Confocal microscopy with structured illumination and increased depth of focus 12th International Workshop on Research an

1. Confocal microscopy with structured illuminationand increased depth of focus12th International Workshop on Research and Education in Mechatronics Dipl.-Ing. (FH) Philipp Heidingsfelder#1, Prof. Dr. Jun Gao*2, Prof. Dr.-Ing. Peter Ott#3 #Institute for Mechatronics, Optical Engineering Lab, Heilbronn University *School of Computer and Information, Hefei University of Technology

2. Contents Motivation Confocal Microscopy Structured Illumination Optical System Results Summaryand Outlook

3. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Confocal Microscopy StructuredIllumination • Inspection of microcircuits and MEMS • Optical sectioning property • Reduction in the presence offlared and scattered light in the image  • High-end applications in biotechnologie • Improved axial and lateral resolution • Great complexity Combine confocal microscopyand structured illumination with moderate effort

4. Principle of Confocal Microscopy Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook BeamSplitter F´T Fobj Detector Sample Sample ExitPupil z Detectorpinhole TubeLens Objective Illumination Pinhole DefocusedPlane Focal Plane Illuminating Beam

5. Principle of Structured Illumination Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Projection of a sinusoidal fringe pattern on the sample Conrast evaluation for defocussing the sample [C. Kranitzky, C. Richter, C. Faber, M.C. Knauer, G. Häusler; Institute of Optics, Information and Photonics, University of Erlangen-Nuremberg]

6. Signal Evaluation Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook • For each z-position 4 shifts of the field stop • 4 irradiance values for each pixel E1, E2, E3, E4  Contrast Evaluation: [C. Kranitzky, C. Richter, C. Faber, M.C. Knauer, G. Häusler; Institute of Optics, Information and Photonics, University of Erlangen-Nuremberg]

7. CCD1 Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Grating Tube Lens Tube Lens Grating Incoherent Illumination Beam Splitter Slit Period 1 4 Exit Pupil Objective Piezo Linear Actor Projected Grating in Focal Plane of CCD1 z Sample

8. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook PatternIllumination For the simulation the Optical Transfer Function (OTF) of the Optical System is needed Exit Pupil Entrance Pupil DetectorPlane The Point Spread Function (PSF) is defined as α' α O1 O1' BeamSplitter S1' S1 ∆z' ∆W ∆z ObjectPlane O2' O2 NA: Numerical Aperturem: Magnitude of the microscope∆W: Optical Path Differnce (OPD) in the exit pupilcirc: Circular-FunctionF : Fourier Transformationλ: Wavelength S2' S2

9. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook F{EP} F {PSF} IF -1{FS·OTF}I

10. CCD1 Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Grating CCD2 Beam Splitter Tube Lens Grating Tube Lens Incoherent Illumination Beam Splitter Slit Period 1 4 Exit Pupil Objective Piezo Linear Actor Projected Grating in Focal Plane of CCD1 z Sample Focal Plane CCD2

11. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Simulated Focal Depth Response for focused (CCD1) and defocused camera (CCD2)

12. CCD_1 Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Grating CCD_2 Beam Splitter Tube Lens Grating Tube Lens Incoherent Illumination Beam Splitter Slit Period 1 4 Exit Pupil Objective Piezo Linear Actor Focal Plane CCD_1 Projected Grating z Sample Focal Plane CCD_2

13. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook

14. Light Source Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook CCD Camera Grating Step Motor and Piezo Actor Tube Lens Beam Splitter Mirror Objective

15. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook

16. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Step Height Standard: 14.33 µm

17. Softmark Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Structured Illumination Mode Confocal Microscopy Mode

18. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Summary Interesting possibility to combine structured illumination with confocal microscopy. Possibility to accelerate the measuring time with second camera.

19. Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook Outlook Realization of the 2-camera-system Downsizing the optical system Analyzing the influence of the pattern illumination and compare it with the waveoptical model

20. Thank you very much for your attention! Motivation ConfocalMicroscopy StructuredIllumination OpticalSystem Results SummaryOutlook