1. SPECTROSCOPY GROUP Photonics West February 3, 2013 Brian C. Smith, Ph.D.,Princeton Instruments Jason McClure, Ph.D. Princeton Instruments Dan Heller, Ph.D. Memorial Sloan-Kettering Cancer Center Ed Gooding, Ph.D. Princeton Instruments An Aberration Free Spectrograph for Improved Imaging and Spectra of Biological Samples

2. SPECTROSCOPY GROUP The Traditional Czerny-Turner (CT) Spectrograph Has seen little design change in ~30 years – Light Path: Collimating Mirror => Grating => Focusing Mirror => Detector – Mirrors were originally spherical, are now toroidal Three primary image distortions observed in CT spectrographs – Field Astigmatism – Coma – Spherical Aberration Caused by the laws of physics Are present in ALL manufacturer’s CT spectrographs

3. SPECTROSCOPY GROUP Traditional CT Spectrographs = Blurred Images The optical aberrations inherent in Czerny-Turner designs cause distorted images Note decent imaging in the center. Blurring gets progressively worse towards sensor edges - Vertical stack of fourteen 200 micron optical fibers stepped across the focal plane of a traditional CT spectrograph. 435 nm light, 1200 groove/mm grating, 300 mm focal length

4. SPECTROSCOPY GROUP 1.Distorted Line Shapes 2.Poor Spectral Resolution 3.Reduced Signal-to-Noise Ratio (SNR) Traditional CT Spectrographs = Poor Spectra An asymmetrically broadened spectral line measured on a traditional Czerny-Turner spectrograph

5. SPECTROSCOPY GROUP The Schmidt-Czerny-Turner (SCT) Spectrograph Focusing mirror Entrance Slit On-axis grating drive Collimating mirror Proprietary Mirror AKA the IsoPlane

6. SPECTROSCOPY GROUP Reduced aberrations = Great Focal Plane Imaging Traditional CT Spectrograph IsoPlane SCT Spectrograph

7. SPECTROSCOPY GROUP IsoPlane = Great Spectroscopy IsoPlane vs. CT Pixis 400BR 1200 gr/mm HVIS grating CT 1 Row

8. SPECTROSCOPY GROUP Interfaces the IsoPlane to an inverted microscope’s UDP Port Olympus, Nikon, and Zeiss microscopes are supported No optics involved The MicroSpec Interface

9. SPECTROSCOPY GROUP NIR Fluorescence of Carbon Nanotubes Single-walled nanotubes 0.6-1.3 nm in diameter ~100-2000 nm long, averaging ~500nm Nanotubes are wrapped in a polymer - Can vary polymer functionality - Proteins and nucleic acids can bind to the polymer

10. SPECTROSCOPY GROUP The NIRvana is PI’s new TE cooled InGaAs focal plane array camera Imaging Nanotubes in Live Cells: NIRvana HeLa Cells, Amine-rich polymer-encapsulated carbon nanotubes, 640 nm ex, 410 um slit. 1 frame/second.

11. SPECTROSCOPY GROUP Nanotubes are Transported Within Living Cells

12. SPECTROSCOPY GROUP NIR Fluorescence Spectrum of a Nanotube 640 nm excitation,20 sec exposure time, 410 micron slits Image of carbon nanotube centered on the IsoPlane slit

13. SPECTROSCOPY GROUP Acknowledgements Trenton Engineering – Bill Asher – Harry Grannis – Bob Bolkus – Bill Hartman Acton Engineering Ed Gooding – Lloyd Wentzell – Bob Fancy – Mike Case – Paulo Goulart – Bob Jarratt Memorial Sloan Kettering Cancer Center – Januka Budhathoki-Uprety

14. SPECTROSCOPY GROUP Outline The Traditional Czerny-Turner (CT) imaging spectrograph and its limitations The Schmidt-Czerny-Turner (SCT) spectrograph: The IsoPlane – Instrumentation – Data showing Reduction or Elimination of image aberrations – Improved imaging – Improved spectroscopy Near Infrared Fluorescence of Carbon Nanotubes in Live Cells

15. SPECTROSCOPY GROUP Field Astigmatism Cause: Using lenses or mirrors to image a source off axis Affects on Imaging: Vertical or horizontal elongation of an image e.g. The dreaded “Bow-Tie” effect Affects on spectroscopy: Limits both spectral and spatial resolution of a spectrograph. Is completely corrected only at the center of the focal plane. Fourteen 200 micron diameter optical fibers, 1200 g/mm grating, 300 mm focal length.

16. SPECTROSCOPY GROUP Coma Cause: Using mirrors to image a source off axis Affects on Imaging: Comet shaped tail on focused images or spectral lines Affects on spectroscopy: spectral lines are asymmetrically broadened Limits spectral resolution of a spectrograph Can only be completely corrected at one grating angle or wavelength An asymmetrically broadened spectral line These are images of optical fibers, not Halley’s Comet!

17. SPECTROSCOPY GROUP Spherical Aberration Cause: Using spherical mirrors to focus light to form an image Affects on Imaging: Diffuse symmetric blur about an image Affects on spectroscopy: Limits both spatial and spectral resolution of a spectrograph Symmetric blur around the image of a 150 micron diameter optical -fiber

18. SPECTROSCOPY GROUP Traditional CT Spectrograph IsoPlane SCT Spectrograph Minus astigmatism, the Dreaded Bow Tie Effect is Gone

19. SPECTROSCOPY GROUP IsoPlane: Better SNR = Increased Sensitivity