tional dark field, 500 600 700 800 900 1000 developer of remote sens- and can effectively Wavelength (nm) Intensity ing software, scatter light from TiO2 spectral profiles supplied the nanoparticles with 2500 sophist icated enough brightness 1000 2000 spectral-anal- to enable the cap- 1500 ysis features ture of spectral 800 1000 of its ENVI information. Vir- 600 500 software. And tually all the light Bruxton (Seat- that is collected has 400 500 600 700 800 900 1000 tle, WA) pro- interacted multiple Wavelength (nm) 200 vided its SIDX times with sample FIGURE 2. The presence of TiO nanoparticles device inter- components and 2 500 600 700 becomes clear following intradermal injection face product to carries the unique Wavelength into mouse skin; bright spots indicate clusters of add instrument spect r a l sig na - FIGURE 3. Hyperspectral microscopy has proven the highly reflective particles (top). The spectral controls, con- tures of the sam- helpful for investigating Anthrax spores, (top) signature of TiO extracted from pixel groups in verting the file- ple constituents. 2, shown at 100X; pixels matching the Anthrax the HSI image with ENVI software, resembles shark input orienta- The smooth spec- library spectra are pseudo colored in red (center). fins, and is distinct from that of other particles tion of ENVI tral output from a Anthrax reference spectra, obtained from (bottom). The spectral peak wavelength of TiO into an inte- halogen source is 2 different regions of the spore, can help to identify nanoparticles appears to depend on the number of gral part of the used to avoid prob- strains and possible sources (bottom). particles in clusters. product. lems with spectral The group analysis when line structure is present, as it is with commonly designed the hyperspectral imagery (HSI) microscope sys- used mercury, xenon, and metal halide sources. tem to work with bright- and dark-field transmission modes, Visible and near-infrared wavelengths between 400 and and with incident light for reflectance and epifluorescence. 1000 nm are resolved with an imaging spectrograph contain- It includes the spectral detector, a second color camera, an ing an original holographic grating, and recorded at 12-bit automated stage, a halogen light source and dual monitors, depth. Hyperspectral images are produced by moving the tar- which are integrated with a research microscope (Fig. 1). An get under the microscope objective to the position sampled by optional live chamber mounts to the microscope stage so the spectrograph slit, and recording the spectra of all points researchers can examine living cells in real time at high res- along the line onto the digital camera. The target is then olution. By November 2008, two CytoViva HSI microscopes moved a very short distance using the automated stage (from were delivered to government research facilities at the FDA Prior Scientific, Boston) to bring the adjacent region of the and USDA. target to the recording position. The process repeats until the The hyperspectral microscope is designed to take full area of interest surrounding the target has been recorded. advantage of its high-intensity dark-field illuminator, giv- The resulting HSI data are represented as a three-dimen-
  • 4. hyperspectral imaging sional structure that holds a stack of spectral imaging can also serve as a tool it is possible to quantify particle abun- conventional two-dimensional images, for “data mining” to determine optimal dance and cluster size. each containing a narrow band of wave- wavelengths and recording conditions Similar methods have been demon- lengths that collectively cover the spec- for specific applications. strated for using captured spectra to tral range. identify strains and possible sources of The result is the ability to acquire sample applications Anthrax spores (Fig. 3). Since there is a spectral information from samples in Applications for the HSI microscopy growing need to increase biofuels pro- a way that allows one to separate the system include nanotoxicology, drug duction from nonfood sources such as unique spectral features of a target mol- delivery, and biomass analytics. At the forest products, microbial activity dur- ecule or other object from background FDA’s National Center for Toxicological ing the fermentation process is being spectra in a sample, or to unmix the Research, Drs. Neera Gopee and Paul investigated with HSI methods. Cellu- spectral information from single pixels. Howard realized the approach could losic, hemicellulosic, and lignin com- Objects such as gold, silver, and TiO provide benefits as a primary detection ponents can be identified spectrally in 2 nanoparticles, carbon nanotubes, flu- tool in research on dermal penetration thin slices of plant materials. orescent probes, quantum dots, and of topically applied formulations includ- Additional uses now being investi- many endogenous components of cells ing nanoparticles. They found HSI help- gated include targeted drug delivery and biomass have their own unique ful for quantifying nanomaterials in tis- with nanoparticles and quantum dots, spectral features that can be identi- sue samples based on their unique spec- and tumor-cell differentiation. In diag- fied. The hyperspectral image contains tral signatures (see Fig. 2) The approach nostic imaging, HSI is being combined these signatures within pixels associ- provided a relatively simple and quick with colonoscopy, ocular funduscopy, ated with distinct objects in the image. quantitative method for screening sam- and body scanning for detection of can- With the image viewer, individual pix- ples prior to using more time-consum- cer and eye disease, and examination of els of objects can be selected and the ing methods such as inductively cou- skin ulcers. We expect HSI microscopy spectra of those pixels saved to a data- pled plasma mass spectrometry or elec- will begin to play a significant role in base. The spectra are used with spectral tron microscopy. By collecting spectra new contributions to basic and applied classification methods in the software to of particles dispersed in liquid media, research, in a wide range of life-science determine the number and locations of similar objects in other samples. Hyper- and then looking for these spectra in hyperspectral images of treated tissues, disciplines.« For More Information: Kelly Marino · 888-737-3130 · kelly.marino@cytoviva.com 300 North Dean Road · Suite 5 – PMB 157 · Auburn, AL 36830 www.cytoviva.com