Transcript Characterizing Autofluorescence in Rodent Tissue Using the Cryo-Imaging Method

Characterizing Autofluorescence in Rodent Tissue Using the Cryo-Imaging Method
Miriam Israelowitz1 and Dr. David L. Wilson2
1Department of Physics, Case Western Reserve University, Cleveland OH, 2Deparment
of Biomedical Engineering, Case Western Reserve University, Cleveland OH
Abstract
Fluorescent microscopy has become the method of choice when
imaging stem cell growth. One of the fastest growing science fields
today, stem cell imaging research exploits the ability of fluorescence
to image the tiny cell amounts. The nature of such imaging
increases the sensitivity to extraneous fluorescence and it becomes
very important to reduce excess signal as much as possible.
Experimental methods can effectively erase external noise sources
such as compounds prone to creating excess fluorescence, however
it cannot compensate for the intrinsic tissue specific fluorescence
present in all organic matter. This project attempts to address
autofluorescent issues by measuring the intrinsic fluorescence for
each specific organ tissue with in a range of light wavelengths.
These calibration curves can be recording into a database which can
be used in signal processing in combination with experimental
techniques. The method of imaging that these calibration curves
will be implemented is a novel new method called Cryo-imaging.
The Cryo-imaging system cryogenically freezes mice that have been
injected with genetically modified stem cells. It then systematically
sections and images the frozen block faces. Two cameras with
optical filters are mounted on a robotic arm to capture the emission
light. This experiment uses a Nuance multi-spectral camera which
has an adjustable emission wavelength capture filter to measure
the spectrum of autofluorescence. The autofluorescent spectrum
collected showed that the stomach, liver, and brain produce the
most autofluorescence while the intensity from the heart and lung
were almost negligible.
Results
In the Cryo-Imaging machine set up, the camera system is positioned
above a mouse-sized Cryo-stat chamber kept at -22°F. The chamber
contains a motorized platform which can be controlled through a panel
or a computer. The platform is positioned under a knife and can be
moved, raised, and lowered to cut slices that are micrometers thin. The
entire imaging system is mounted on an xyz robotic arm. The arm is
able to slide the microscope and camera system over the Cryo-stat
chamber and lower the apparatus in.
Figure 2: Scope of the Nuance
Imaging system. After the block
face of the mouse has been
exposed, the sample can be imaged
in brightfield and fluorescence. The
upper left hand image is the
brightfield image taken in white
light. The upper right hand image is
the sum of the images taken
through fluorescent imaging over a
range of emission side wavelengths.
The lower left hand image shows
how the Nuance Imaging system
is able to separate the distinct organ fluorescence wavelengths from the
background image. The final lower right hand image shows the separated
wavelengths that are fluoresced, indicated through different colors. In this image, it
can be seen that the internal organ tissue fluoresce at a higher wavelength than
skin tissue.
Spectrum of Excitation, Emission, and Long Pass Filter Wavelengths
The autofluorescent spectrums were captured through a system
consisting of a microscope, optical filter cube, and Nuance MultiSpectral Imaging camera.
change its allowed wavelength in increments of 10nm. The camera is powered
by a 5V supply. The images taken are transferred to the Nuance software
program for analysis.
1000
900
Ref. (GFP)
Heart
Lungs
Stomach
Liver
Spleen
Brain
800
700
600
500
Methods
Figure 1: Schematic of Imaging
system. A fluorescent 100V light
supply is directed through a
coaxial light cable to a blue
wavelength excitation filter. The
filter allows light ~488nm to
enter the optical filter cube. A
dichroic mirror directs light to the
sample and allows only light
above 488nm to pass through to
the emission side. The emission
side filter is a long pass filter
which allows only light above
540nm to pass through to ensure
only the fluorescently produced
light is recorded. The Nuance
Multi-spectral Imaging Camera is
equipped with a filter which can
Organ Spectrum (Un-Normalized)
Figure 3: Long pass filters at the excitation side and emission side of the filter cube
limit the range of wavelengths that may pass through to the camera. The range of
wavelengths allowed through the excitation filter is shown as blue colored area.
Only the range above 488nm is allowed. The range of wavelengths allowed through
the green emission side filter is shown as the area colored green. The cutoff
wavelength is 540nm. The filters ensure that only fluorescent light will enter the
camera.
The autofluorescent spectrums were taken from 500nm to 950nm in
increments of 10nm. The imaging time for each wavelength was 20ms.
400
300
200
100
0
500
550
600
650
700
750
Wavelength (nm)
800
850
900
950
Figure 4: Experimentally found spectrum of the autofluorescence of various
organs. The organ spectrums are compared to the spectrum of a reference
slide which emulated the fluorescence of a GFP cell. The intensity of the
signal is dependent on the duration of the camera capture time. It can be
seen that the stomach, as expected, has the highest amount of
autofluorescence present. The liver and the brain are the next two organ
tissues to emit a high autofluorescent spectrum.
Conclusion
The measurements of the autofluorescent spectrums showed that
the stomach had the highest intensity of fluorescence. This is
because of the high concentration of fluorescent molecules that
pass through the digestive system, such as chlorophyll. Placing the
mouse on a chlorophyll-free diet helps reduce the amount of
extraneous molecules, but it cannot completely eliminate the
autofluorescence. Other tissues that have high fluorescent
intensities are connective tissues. The heart and the lung have
such a low spectrum of intensities and it can be assumed that the
autofluorescence is negligible in these tissues. The stomach has a
peak value at 580±10nm. Other studies have shown the peak
intensity of the stomach as 560nm. The measured value, although
out of range of comparative studies, is within a reasonable
distance. The spectrum of autofluorescence can be used in
further contrast enhancement techniques.
Future Work
The spectrum of autofluorescence can be used in combination with
imaging techniques to give images of the mouse block faces. The
true signal of the GFP cells can be found by subtracting off the
intensity caused by autofluorescence.
Acknowledgements
I would like to thank Debashish Roy and Grant Steyer for all their help. I
would especially like to thank Debashish Roy for his patience and willingness
to help make the project possible.