Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time,
as an adjunct to biopsy and pathology. Current confocal
point-scanning systems are large, complex, and expensive.
A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and
may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be
implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a
beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high
resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner.
We have developed a confocal line-scanner that combines both
divided-pupil and full-pupil configurations. This
combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for
imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning
microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast,
signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions.
Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging
performance. Both results will be compared to theoretical calculations using our previously reported Fourier
analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of
confocal reflectance scanning microscopes for clinical and surgical dermatology.
Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time,
as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal
line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical
dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the
pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided
pupil may offer better sectioning and contrast.
We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in
both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We
repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fillfactor,
h, the ratio of the 1/e2 diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02
(full) and 0.52 (half).
Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil
configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil
configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in
comparing the experimental performance of the different configurations.
Several quantitative phase imaging techniques, such as digital holography, Hilbert-phase microscopy, and phase-shifting
interferometry have applications in biological and medical imaging. Quantitative phase imaging measures
the changes in the wavefront of the incident light due to refractive index variations throughout a 3-D specimen. We
have developed a multimodal microscope which combines optical quadrature microscopy (OQM) and a Shack-
Hartmann wavefront sensor for applications in biological imaging. OQM is an interferometric imaging modality
that noninvasively measures the amplitude and phase of a signal beam that travels through a transparent specimen.
The phase is obtained from interferograms with four different delayed reference wavefronts. The phase is then
transformed into a quantitative image of optical path length difference. The Shack-Hartmann wavefront sensor
measures the gradient of the wavefront at various points across a beam. A microlens array focuses the local
wavefront onto a specific region of the CCD camera. The intensity is given by the maximum amplitude in the
region and the phase is determined based on the exact pixel position within the region.
We compare the amplitude and quantitative phase information of poly-methyl-meth-acrylate (PMMA) beads in oil
and one-cell and two-cell mouse embryos with micrometer resolution using OQM and the Shack-Hartmann. Each
pixel in OQM provides a phase measurement, whereas multiple pixels are used in Shack-Hartmann to determine the
tilt. Therefore, the simple Shack-Hartmann system is limited by its resolution and field-of-view. Real-time imaging
in Shack-Hartmann requires spatial averaging which smoothes the edges of the PMMA beads. The OQM has a
greater field-of-view with good resolution; however, it is a complex system requiring multiple optical components
and four cameras which may introduce additional artifacts in processing quantitative images. The OQM and Shack-
Hartmann has certain advantages depending on the application. A combination of these two systems may provide
improved quantitative phase information than either one alone.cHJl
Precise removal of basal cell carcinomas (BCCs) with minimal damage to the surrounding normal skin is guided by the examination of frozen histology of each excision during Mohs surgery. The preparation of frozen histology is slow, requiring 20 to 45 min per excision. Confocal reflectance mosaicing may enable rapid detection of BCCs directly in surgical excisions, with minimal need for frozen histology. Soaking the excisions in acetic acid rapidly brightens nuclei and enhances BCC-to-dermis contrast. Clinically useful concentrations of acetic acid from 10 to 1% require 30 s to 5 min, respectively. A tissue fixture precisely controls the stability, flatness, tilt, and sag of the excisions, which enables mosaicing of 36×36 images to create a field of view of 12×12 mm. This simulates a 2× magnification view in light microscopes, which is routinely used by Mohs surgeons to examine frozen histology. Compared to brightfield, cross-polarization enhances contrast and detectability of BCCs in the papillary dermis but not in the reticular dermis. Comparison of mosaics to histology shows that nodular, micronodular, and superficial BCCs are easily detected. However, infiltrative and sclerosing BCCs tend to be obscured within the surrounding bright dermis. The mosaicing method currently requires 9 min, and thus may expedite Mohs surgery.
Mohs surgery is a procedure for microscopically excising basal cell carcinomas (BCCs)
while preserving maximal surrounding normal skin. Each serial excision is guided by
examination of the frozen histology of the previous excision. Because several (2-20)
excisions must be made and frozen histology prepared for each excision. Mohs surgery
is time-consuming (15-45 minutes per excision) and tedious. Real-time confocal
reflectance mosaicing enables detection of BCCs directly in fresh excisions, following
contrast-enhancement by acetowhitening. A confocal mosaic allows rapid observation of
15x15 mm2 of tissue, which is equivalent to a low magnification, 2X view of the
excision. Relatively large superficial nodular and micronodular BCCs are rapidly
detectable in confocal reflectance mosaics, whereas detection of much smaller infiltrative
and sclerosing BCCs is a challenge due to the lack of sufficient nuclear/dermis contrast in
acetowhitened excisions. Multimodal contrast, combining reflectance with either
fluorescence or autofluorescence may make it possible to detect infiltrative and
sclerosing BCCs. A reflectance image shows both nuclei and the surrounding dermis,
whereas an autofluorescence image (excitation at 488nm, detection 500-700nm) shows
only the dermis. Thus, ability of a composite (i.e., reflectance-less-autofluorescence)
image shows significantly darkened dermis, with stronger enhancement of nuclear/dermis
contrast. Preliminary results illustrate that this may enable detection of infiltrative and
sclerosing BCCs. The use of reflectance and autofluorescence parallels the use of two
stains (hematoxylin and eosin) in histology, thus allowing a more complete optical
detection method.
Mohs surgery is a staged procedure for microscopically excising basal cell carcinomas (BCCs) while preserving the surrounding normal skin. Serial excisions are performed with each excision being guided by examination of the frozen histology. Mohs surgery is a meticulous and time-consuming (15-45 minutes per excision) procedure requiring several (2-20) excisions and frozen histology prepared for each excision. Real-time confocal reflectance microscopy may make Mohs surgery more efficient by enabling rapid detection of BCCs directly in fresh, unprocessed excisions, and thereby possibly avoiding frozen histology. As previously reported, we are developing an acetowhitening-and-cross polarized method to detect BCCs with a confocal reflectance microscope. Acetowhitening compacts the chromatin within the nucleus, increasing nuclear backscatter, and brightening the nuclei in the confocal images of the tissue. Our experiments to optimize acetowhitening, using acetic acid concentrations from 1% to 30% and treatment times from 30 seconds to 5 minutes, show that a minimum concentration of 2% with minimum washing time of 2 minutes is required for enhancing nuclear morphology. Increased depolarization is observed within the compacted chromatin relative to the surrounding collagen, and imaging in brightfield or crossed polarization brightens or darkens the cellular cytoplasm and birefringent dermis; thus, we may potentially vary nuclear/cytoplasm and nuclear/dermis contrast. Images are collected, oriented, and tiled to create mosaics and sub-mosaics to view large excisions at variable 2X - 10X magnifications. To create and display mosaics, adequate pixelation relative to resolution must be maintained and precise mechanical fixturing is necessary to control tilt, sag, flattening and stability of the excised tissue specimen.
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