Low-Cost UV/VIS/NIR Microscope-Spectrophotometer for Medical Decision Making and Documentation

1. SETTING-UP A LOW-COST UV/VIS/NIR MICROSCOPE -SPECTROPHOTOMETER SUPPORTING MEDICAL DECISION MAKING AND DOCUMENTATION B. Spyropoulos, A. Tzavaras, P. Koudounas Biomedical Technology Laboratory Medical Instrumentation Technology Department Technological Education Institute of Athens Athens, Greece Email: basile@teiath.gr

2. Aim of the project • A microscope-spectrophotometer combines an optical microscope and a highly sensitive spectrophotometer, as an external additional component, measuring the intensity of light versus its wavelength. • The purpose of the project is the development of a low-cost UV-VIS-NIRmicroscope-spectrophotometerto support Medical Decision Making and Documentation. IFCC WorldLab

3. Starting point of the system is a simple Zeiss-Jena Laboval 30-G062 Microscope employed in our Lab for 23 Years. Following components have been integrated to the system: A modern USB-connected HR4000CG composite-grating Ocean-Optics spectrophotometer, employing: A HC-1 variable-blazed grating. A Toshiba TCD1304AP linear Charge-Coupled Device (CCD)-array, providing for full spectral output (200-1100 nm). A Bresser eyepiece-insertable PC-USB digital imaging camera including ΟΕΜ digital imaging software. A Deuterium-Wolfram-Halogen light-source. Optical fiber transporting light from a Region of Interest (ROI) of a sample to the spectrophotometer. Materials IFCC WorldLab

4. The HR4000 CG spectrophotometer the TCD1304AP detector and the relative spectral response of reflexion efficiency and power IFCC WorldLab

5. Animation representing CCD operation IFCC WorldLab

6. Spectrometer Operating Principles (I) • The animation shows how Ocean Optics CCD detector-based Spectrometers operate. • Light enters the optical fiber and is efficiently transmitted to the spectrometer. • Once in the spectrometer, the divergent light emerging from the optical fiber is collimated by a spherical mirror. • The collimated light is diffracted by a plane grating, and the resulting diffracted light is focused by a second spherical mirror. • An image of the spectrum is projected onto a linear CCD array, and the data is transferred to a computer through an onboard A/D converter. IFCC WorldLab

7. Spectrometer Operating Principles (II) • Light impinges on photodiodes with the CCD pixels. • These reverse-biased photodiodes discharge a capacitor at a rate proportional to the photon flux. • When the integration period of the detector is complete, a series of switches closes and transfers the charge to a shift register. • After the transfer to the shift register is complete, the switches open and the capacitors attached to the photodiodes are recharged and a new integration period begins. • At the same time that light energy is being integrated, the data is read out of the shift register by an A/D converter. • The digitized data is then displayed on your computer. IFCC WorldLab

8. Grating Selection Chart IFCC WorldLab

9. The insertable Bresser eyepiece USB- digital-camera & ΟΕΜ imaging software IFCC WorldLab

10. The Deuterium-Wolfram-Halogen light-source and the optical fibers Laboratory-grade Patch Cord Optical Fiber Assemblies IFCC WorldLab

11. Method (Ι) • The condenser-lens light, or that from an external-source, is focused onto the sample that absorbs, reflects or emits (fluorescence), some wave-lengths better than others, depending upon the sample’s biochemical structure, staining or tagging. • Most of this light is reflected through the microscope’s bending-mirror, into the oculars and/or the digital imaging camera. Bending Mirror IFCC WorldLab

12. Method (ΙΙ) • Part of the light is collected by an optical-fiber probe, proximal to the bending-mirror, and is guided into the spectrophotometer-aperture. • Thus, the aperture of the spectrophotometer is virtually overlaid on the sample, which can easily be appropriately positioned, for spectra-uptake. Digital Camera Fiber Optics Sample ToHR4000 CG IFCC WorldLab

13. Details of the Fiber Mounting Digital Camera Fiber optics to the Spectrophotometer IFCC WorldLab

14. Block diagram and optical paths Fiber-optic to Spectrophotometer To digital imaging CCD Detector Grating Mirror Eyepiece Objective lens Sample Light Source Mirror IFCC WorldLab

15. Overview of the modified microscope and the of the whole system IFCC WorldLab

16. Method (ΙΙΙ) • The light is then analyzed by the optical grating and it its spectral-intensity is measured on the CCD-detector. • The data processing, display and documentation is performed by, both, Original Equipment Manufacturer (OEM) and custom-made software. IFCC WorldLab

17. Absorption spectra of test samples control sample 4 Baseline (noise) control sample 8 IFCC WorldLab

18. Conclusions • The presented approach offers low-cost upgrading of existing microscopes. • The system is being presently experimentally tested in the Biomedical Technology Laboratory for: • NAD(P)H skin auto-fluorescence under wide-spectrum UV illumination. • Immunofluorescence applications. • IR-absorption based subcutaneous tissue-O2 detection. IFCC WorldLab