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

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