Dynamic Molecular Imaging of Single Cells in Their Native Environments
Three concurrent projects we have investigated over the past five years include the use of
attenuated total internal reflectance (ATR) infrared microspectroscopy as a detection method for
chromatographic systems (1-3), the use of infrared imaging to study the pathology of kidney
disease (4-10), and the use of infrared array based spectrographs for real-time infrared imaging
(11,12). Our goal over the next five years is to combine all of these methods in an effort to
conduct dynamic molecular imaging of single cells in their native environments. The ability to
do so could provide a wealth of information on chemical events that take place during a cells
growth cycle, its defense mechanism and its interaction with specific drugs. In addition to the
study of single cells, the developed method should yield significant benefits in the fields of
geology, bio-geology, medicine and chemistry.
Attenuated total internal reflectance infrared microspectroscopy is a combination of immersion
microscopy and evanescent wave infrared spectroscopy. Microscopists often employ immersion
methods to improve the spatial resolution of optical microscopes. In these cases the immersion
medium is an oil, which in effect reduces the wavelength of the light by a factor equal to the
refractive index of the immersion medium (noil =1.48). In our infrared studies we have been able
to immerse the sample in germanium (nGe =4.0) effectively increasing the spatial resolution by a
factor of four. Figure 1 shows the experimental setup, which involves placing the sample in
intimate contact with and beneath a germanium hemisphere. In addition, the infrared radiation is
directed into the hemisphere beyond the critical angle such that it experiences total internal
reflection at the Ge/sample interface. In doing so, an evanescent wave is created that extends
into the sample only ~ 0.2 to 1 µm (dp). The three dimensional shape of this evanescent wave is
a cone whose diffraction limited volume is 0.2 femto-liters at 6.0 µ wavelength.
Most optical detection schemes employed for liquid chromatography and capillary
electrophoresis suffer from the fact that the detector volume is significantly larger than the
sample volume. In our detector, the volume is equal to or smaller than the analyte volume. By
exploiting this very small sample volume we have been able to identify and detect moderate
infrared absorbing compounds in aqueous solution down to 20 ppm. Corresponding mass
detection limits are in the femto-gram regime.
Infrared in
Infrared out
dp
Analyte
HPLC
column
Mobile
phase flow
Figure 1. ATR Infrared Microspectroscopy for Chromatographic Detection, terminus of the LC or CE capillary is
placed at the sample position.
Another major effort in our laboratory has been the elucidation of the chemistry associated with
kidney stone formation. This study is being done in collaboration with pathologists at Indiana
University Medical School in Indianapolis. The major benefit in this study is that infrared
analysis provides direct molecular identification of the materials under investigation. In
addition, the method is not subjective like many of the current disease detection methods (visual
optical analysis) used by pathologists. Figure 2 shows a cross sectioned-kidney stone and the
analyzed area (red box) in which over 130,000 spectra were collected. These spectra were then
employed to generate molecular specific images (chemical maps) of hydroxyapatite and calcium
oxalate. Pathologists initially identified the stone composition as hydroxyapatite. However,
infrared analysis identified two additional components which included the monohydrate and
dihydrate forms of calcium oxalate. The images clearly show that the individual’s chemistry was
modulated due to the fact the nucleus of the stone is hydroxyapatite, which changes to calcium
oxalate and back on progressing radially outward to the edge of the stone. Since we know the
patient’s dietary history and blood chemistry, we are beginning to unravel the chemistry of stone
growth. This very simple example demonstrates the power of infrared microspectroscopy to
provide molecular markers for disease detection, which is currently a major thrust in many NIH
programs (13).
Figure 2. Photomicrograph of a cross-sectioned kidney stone (left) and molecular images based on hydroxyapatite
(center) and calcium oxalate (right).
The above example, although very elegant, required 8 hours of data
collection. By using array detectors and the ATR method mentioned
previously, we have the ability to generate molecular specific images
much more rapidly and in native environments (14). Figure 3
illustrates the surface image of a single human red blood cell
collected in ~ 10 minutes. The image is based on the peak area of
the amide I absorption located near 1650 cm-1 in the infrared
spectrum. There are approximately 12 pixels across the cell each
containing a full range spectrum. In theory, one could monitor the
Figure 3. Infrared Surface
Image of a Single Human Red
chemistry across the cell surface as it responds to different
physiological environments and/or adverse conditions. For example Blood Cell
there are several known infrared markers for breast, prostate and cervical cancers (15).
Although the above methods are relatively mature from an instrumental standpoint, one
drawback is the temporal resolution. Measurements in the mid-infrared spectral region continue
to be dominated by interferometers whose temporal resolution is directly tied to the modulation
efficiency of the detector and the interferometer mirror drive. For example, a typical mercury
cadmium telluride (MCT) detector operates best at an optical path difference velocity of 1-2
cm/sec. If 4 cm-1 resolution is required, the fastest spectral acquisition rate would be one
spectrum every 250-125 milliseconds.
More recently we have begun investigating the use of array detectors in combination with a
spectrograph and a microscope. In this system, the sample area defining aperture of the
microscope (confocal aperture) also serves as the entrance slit to the spectrograph (Figure 4).
The horizontal dimension of the slit defines the spectral resolution of the spectrograph and the
vertical dimension defines the area of the sample studied. A typical infrared image of a crosssectioned photographic film is shown in the right hand side of Figure 4. The cross-section is
composed of a 9 µm thick gelatin layer and a ~54 µm thick layer of polyvinylacetate. In this
image spectra are spread across the rows of pixels on the array and spatial information about the
sample is spread up and down a column of pixels. The color of a given pixel conveys the
intensity of an absorption at a specific wavelength. The image clearly shows the gelatin layer
with its characteristic amide I, II and A absorptions located at 1650, 1550 and 3300 cm-1,
respectively. In addition, one can clearly see the C=O and C-O-C stretch associated with the
polyvinyl acetate located at 1730 and 1240 cm-1, respectively. Spectra of each material have
been overlayed for clarity.
Figure 4. Optical view of spectrograph entrance slit and microscope area defining (confocal) aperture (left) and
infrared image of cross-sectioned photographic film (right).
What is most impressive about the image is that it is composed of 150,000 spectra collected in a
total of 10 seconds. The current version of the array detector we are using allows one to collect
data at frame rates (256 spectra/frame) of 1 KHz (i.e. 1 millisecond). New versions of detectors
allow frame rates of 1 MHz, i.e. 256 spectra per microsecond. In order to demonstrate the
fidelity of the current method, we have provided a real-time infrared spectral movie of the above
laminate being moved in and out of the focal region of the microscope (LAM2). No time
expansion has been used. It should also be noted that images (spectra) in the movie are raw
single beam images, which have not been ratioed against a background image. The current
version of software does not allow this option, which would dramatically improve the spectral
contrast. The movie is presented as collected and it clearly demonstrates the capabilities of the
method and the future possibilities.
One final movie that is included is that labeled “Flow”. The instrumental setup for this movie
was a flow cell similar to that described in the first page of this proposal. At the beginning of the
movie you will see three dark bands on the left of the image, which correspond (left to right) to
the O-H stretch of water, the C-H stretch of hexane, and the CO stretch of CO2. The cell is
initially filled with hexane and the water and CO2 are from atmospheric gases. As the movie
progresses, an injection of water is applied and as a result the C-H band disappears and the O-H
band darkens. Keep in mind that the volume of the cell is 10 nano-liters.
The focus of future research to be will be to combine the above methods with both laser trapping
and hydrodynamic trapping of single particles (ref. Figure 5). By trapping a single particle in the
evanescent wave at the focus of the infrared microscope, spectra can be collected in real-time to
reveal the chemistry taking place at the particles surface. Cherney et al. have done similar
studies using Raman microspectroscopy (16). In the case of a single cell, chemical events that
take place during a cells growth cycle, its defense mechanism, and its interaction with specific
drugs could readily be elucidated. Due to the fact that we are able to collect real-time
information, we can study the kinetic relationships associated with each process. In addition to
the study of single cells, the developed method should yield significant benefits in the fields of
geology, bio-geology, medicine and chemistry.
Our model system will focus on the processes associated with the nucleation of kidney stones.
With our current technology we have provided a wealth of information regarding the type of
stone and the general locations of stone formation within the kidney tissue. We have also
provided a wealth of information to those providing knock–out models for specific kidney
diseases. What we are lacking and what the new technology will be able to provide is, what
conditions actually trigger kidney stone growth. The study would involve trapping a model
tissue particle or mineral inclusion, after which the chemical environment of those particles
could be systematically changed. Infrared images and spectra would then be collected in real
time to reveal the surface chemistry as a function of time. Due to the fact that we are studying
the particles as they grow in their native environment, we eliminate any post processing
problems (e.g. sectioning etc.). More importantly, since the evanescent wave is only penetrating
the particle to a depth of at most 1 µm, we will be able to get spatial information at a resolution
which is better than most optical microscopes based in the visible region of the electromagnetic
spectrum.
Infrared in
Infrared out
Infrared in
Infrared out
Fluid
flow
Fluid
flow
Fluid
flow
Focused laser
Figure 5. Particle Trapping Schemes, hydrodynamic trapping (left) and laser trapping (right).
References
1. "Attenuated Total Internal Reflectance Infrared Microspectroscopy as a Detection Technique for Capillary
Electrophoresis", B. M. Patterson, N. D. Danielson and A. J. Sommer, Anal. Chem., 76(13), 3826-3832 (2004).
2. "Attenuated Total Internal Reflectance Infrared Microspectroscopy as a Detection Technique for High
Performance Liquid Chromatography", B. M. Patterson, N. D. Danielson and A. J. Sommer, Anal. Chem., 75(6),
1418-1424 (2003).
3. "Attenuated Total Internal Reflection (ATR) Infrared Microspectroscopy for the Analysis of Trace Solutes in
Aqueous Solutions", A. J. Sommer and Mark Hardgrove, Prominent Young Vibrational Spectroscopists, Vib.
Spectrosc., 24(1), 93-100 (2000).
4. "Analysis of Renal Stone Samples Using an Infrared Microspectroscopic Reflectance Imaging Technique", J. C.
Anderson, J. C. Williams, Jr., A. P. Evan, K. W. Condon, and A. J. Sommer, submitted to Urological Research,
August 2006.
5. "Nephrolithiasis and Nephrocalcinosis in Rats with Small Bowel Resection", R. C. O'Connor, A. P. Evan, S.
Meehan, E. Worcester, D. Kuznetsov, B. Laven, A. J. Sommer, S. B. Bledsoe, J. H. Parks, F. L. Coe and G. S.
Gerber, Urological Research: 33, 105-115 (2005).
6. "A Concerted Protocol for the Analysis of Mineral Deposits in Biopsied Tissue Using Infrared Microanalysis", J.
Anderson, J. Dellomo, A. J. Sommer, A. P. Evan, and S. Bledsoe, Urological Research 33: 213-219 (2005).
7. "Crystal Associated Nephropathy in Patients with Brushite Nephrolithiasis", A. P. Evan, J. E Lingeman, F. L.
Coe, Y. Shao, J. H. Parks, S. B. Bledsoe, A. J. Sommer, R. F. Paterson, R. L. Kuo, S. Kim and M. Grynpas, Kidney
International 67, 576-591 (2005).
8. "Helical Computed Tomography Accurately Reports Urinary Stone Composition using Attenuation Values: In
Vitro Verification using High-Resolution Micro-Computed Tomography Calibrated to Fourier Transform Infrared
Microspectroscopy", C. A. Zarse, J. A. McAteer, M. Tann, A. J. Sommer, S. C. Kim, R. F. Paterson, E. K. Hatt, J. E.
Lingeman, A. P. Evan, and J. C. Williams, Jr., Urology 63(5), 828-833 (2004).
9. "Nondestructive Analysis of Urinary Calculi Using Micro Computed Tomography", C. A. Zarse, J. A. McAteer,
A. J. Sommer, S. C. Kim, E. K. Hatt, J. E. Lingeman, A. P. Evan, J. C. Williams, Jr., BMC Urology, 4:15, 2004.
10. “Randall Plaque of Patients with Nephrolithiasis Begins in Basement Membranes of Thin Loops of Henle”, A.
P. Evan, J. E. Lingeman, J. H. Parks, S. M. Bledsoe, Y. Shao, A. Sommer, R. Paterson, R. L. Kuo, M. Grynpas
and F. L. Coe, The Journal of Clinical Investigation 111(5), 607-616 (2003).
11. “Infrared Microspectroscopy using a Planar Array Infrared Spectrograph”, Zachary Keltner, Katherine Kayima, Luis
Lavalle, Marina Canepa, Adam Lanzarotta, Andre’ J. Sommer, Gloria M. Story, Anthony E. Dowrey and Curtis Marcott,
Oral Presentation at the Pittsburgh Conference and Exposition, Orlando, Florida 2006.
12. “Rapid Infrared Microspectroscopy using an Infrared Spectrograph Based on a Prism Dispersing Element and
an MCT Array Detector” Zachary Keltner, Katherine Kayima, Luis Lavalle, Marina Canepa, Adam Lanzarotta,
Anthony E. Dowrey, Gloria M. Story, Curtis Marcott and Andre’ J. Sommer.
13. http://imaging.cancer.gov/researchfunding/current/currentfunding.
14.“Attenuated Total Internal Reflection Infrared Mapping Microspectroscopy Using an Imaging Microscope", A. J.
Sommer, L. G. Tisinger, C. M. Marcott and G. Story, Appl. Spectrosc., 55(3), 252-256 (2001).
15. “High Throughput Assessment of Cells and Tissues: Bayesian Classification of Spectral Metrics from Infrared
Vibrational Spectroscopic Imaging Data”., Bhargava, Rohit; Fernandez, Daniel C.; Hewitt, Stephen M.; Levin, Ira
W., Biochimica et Biophysica Acta, Biomembranes (2006), 1758(7), 830-845.
16. “Optical-trapping Raman Microscopy Detection of Single Unilamellar Lipid Vesicles”., Cherney, Daniel P.;
Conboy, John C.; Harris, Joel M.. Department of Chemistry, University of Utah, Salt Lake City, UT, USA.
Analytical Chemistry (2003), 75(23), 6621-6628.