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Bio-optics & Microscopy (MEDS 6450) 11/16/2010 Presented by: Mathilde Bonnemasison Leia Shuhaibar Steve Pirnie Ronghua (Ronnie) Yang Neil MAA, Juskaitis.

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Presentation on theme: "Bio-optics & Microscopy (MEDS 6450) 11/16/2010 Presented by: Mathilde Bonnemasison Leia Shuhaibar Steve Pirnie Ronghua (Ronnie) Yang Neil MAA, Juskaitis."— Presentation transcript:

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): (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, 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


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