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ADVANCED ADVANCED METHODS METHODS FOR FOR CONFOCAL CONFOCAL MICROSCOPY MICROSCOPY III III Jean-Yves Chatton / Yannick Krempp / Sept. 2006 Workshop outline • • Simultaneous fluorescence and transmitted images (DIC, Nomarski) on a confocal microscope 3D and confocal microscopy Interference of electromagnetic waves A fraction of the light is deviated when it encounters certain parts of the specimen. This deviated light (i.e. DIFFRACTED) becomes 1/2 wavelength out of phase with respect to the non-diffraced light (1/4 for non-absorbing objects) Image formation (transmission microscopy) Intermediate image The image produced by an objective is congugated with the specimen, i.e. each point is geometrically related to a corresponding point in the specimen Back focal plane Objective Æ Each point in the specimen is represented by a correponding point in Specimen the image Condenser Light source Principles of phase contrast Resulting wave Undeviated light Resulting wave Wave accelerated by λ/4 Diffracted wave Diffracted wave λ/4 out-of-phase Differential interference contrast microscopy (DIC, Nomarski) Georges Nomarski (1919 – 1997) A Polish born physicist and optics theoretician, who adopted France as his home after World War II. Nomarski is credited with numerous inventions and patents, including a major contribution to the wellknown differential interference contrast (DIC) microscopy technique. Contrast technique that enhances the gradient of optical path in the specimen (i.e. gradient of thickness and refractive index) Bright field Human buccal epithelial cells, Obj. 100X DIC contrast Differential interference contrast microscopy (DIC, Nomarski) William Hyde Wollaston (1766 – 1828) One of the most influent scientist of his time. Although trained as a physician, he studied and made advances in several fields as chemistry, physics, botany, crystallography, optics, astronomy, and mineralogy. He is at the origin of the major invention of the so-called Wollaston prism, that is fundamentally important to interferometry and differential interference (DIC) contrast microscopy. Principles of differential interference contrast Analyzer Wollaston prism Wollaston prism Polarizer From: Inoué et Spring, "Video Microscopy", Plenum Press, 1997 Shading and relief effect in DIC microscopy Shear effect Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson Comparison phase contrast and DIC microscopy Phase contrast DIC contrast • Phase contrast yields image intensity values as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background • Optical path length gradients (thickness, refractive index), i.e. the rate of change in the direction of wavefront shear) are primarily responsible for introducing contrast into specimen images. • Specimen features that have relatively low thickness, or a refractive index less than the surrounding medium, are rendered much lighter when superimposed on the medium gray background. • Steep gradients in path length generate excellent contrast, and images display a pseudo three-dimensional relief shading that is characteristic of the DIC technique. • Regions having very shallow optical path slopes, such as those observed in extended, flat specimens, produce insignificant contrast and often appear in the image at the same intensity level as the background. Comparison phase contrast and DIC microscopy DIC Phase Buccal Epithelium Section of rodent renal tissue Obelia (marine polypus) Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson Halos in phase contrast and DIC microscopy Erythrocytes HeLa cells Algae, Zygnema Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson Depth of field in phase contrast and DIC microscopy Phase DIC Obelia (polypoid) Butterfly scales Eggs of canine cucumber tapeworm Adapted from Douglas B. Murphy - Ron Oldfield - Stanley Schwartz - Michael W. Davidson Comparison phase contrast and DIC microscopy Characteristics Phase Contrast DIC Image Brightness (Brightfield = 100 Percent) 1.3 Percent 0.36 - 2.3 Percent Epi-Fluorescence Light Loss (Brightfield = 0 Percent) 28 Percent 73 Percent Lateral Resolution Condenser Annulus Restricted Superior Axial Resolution (Depth Discrimination) Poor Superior Illuminating Aperture 10 Percent of Objective NA Variable Phase Shift Detection Limit < λ/100 < λ/100 Utility at High Phase Shifts Not Useful Useful Azimuthal Effects No Yes Halos and Shade-Off Yes No Stained Specimens Not Useful Useful Birefringent Specimens Useful Not Useful Birefringent Specimen Containers Yes No Brightfield Image Deterioration Slight None Cost Moderate High Köhler illumination • Production of a magnified image of the lamp filament that is focused at the level of the aperture plane of the condensor. • Opening or closing this diaphragm controls the angle of the light cone that reaches the specimen, which is determining the image resolution along with the numerical aperture of the objective (i.e. the global numerical aperture of the optical system). • Since the light source is not focused at the level of the specimen, the light is diffuse and speads evenly at the level of the speciment and does not suffer from deteriorations due to dust and glass defects in the condensor. Conventional DIC Observation DIC observation in confocal mode Laser (polarized light) This DIC image is for free as it is formed using the light that has not been absorbed by the fluorescence specimen Photomultiplier Setting up the microscope for DIC observation 2 1 3 Transmitted light detector Simultaneous fluorescence and DIC acquisition Cortical neurons With GFP expressing cell Simultaneous fluorescence and DIC acquisition Living brain slice (rat hippocampus) Sulforhodamine 101 (astrocyte stain) Next... ADVANCED METHODS FOR CONFOCAL MICROSCOPY III Arnaud Paradis 26 Septembre 2006 Adapted by Yannick KREMPP Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK ACQUISITION Standard Z-Stack acquisition HOW TO SET UP THESE PARAMETERS ? THERE IS NO RULE. AS ALWAYS IT IS A TRADE-OFF BETWEEN SPEED AND QUALITY Z-STACK ACQUISITION Standard Z-Stack acquisition SOME CLUES… If SPEED is what you need (fragile samples, you’re in a hurry, etc…): -Decrease the number of slices (or increase the size of the interval) with « keep slices » enabled, WITH A CONSTANT SLICE THICKNESS +SPEED THE TRADE-OFF: You have now gaps in your stack. -Increase the slice thickness (or decrease the slice number) with « keep interval » enabled. +SPEED THE TRADE-OFF: The pinhole opens more, the Z resolution is poor. Z-STACK ACQUISITION Standard Z-Stack acquisition SOME CLUES… If QUALITY is what you need (fixed samples, you want the cover of Nature magazine, etc…): Try to fullfill the Nyquist-Shannon criterium (signal sampling theorem). +QUALITY YOU NEED AT LEAST AN OVERLAY OF 50% OF 2 CONSECUTIVE IMAGE PLANES THE TRADE-OFF You need plenty of time, and a very resistant dye regarding bleaching 50% overlay Z-STACK ACQUISITION Standard Z-Stack acquisition THE EASY WAY NB : In the case of multiple channel acquisitions, you have to verify that you have the same optical slice thickness for the different channels. Change the pinhole size if necessary. Z-STACK ACQUISITION Standard Z-Stack acquisition Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK ACQUISITION Z-Line A Z-Line acquistion allows you to scan a line (x) at the focal plane along the z axis. As for the conventional XYZ stack you can change the number of focal planes in the interval. Z-STACK ACQUISITION Z-Line – drawing the line X y Z-STACK ACQUISITION Z-Line – The result X Higher limit Z current line. Lower limit Z-STACK ACQUISITION Z-Line – Intensity profile Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK VIEWING Gallery Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK VIEWING Depth Coding Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK VIEWING Projections – Orthogonal projections Z-STACK VIEWING Projections – Cut view Z-STACK VIEWING Projections - MIP MIP Maximum Intensity Projection Z-STACK VIEWING Projections - Transparency Transparency tweaking Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris Z-STACK VIEWING Projections – Rotating the projection Rotation around the Y axis Part I Optical sectioning series acquisition (z-stack) - Standard acquisition (XYZ scan) - Z-line (scan xz) Part II 3D views, capabilities with the LSM Software -Gallery -3D Depth Code -3D Projections xyz -3D Animations Part III Zeiss LSM Software vs Imaris IMARIS Section mode Z X Y Y X Z Z-stack of mouse kidney in Section display with MIP mode IMARIS Section mode dx dy Extended view IMARIS Section mode Extended view – Blend rendering IMARIS Surpass mode (Real-time 3D) Blend IMARIS Surpass mode (Real-time 3D) MIP IMARIS Surpass mode (Real-time 3D) MIP+ Isosurface + Grid IMARIS Surpass mode (Real-time 3D) Isosurface only - Low threshold Isosurface only - High threshold IMARIS Surpass mode (Real-time 3D) Isosurface – Low details Isosurface – High details IMARIS Animation mode – movie recording