SKY super resolution microscope ppt.pptx

Srrf microscopy

1. An Introduction to the Light Microscope, Confocal Microscopy Techniques

2. A single lens as a magnifying glass by creating a magnified, virtual image of an object placed close to it. Magnification Magnification = Image size ÷ Actual size

3. Simple Microscope Compound Microscope

4. Compound microscope bodies. a) Standard upright microscope indicating (1) eyepiece (ocular lens), (2) objective turret, revolver, or revolving nose piece (to hold multiple objective lenses), (3) objective lenses, focus knobs (to move the stage) (4) coarse adjustment, (5) Fine adjustment, (6) Stage (to hold the specimen), (7) Light source (a light or a mirror), (8) Diaphragm and condenser, (9) Mechanical stage. b) Stereo microscope. c) Inverted microscope. d) Comparison microscope. Compound Microscope Components

5. Light Microscopy

6. Confocal Microscopy

7. Region of out-of-focus information Widefield blurred &large Confocal very small Fundamental Set-up of Fluorescence Microscopes: confocal vs. widefield Widefield 2 - 3 µm Confocal 0.5 µm Focus area

8. Confocal Microscopy Principle In confocal microscopy two pinholes are typically used: A pinhole is placed in front of the illumination source to allow transmission only through a small area. This illumination pinhole is imaged onto the focal plane of the specimen, i.e. only a point of the specimen is illuminated at one time. Fluorescence excited in this manner at the focal plane is imaged onto a confocal pinhole placed right in front of the detector. Only fluorescence excited within the focal plane of the specimen will go through the detector pinhole. Scanning of small sections is done and joined them together for better view.

9. Working Confocal microscope incorporates two ideas: Point by point illumination of the specimen. Rejection of out of focus of light. Light source of very high intensity is used-Zirconium arc lamp in Minsky’s design and laser light source in modern design. a)Laser provides intense blue excitation light. b)The light reflects off a dichroic mirror, which directs it to an assembly of vertically and horizontally scanning mirrors. c)These motor driven mirrors scan the laser beam across the specimen. d)The specimen is scanned by moving the stage back and forth in vertical and horizontal directions and optics are kept stationary.

10. Advantages The specimen is everywhere illuminated axially , rather than at different angles, thereby avoiding optical aberrations. Entire field of view is illuminated uniformly. The field of view can be made larger than that of the static objective by controlling the amplitude of the stage movements. Image formed are of better resolution. Cells can be live or fixed. Serial optical sections can be collected. Taking a series of optical slices from different focus levels in the specimen generates a 3D data set.

11. Drawbacks Resolution: It has inherent resolution limitation due to diffraction . Maximum best resolution of confocal microscopy is typically about 200nm. Pin hole size: Strength of optical sectioning depends on the size of the pinhole. Fluorophores: a)The fluorophores should tag the correct part of the specimen. b)Fluorophore should be sensitive enough for the given excitation wavelength. c)It should not significantly alter the dynamics of the organism in the living specimen. Photobleaching: Photochemical alternation of a dye or a fluorophore molecule such that it permanently is unable to fluorescence .