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IPM Progress Report
System design
Spatial resolution
Temporal resolution
Concentration measurements in water
Concentration measurements in vaginal simulant
System Photos
Additional notes for Katz
1. System Design
The endoscopic confocal system is designed to deliver laser illumination to a sample and collect
the fluorescent signal from a thin depth section. Figure 1 shows a schematic of the optical system. Light
from the laser (λ = 532nm) is formed into a line by the illumination pathway optics and is then scanned
laterally across the proximal face of the endoscopic imaging fiber bundle. The distal imaging optics
consist of a 10x and 40x microscope objective and produce a magnification of 4x from the sample to the
face of the fiber bundle. This system delivers the illumination line from the bundle to the sample plane,
and then collects the fluorescent signal. Each fiber core has a 7.8μm diameter and acts as a pinhole to
reject out-of-focus light. The returning fluorescence line is descanned by the scanning mirror and
transmitted to the EMCCD camera (PhotonMax:512B, Princeton Instruments) by the fluorescence
imaging pathway optics.
Only one line of fiber cores is illuminated at a given time, and therefore only one line of fiber
cores contains in-focus fluorescence signal. Since the active line of the fiber bundle is descanned onto
the EMCCD, the same line of pixels is captured for each scan position. Sequentially capturing 512 lines
at different scan positions produces a 512x512 fluorescence confocal image of the sample.
Figure 1: Schematic of the line-scan endoscopic confocal system. The illumination optics shape the beam to produce a narrow
line that is scanned across the sample plane. The fiber core size provides a confocal advantage, confining the fluorescence
signal to a narrow focal depth.
2. System Resolution
A. Lateral Resolution
The lateral resolution of the optical system is typically limited by the numerical aperture of the
imaging optics. Since the image is transmitted through the fiber bundle by the individual fiber cores, the
maximum possible spatial resolution of the system is limited by the fiber core spacing within the bundle.
To measure lateral resolution, a USAF 1951 test target was placed in the image plane and backilluminated with a white light source. The acquired image, shown in figure 2A, displays elements 1-6 of
group 7 on the target. The smallest element has a bar width of 2.19µm. Using the element dimensions
as a calibration, we calculate the core-to-core spacing to be 1.87µm on the sample. Once the
magnification factor (4x) is taken into account, this agrees closely to the theoretical core-core spacing on
the sample (1.95µm).
The lateral resolution of our system is twice the core-to-core spacing, or 3.75µm. Since the
image information is transmitted only by the fiber cores the images can be filtered to remove the
hexagonal pattern of the cores without degrading the system resolution. Figure 2B shows the filtered
image of the test target.
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Figure 2: Full field image of group 7 on a USAF 1951 test target. The target provides a resolution calibration measurement. A)
The raw image has a hexagonal pattern of fiber cores. B) Filtering the image removes the core pattern while preserving image
resolution.
B. Axial Resolution
To measure the axial resolution of the endoscopic confocal system, a volume of Rhodamine 6G
(Rho-6G) dye with a finite axial thickness was translated through the sample plane of the distal imaging
optics. Intensity measurements were made at a single point as the sample was scanned axially, and the
full-width half maximum (FWHM) of the scan was taken to be the axial resolution. Figure 3 shows the
relative intensity of a scan performed with a 1.55µm-thick volume of 0.1% aqueous Rho-6G. The
measured FWHM of the system was found to be 5.7µm. Figure 4 shows that for a 100µm-thick sample
the peak is distinctly broadened.
Axial response of a 1.55m-thick sample of 0.1% Rho-6G
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Figure 3: Axial scan of a 1.55µm-thick volume of 0.1% aqueous Rho-6G dye. This volume approximates an infinitesimally thin
sheet of fluorescence. The axial FWHM of the measured intensity is 5.7µm.
Axial response of a 100m-thick sample of 0.1% Rho-6G
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Figure 4: Axial scan of a 100µm-thick volume of 0.1% aqueous Rho-6G. The shape of the intensity peak is broadened and
distorted due to focusing, absorption, and dispersion effects that arise from imaging through a thick medium.
3. Temporal Resolution
Confocal scans are produced by acquiring 512 sequential lines of a 1x512 ROI on the EMCCD camera
(PhotonMax:512B, Princeton Instruments). The 512 lines are then stitched together into a single 512 x
512 grayscale image. Currently images can be acquired at a rate of 2.65 frames per second.
4. Concentration Measurements
In order to evaluate sensitivity of the system, various concentrations of fluorophores (40nm diameter
FluoSpheres and Rhodamine 6G) were diluted in water and detected by the confocal system. 35μL of
each concentration was injected into a square capillary of 0.4mm path length. The capillary containing
the solution was placed in the focal plane of the distal optics. A background image B was first taken
where no fluid was present in the capillary, before taking a fluorescent image F of the fluid itself. A
background subtraction of F – B was processed using the image math function of ImageJ. A region of
interest was taken of focused pixels and averaged, a process repeated for each concentration.
Figure 5. A log-log plot of intensity counts after background subtractions versus FluoSphere bead concentration (percent
weight in volume of water). A double-log regression model is shown in red, with the noise floor shown in cyan. Note that
while measureable, the means of the lowest concentrations fell below the noise floor.
The noise floor of the system was calculated using the square-root of the background intensity. For the
FluoSphere beads the noise floor was 65 intensity counts. A double-log fit of the data points yielded an
R-squared goodness-of-fit of 0.971. From this model, it can be seen that we have a sensitivity that can
detect as low as 0.0004% concentration (w/v) for the 40nm fluorescent beads.
Figure 6. A log-log plot of intensity counts after background subtractions versus Rhodamine 6G concentration (percent
weight in volume of water). A double-log regression model of the non-saturated points is shown in red, with the noise floor
shown in cyan.
The noise floor for the Rhodamine 6G was 49 intensity counts. Due to the high absorption of
Rhodamine 6G, three data points exhibited a saturation effect. A double-log fit of the data points that
were not saturated yielded an R-squared goodness-of-fit of 0.967. From this model, it can be seen that
we have a sensitivity that can detect as low as 0.00005% concentration (w/v) for the Rhodamine 6G.
5. Concentration Measurements in Vaginal Simulant
The experiment in section 4 was repeated by diluting the fluorophores in vaginal simulant in place of
water in order to determine if there is any quenching effect by the vaginal simulant.
Figure 7. A log-log plot of intensity counts after background subtractions versus FluoSphere bead concentration (percent
weight in volume of vaginal simulant). A double-log regression model is shown in red, with the noise floor shown in cyan.
Note that while measureable, the means of the lowest concentrations fell below the noise floor.
The noise floor of the system was calculated using the square-root of the background intensity. For the
FluoSphere beads in vaginal simulant the noise floor was 65 intensity counts. A double-log fit of the
data points yielded an R-squared goodness-of-fit of 0.964%. From this model, it can be seen that we
have a sensitivity that can detect as low as 0.0005% concentration (w/v) for the 40nm fluorescent
beads.
Figure 8. A log-log plot of intensity counts after background subtractions versus Rhodamine 6G concentration (percent
weight in volume of vaginal simulant). A double-log regression model of the non-saturated points is shown in red, with the
noise floor shown in cyan.
The noise floor for the Rhodamine 6G in vaginal simulant was 49 intensity counts. Due to the high
absorption of Rhodamine 6G, two data points exhibited a saturation effect. A double-log fit of the data
points that were not saturated yielded an R-squared goodness-of-fit of 0.967%. From this model, it can
be seen that we have a sensitivity that can detect as low as 0.000025% concentration (w/v) for the
Rhodamine 6G.
6. Photos of the system
7. Progress Notes to Katz
Lateral Resolution
The lateral resolution was measured using wide-field reflectance, not confocal fluorescence
scans. This was due to the fact that the 40X objective has a very short working distance. In order to do
a fluorescence resolution measurement, a sheet of fluorescence must be masked by the USAF resolution
target. This target is too thick to fit within the 40X objective’s working distance. We are fabricating
copies of the USAF resolution target on thin microscope coverslips so that we can make this
measurement next week.
Axial Resolution
Axial resolution was not part of our deliverables for the IPM milestone, but the 5.7µm FWHM of
the thin sheet response demonstrates good axial resolution. Theoretically, our confocal system is not
completely optimized: the optimal pinhole size for the distal optics is ~2.5µm, which is much smaller
than the 7.8µm cores of our current fiber bundle (Schott leached fiber bundle). We have fiber bundles
from Fujikura that have cores with a 3µm mean diameter, which should give much better out-of-focus
light rejection than our current fiber bundle. This will result in better localization of concentration
measurements. We plan to test this one of the Fujikura imaging fiber bundles in our system next week.
Distal Optics
Our distal optics currently consist of an imaging system of a 10x and a 40x microscope objective.
This produces a magnification of 4x from the sample to the distal end of the fiber bundle. While
replacing the current fiber bundle with the Fujikura fiber bundle with smaller cores will increase our
axial and lateral resolution, we are redesigning our distal end optics to increase illumination delivery
efficiency as well. This will result in higher sensitivity and a decrease in the minimum concentration of
detectable fluorophore.
Temporal Resolution
Currently our maximum frame rate is 2.65 frames per second. This corresponds to an exposure
time of less than 1ms for each line. We are working with Princeton Instruments to figure out how to
improve this frame rate. However, a higher frame rate will mean a shorter exposure time and will likely
require better optical delivery and collection efficiency.
Concentration Measurements
Since this data has been taken, we have found that we can reduce the noise floor even further
by removing the ADC offset to the camera. By optimizing this, we can further improve the sensitivity of
the system. Furthermore, by swapping out the fiber bundle, we may be able to reduce
autofluorescence in our detected wavelengths. This will also drive the noise floor down and allow us to
pump with higher laser intensity, and in turn improve the sensitivity of the system.