Confocal Raman Microspectroscopy on Cornea

Jenkins He

University of Rochester Dr. Andrew J. Berger Biomedical Optics Lab

Human Cornea • Raman Spectroscopy is a powerful optical technique for the biochemical characterization

tissues and other biologic media.

• For human cornea, referring from Dr.Bauer ‘s paper, the typical Raman spectra of a normal human cornea in the higher Raman shift range from 2900 to 3600/cm-1

What is Confocal Microscopy?• First developed by Marvin Minsky and patented as a “microscopy apparatus” in 1961.• • A well designed confocal system can be described as “double focusing” system.

• Instead of focusing the light through a pinhole, we focus into a central fiber which has fibers surrounding it.

• Block out as much unfocused light as possible also minimize as much unfocused light into the central fiber as possible.

Our basic alignment

•Using 50 microns pinhole in spatial filter

•Beam size ~ 7mm ( Back aperture of microscope objective is around 7 mm)

MODE1 MODE 2 MODE 3 MODE 4

Intensity of light Onto camera Strongest Weakest N/A Stronger

than 2

Signal to spectrometer Weakest Strongest N/A Stronger

than 1

The spectrometer we used HoloSpec F/1.8 Imaging Spectrometer

Scaling and Testing Results

USAF image with50X Objective

USAF image with10X Objective

Group Number /Element Number

Line Pairs/ mm Line Width (micros)

Estimated Laser Spot Size (microns)

50X 7/2 143.7 ~3.5 ~ 2

10X 5/1 32 ~15 ~ 10

Scaling and Testing Results

•Tube Length = 200mm with 50X objective gives us the focal length of objective is 4 mm

•The lens focusing beam into fiber has focal length of 200 mm

•So the confocal spot size is magnified by 50X to ~ 100 microns

•Center core fiber has a size of 100 microns

Scaling and Testing Results

Image of fibers for microscope glass slide with 50s exposure time with 50X objective

Use Nikon 60X water immersion objective for Bio sample like cornea Test 1: Plastic

Scaling and Testing Results

Plastic

Water drop

60X Objective

Test1: Plastic

FWHM ~ 20 microns

Test 2: Teflon

Teflon

Water drop

60X Objective

Test 2: Teflon

FWHM ~20 microns

plastic

Finger nail

Cornea(test 1)

Berger’s Rugby League

Thank you for coming!