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Brightfield microscopy

Brightfield microscopy . Generally only useful for stained biological specimens Unstained cells are virtually invisible. Oblique Illumination. Phase Contrast. http://microscopy.fsu.edu/primer/techniques/ phasegallery/chocells.html. Aberrations. Spherical aberration Most severe

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Brightfield microscopy

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  1. Brightfield microscopy • Generally only useful for stained biological specimens • Unstained cells are virtually invisible

  2. Oblique Illumination

  3. Phase Contrast http://microscopy.fsu.edu/primer/techniques/ phasegallery/chocells.html

  4. Aberrations • Spherical aberration • Most severe • Immersion fluid • Field curvature • Chromatic aberration • Astigmatism, comma • http://micro.magnet.fsu.edu/primer/lightandcolor/opticalaberrations.html

  5. Phenomenon of fluorescence Probes.invitrogen.com Jablonski diagram: Absorption of photon elevates fluorophore to excited singlet state S1’ Nonradiative decay to lowest energy singlet excited state S1 Decay to ground state by emission of a photon

  6. Non-radioactive decay to triplet state leads to photobleaching Molecular Expressions website

  7. Ideal fluorophore characteristics • High quantum efficiency • Slow photobleaching • For live cells: excitation wavelengths non-phototoxic to cells • Little overlap with autofluorescence • Mammalian cells: Flavoprotein, pigment • Plant cells: chlorophyll

  8. Fluorescent proteins: GFP Tsien Lab (UCSD)

  9. How we observe fluorescence • Black light • Not enough sensitivity • Filters • Bleed-through • Darkfield fluorescence microscopy

  10. Epifluorescence microscopy Nikon Microscopy U

  11. Essence of epifluorescence microscope: Dichroic mirror www.microscopyu.com

  12. Examples of Fluorescence

  13. Confocal Microscopy The term “confocal” means “having the same focus” This is accomplished by focusing the condensor lens to the same focal plane as the objective lens. • Reduced blurring of the image from light scattering • Increased effective resolution • Improved signal to noise ratio • Clear examination of thick specimens • Z-axis scanning • Depth perception in Z-sectioned images • Magnification can be adjusted electronically

  14. Laser • Acronym: Light Amplification by Stimulated Emission of Radiation • Ordinary light emission: Comes from spontaneous decay of excited state to ground levels • Stimulated emission: molecule remains in excited state until stimulated to emit by incoming light that is insufficient to raise it to the next higher excited state

  15. Conventional Fluorescence Confocal The difference in resolution can be significant in those specimens which are too thick to fit entirely within the focal plane of the lens.

  16. Optical section of an aphid showing internal structure of an intact animal

  17. If this is coupled with a point source of illumination and a matching point source of detection Pinhole 1 Pinhole 2 Specimen Detector Objective Lens Condenser Lens Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989

  18. Arc Lamp Fluorescent Microscope Excitation Diaphragm Excitation Filter Ocular Objective Emission Filter

  19. Laser Confocal Principle Excitation Pinhole Excitation Filter PMT Objective Emission Filter Emission Pinhole

  20. In a confocal microscope only a relatively small portion of the specimen is illuminated at a time whereas in a conventional fluorescence microscope a much broader area is illuminated.

  21. If the source of illumination is truly a point and it is focused to a point then only a single point in the specimen will imaged at any one time. Either the specimen must be moved to create a complete view or the beam must be scanned in a raster pattern.

  22. One way to accomplish this is to pass the illumination through a series of pinholes that have been arranged in a pattern. As this disk is spun it will create a raster pattern and the light coming back through the pinholes will be confocal. The pinholes in a spinning disk system act as both the point sources and confocal apertures.

  23. Spinning disk confocals: • Can image in “real” time provided that the disk is spun quickly enough • Can use a variety of light sources • Can be retrofitted to many existing fluorescence microscopes

  24. Spinning disk confocals: • Are inefficient and require a very bright illumination and fluorescence • Cannot use sensitive light detectors such as photomultiplier tubes

  25. Scanning Galvanometers Point Scanning Mirrors control beam movement in X/Y raster pattern x y Laser out To Microscope Laser in

  26. Some scan mirror systems are able to be rotated which can result in a rotation of the raster pattern

  27. By creating an image in a point by point manner the confocal microscope functions as a point scanning/signal detecting device and like an SEM magnification can be increased by scanning a smaller portion of the specimen

  28. Imperfections in conventional light optics usually restrict useful zoom to 6X or less.

  29. A modern confocal system consists of the microscope, associated lasers, the scan head with detectors and confocal apertures and a computer system that controls the scanning, adjusts the illumination, collects the signal, displays the images and stores the data for later image processing and analysis.

  30. A laser scanning confocal microscope has many components including a way for several different lasers to provide excitation wavelengths and several separate detectors for various emission wavelengths

  31. Although confocal microscopy can be done in the reflected (light backscattered) or even transmitted mode most systems are optimized for fluorescence.

  32. Light Sources - Lasers • Argon UV ArUV 351-364 nm • Solid State Violet 405 nm • Argon Ar 488-514 nm • Krypton-Ar ArKr 488-568-648 nm • Helium-Cad HeCd 442 nm • Helium-Neon GreNe 543 nm • Helium-Neon HeNe 633 nm

  33. Excitation - Emission Peaks % Max Excitation at 488 568 647 nm Fluorophore EXPeak EM peak DAPI 358 461 0 0 0 FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0 Texas Red 592 610 3 45 1 CY5 649 666 1 11 98

  34. Since the illumination wavelengths available are often limited the selection of matching fluorochromes is very important.

  35. In a conventional confocal scan head the photons returning from the specimen are separated based on their energies (color) by passing them through a series of filters and collecting each on separate PMTs

  36. One significant advance is how some systems separate the emission spectrum (signal) by wavelength and using slits sample those specific wavelengths using separate PMTs.

  37. One of the advantages of having separate control over the collection of different emission spectra is the ability to create evenly balanced double, triple, and even quadruple labeled images. Each of the signals can be collected simultaneously and merged afterwards.

  38. Spectral properties of the available dyes limit the experimental freedom. • Often it is even difficult to clearly separate two fluorescence markers. • With more markers, the problem grows increasingly complex. Cross-talk between the FP variants at the excitation and emisson level

  39. Fluorescent Proteins are essential for life science studies. However, overlapping emission AND excitation spectra and corresponding crosstalk makes combinations difficult for imaging! (especially true for multiphoton imaging) Heavy overlap!

  40. GFP and YFP (Distance of emission peaks ca. 12nm) A431 cells expressing GFP, Rab11-YFP GFP YFP overlay This type of separation is nearly impossible to accomplish with conventional filters, especially for weakly fluorescent samples

  41. By collecting a series of images of the specimen at different distances from the lens (focal planes) a through-focus series or “Z-series” can be created.

  42. The ability to collect data in the X,Y, and Z dimensions enables one to create an image of the specimen as if it were be observed from an orthogonal plane.

  43. Stereo pair images can be created from a stack of confocal images by a technique known as “pixel shifting” In pixel shifting two separate 2-D projections of the data set are created by shifting adjacent image planes slightly out of registry creating a pseudo-left and pseudo-right projection.

  44. The two images can be colorize to produce an anaglyph stereo pair. (PC12 cell stained for microtubules)

  45. Stereo Anaglyph Two dimensional projection of focus series

  46. Pacific coral in backscattered light mode

  47. Zebrafish embryo

  48. Muscle cells

  49. 3D Image Reconstruction y z x By accessing information in all three dimensions a 3-D reconstruction of the data is possible

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