Bio-optics & Microscopy (MEDS 6450) 11/16/2010 Presented by: Mathilde Bonnemasison Leia Shuhaibar...
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Transcript of Bio-optics & Microscopy (MEDS 6450) 11/16/2010 Presented by: Mathilde Bonnemasison Leia Shuhaibar...
Method of obtaining optical sectioning by using structured light in a
conventional microscope
Bio-optics & Microscopy (MEDS 6450) 11/16/2010
Presented by: Mathilde BonnemasisonLeia ShuhaibarSteve PirnieRonghua (Ronnie) Yang
Neil MAA, Juskaitis R, Wilson T. Optics Letters. 22 (24):1905-1907 (1997)
ObjectivesOverview of the techniquePresentation of the paperInteresting images created with Structure
IlluminationComparison with Laser scanning Miscroscopy
Methods of Optical Sectioning1. Confocal laser-scan microscopy2. 3D deconvolution3. Nipkow disk4. Structured Illumination
Goal: Improve contrast & resolution
Structured IlluminationComponents:
Fluorescence microscopeCooled CCD cameraComputer plus monitorSoftwareSlider – inserted into the plane of the field
diaphragm of the illumination beam pathContains a grid structure with grid lines of
defined width
Sampleplane
Objective
Condensor
FieldDiaphragm
plane
Tube Lens
Intermediate Imageplane
Image Planes
CCD chip
Field diaphragm matched to the focal plane
Figure 1. Schematic of the optical arrangement
Tube Lens
Objective Lens Condensor
Lens
Optical Arrangement
3 Images 120° apart
Langhorst MF, Schaffer J, Goetze B. Biotechnology Journal 2009, 4,858-865
Acquired Images Reconstructed Image
Widefield Image: I0=I1+I2+I3
Reconstructed Image:Ip=[(I1-I2)2+(I1-I3)2+(I2-I3)2]1/2
Mirror Experiment
µ =
ύ is normalized spatial frequency of the grid
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)MM= magnification of the objective lens
More cool images
Source of ArtifactsImperfect grid movement perceivable grid lines
in the resulting imageFluctuations 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 ConsSequential image acquisition not well-suited for
fast moving sample
Out of Plane Rejection of Light
Fewer photons collected thanWidefield fluorescence•only from plane in focus•Also losses from optical path
Worse S/NBut Better Resolution thanWidefield fluorescence~30% lateralWidefield does not haveAxial resolution
Pinhole aperture blocks out-of focus light
Comparing against Confocal Coarser Grid Finer Grid