confocal reflectance theta line-scanner for intra-operative imaging confocal reflectance theta...
TRANSCRIPT
Confocal Reflectance Theta Line-Scanner for Confocal Reflectance Theta Line-Scanner for Intra-operative ImagingIntra-operative Imaging
Peter J. Dwyer*, Charles A. DiMarzio*, and Milind Rajadhyaksha **The Center for Subsurface Sensing and Imaging Systems (CenSSIS),
Northeastern University, Boston, MA Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY
Acknowledgement: This work is supported in parts by NIH/NCI (Award Number 1R43CA93106 and 2R44CA93106) and the NSF Partnerships in Education and Research Program (Award Number EEC-012931).
• Confocal microscope performs “optical sectioning:” - A thin section within tissue is imaged non-invasively, - m-level three-dimensional resolution and high contrast is achieved (Figure 1).
• Line scanning is simpler and may offer resolution, contrast and depth of imaging performance comparable to point scanning. • Basal cell cancers (BCCs) are among the fastest growing skin cancers (>1.2 million new cases every year in the USA alone);
- Precise microsurgical excision is performed by a procedure known an Mohs micrographic surgery. - Mohs surgery is guided by frozen histology of multiple excisions, and is thus inefficient and slow.
• The confocal theta line-scanner may enable- Imaging of BCCs directly on patients in real-time, - Guidance of Mohs surgery, thus reducing the number of excisions and avoiding frozen histology.
IntroductionIntroduction
A confocal reflectance theta line-scanner is being developed for imaging nuclear, cellular and architectural detail in human tissues in vivo. Preliminary design and experimental results show lateral resolution of 1-5 m and sectioning (axial resolution) of 2-5 m is possible within living human skin; thus, nuclear and cellular detail has been imaged in epidermis in vivo. Experimental results are being verified with an analytic model and optical design analysis. An immediate clinical application will be intra-operative imaging of basal cell cancers to guide Mohs micrographic surgery for the precise excision of these cancers.
AbstractAbstract
• Confocal theta microscopy was developed by Koester (1980), Stelzer (1995) and Webb (1999).• We designed and built a theta line-scanning confocal microscope (Figure 2) with specifications as shown in Table 1.• Optimized design based on diffraction for best lateral and axial (section) resolution.• The optical design and system set-up are shown in Figure 3.• In vivo and excised skin specimens were imaged (Figure 4 - 6).• Measurements of the axial and lateral resolution were made.
Optical DesignOptical Design
Line Scanner SpecificationsImaging: 5-20 frames/sec (real-time)
Lateral Resolution: 1-3 mVertical Section Thickness: 2-5 m
Max Depth: 300 mObjective: 10x, NA = 0.80
Field of view: 1.0 mmLaser illumination: 830 nm (Diode)
• Resolution (sectioning) of line-scanner compares well to that provided by current point-scanning technology.• Experimental results of axial and lateral line spread function measurements agree well with theoretical values.• Further work needed;
- Aberrated LSF at pupil edges and within skin- Contrast and SNR- Illumination scan / detection de-scan mismatch.- Signal drop-out (illum./detect. path mismatch)- CMOS Detector Sensitivity- Correlation of images to histology
Conclusions/Future WorkConclusions/Future Work
Figure 3. – Confocal Theta Line-Scanning. The intersection of the illumination and detection paths creates confocal probe volume (PSF), (a). Figure 3b illustrates the system layout.
Figure 5. - Line scanner confocal images in human skin in vivo using a 50 m slit. The middle layers of the epidermis containing the spinous cells. The dark nuclei are surrounded by the bright, grainy cytoplasm.
Figure 4. – Line scanner confocal images in human skin in vivo using a 50 m slit. The upper layers of the epidermis contain the stratum corneum (a) and the granular cells (b).
Figure 6. - Line scanner confocal images in human skin in vivo using a 50 m slit. The deeper layers of the epidermis contain basal cells. The bright basal cells are clustered around dermal papillae (dark circles).
Figure 2. – Confocal RTLS Microscope
Table 1. - Instrument Specifications
Figure 1. – Confocal Microscopy, “Optical Sectioning”
ur
FOCAL (OBJECT) PLANE
CONFOCALSECTION 2hTHICKNESS
ILLUMINATION DETECTION/ PATH IMAGING PATH
max
min
wdet
OBJECTIVE LENS
DIVIDER STRIP
diode laser
linear polarizer
analyzer
focussing lens f = 125 mm
cylindrical lensf = 200 mm quarter-wave
plate
objective lensf = 17.5 mm
object plane
linear detectorpixel size 7.5 m 1024 pixels
detection slitwidth 7.5 m
divider strip (divides objective lens aperture into two halves)
scanner
• Design and Development;- Axial line spread function measurements show that the sectioning is 1.6 – 9.8 m for detection slits of width 5-100 um under nominal conditions (Figure 7).- Lateral resolution of 0.8 – 2 m have been measured with 25, 50, 100 m and no slit widths (Figure 8). Note: Lateral resolution = 5 m at the detector.- Preliminary images of epidermis appear comparable to those with point scanning confocal microscopy.
ResultsResults
Figure 4a – Stratum Corneum Figure 4b – Granular Cells
Figure 3a. – Geometrical Optics Concept
Plane in Focus
DETECTOR
BEAMSPLITTER
OPTICALSYSTEM
Small Aperture
TISSUE
Legend:In-focus lightOut-of-focuslight
SmallAperture
Figure 3b. – Optical Layout Figure 7. – (a) Axial LSF measurement; 10 m Slit. (b) Axial line spread function measurements for various detection slit widths.
• Present: Point scanning confocal microscopes are complex, expensive and difficult to manufacture.• In-progress Research: Theta line-scanner may be simple, robust, inexpensive and easy to manufacture.• Present Standard of Care: Surgical removal of skin cancers with unknown margins and potentially more scarring; surgery is guided by frozen histology performed parallel: processing requires 45 mins per excision, 2-20 excisions per patient.• Proposed: Confocal theta line-scanner for real-time, intraoperative imaging and surgical guidance.
State of the ArtState of the Art
Figure 8. – (a) Lateral LSF Measurement; 50 m slit. (b) Lateral line spread function measurements for various detection slit widths.
Laser Diode
Scanning Mirror
Detector BoardObjective Lens
Fold Mirror
Slit Mount
100 um
100 um 100 um
100 um
100 um 100 um