1. Bio-optics & Microscopy (MEDS 6450) 11/16/2010 Presented by: Mathilde Bonnemasison Leia Shuhaibar Steve Pirnie Ronghua (Ronnie) Yang Neil MAA, Juskaitis R, Wilson T. Optics Letters. 22 (24):1905-1907 (1997)

2. Objectives Overview of the technique Presentation of the paper Interesting images created with Structure Illumination Comparison with Laser scanning Miscroscopy

3. Methods of Optical Sectioning 1. Confocal laser-scan microscopy 2. 3D deconvolution 3. Nipkow disk 4. Structured Illumination Goal: Improve contrast & resolution

4. Structured Illumination Components: Fluorescence microscope Cooled CCD camera Computer plus monitor Software Slider – inserted into the plane of the field diaphragm of the illumination beam path Contains a grid structure with grid lines of defined width

5. Sample plane Objective Condensor Field Diaphragm plane Tube Lens Intermediate Image plane Image Planes CCD chip Field diaphragm matched to the focal plane

6. Figure 1. Schematic of the optical arrangement Tube Lens Objective Lens Condensor Lens Optical Arrangement

7. 3 Images 120° apart Langhorst MF, Schaffer J, Goetze B. Biotechnology Journal 2009, 4,858-865 Acquired Images Reconstructed Image

8. Widefield Image: I0=I1+I2+I3 Reconstructed Image: Ip=[(I1-I2) 2 +(I1-I3) 2 +(I2-I3) 2 ] 1/2

9. Mirror Experiment µ = ύ is normalized spatial frequency of the grid

10. Lily Pollen Grid: 40-line/mm saw-tooth movement synchronized to the camera frame rate  successive camera images corresponded to a spatial shift of 120 degrees in the position of the projected image of the grid 15 W tungsten halogen lamp as light source Green filter (bandwidth 100nm) 30um axial scan with 50X, 0.75 NA objective Effective magnification of (50/180)M M= magnification of the objective lens

11. More cool images

12. Source of Artifacts Imperfect grid movement  perceivable grid lines in the resulting image Fluctuations in light intensity leads to Δ in intensity (compensate by normalizing using average image intensity) Bleaching  intensity losses that have to be taken into account during calculation Thicker specimen giving more fluorescence volume  use finer grid Other Cons Sequential image acquisition  not well-suited for fast moving sample

13. Out of Plane Rejection of Light Fewer photons collected than Widefield fluorescence only from plane in focus Also losses from optical path Worse S/N But Better Resolution than Widefield fluorescence ~30% lateral Widefield does not have Axial resolution Pinhole aperture blocks out-of focus light

14. Comparing against Confocal Coarser GridFiner Grid