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Quantitative Imaging

Quantitative Imaging . Using imaging to analyze molecular events in living cells. Ann Cowan. FUNCTION OF MICROSCOPY. Function of any microscopy is NOT simply to magnify! Function of the microscope is to RESOLVE fine detail. Magnification makes objects bigger. Magnification.

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Quantitative Imaging

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  1. Quantitative Imaging Using imaging to analyze molecular events in living cells Ann Cowan

  2. FUNCTION OF MICROSCOPY • Function of any microscopy is NOT simply to magnify! • Function of the microscope is to RESOLVE fine detail.

  3. Magnification makes objects bigger Magnification

  4. Magnification in the microscope is not perfect; the magnified image is blurred by diffraction Magnification

  5. RESOLUTION means objects can be seen as separate objects Resolution

  6. RESOLUTION l  d N.A. The resolution of a microscope is the shortest distance two points can be separated and still be observed as 2 points. Not resolved just resolved Well resolved MORE IMPORTANT THAN MAGNIFICATION !!

  7. How to get better resolution? Image plane Objective lens specimen

  8. specimen How to get better resolution? Image plane Objective lens

  9. specimen How to get better resolution? Image plane Objective lens

  10. WHAT DETERMES RESOLUTION? • Contrast is necessary to detect detail (edges) from background • Diffraction fundamentally limits resolution diffraction occurs at the objective lens aperture

  11. IMAGE OF A SELF-LUMINOUS POINT IN THE MICROSCOPE maximum First minimum Light from each point of the object is spread out in the microscope because light diffracts at the edges of the lens = Airy Disk Objective lens

  12. RAYLEIGH CRITERIONGenerally accepted criterion of resolution Single point sourcce Just resolved Just resolved Wel resolved Intensity Central maximum of one peak overlies 1st minimum of neighboring peak

  13. What determines the distance between Peaks? Objective θ θ specimen The maximum angle of light collected by the objective lens. Larger angle of collection = Better resolution

  14. Maximum angle of light collected from a point determines width of Airy Disk q specimen Objective lens Image plane Min distance between points: wavelength refractive index λ  d sinq n Numerical Aperture (N.A.) = n sinq

  15. Resolution therefore is given by: l  d N.A. • To reduce d, and therefore achieve better resolution: •  wavelength •  N.A. • Light microscope: • maximum N.A. is 1.4, • for visible (e.g. green light),  = 500 nm • thus best resolution is 0.2 um. Useful magnification is limited to 500-1000 X N.A., so about 1,000 X

  16. Contrast is required to see objects Increasing Contrast light from an object must either be different in intensity or color (= wavelength) from the background light

  17. Airy Disk

  18. AIRY DISK

  19. AIRY DISK 255 INTENSITY 0 Z-POSITION

  20. AIRY DISK 255 INTENSITY 0 Z-POSITION

  21. AIRY DISK 255 INTENSITY 0 Z-POSITION

  22. PSF Z

  23. Z psf

  24. FWHM Z-POSITION INTENSITY Z resolution Z Resolution defined as FWHM = the full width at half maximal intensity of a z line of a point source For 1.4 N.A. lens, Z resolution ~ .5 um By Nyquist theorem, need to collect at 0.25 um Z steps

  25. NA NA4 mag2 OBJECTIVE LENS • Resolution • Intensity  • > corrections Intensity (For epiflourescence; for transmission it is NA2 of objective time NA2 of condenser)

  26. Digital Images Are Arrays of Numbers Value at each point is the amount of light collect from each point in an image 2-D Image becomes array of intensity values (grey levels) from 0 -255 (for 8 bit image) or 0-4,126 for 12 bit image. Each point in the array is a pixel

  27. How CCD cameras Make an image Figure 1. The pixels of a CCD collect light and convert it into packets of electrical charge Figure 2. The charges are quickly moved across the chip. Figure 3. The charges are then swept off the CCD and converted to analog electrical impulses, which are then measured as digital numerical values.

  28. RGB (color ) IMAGE Display Red channel Green channel Blue channel

  29. VOXELS ARE 3D PIXELS 2-D Image becomes array of intensity values (grey levels) from 0 -255 (for 8 bit image) or 0-4,126 for 12 bit image. Each point in the array is a pixel For successive Z section, 2D arrays are stacked into 3D arrays of values, each element is called a “voxel”

  30. DIGITAL IMAGE MANIPUTATIONS (manipulating arrays of numbers in meaningful ways) • Frame averaging • (time averaging on CCD) 2 + =

  31. DIGITAL IMAGE MANIPUTATIONS Output value Input value LUT (manipulating arrays of numbers in meaningful ways) • look up table (LUT) manipulations e.g. contrast stretching

  32. DIGITAL IMAGE MANIPUTATIONS (manipulating arrays of numbers in meaningful ways) • image math e.g. ratio imaging =

  33. Image enhancement

  34. Original image enhanced image background image enhanced - background image frame averaged enhanced - background

  35. FLUORESCENCE MICROSCOPY

  36. FLOURESCENCE Excited Energy States E Ground State lifetime t

  37. Stokes Shift

  38. EPIFLUORESCENCE First barrier filter Second barrier filter dichroic mirror objective lens specimen

  39. Flourescence detection is linear and can be used to quantify relative or absolute amounts of molecules • If conditions are identical, 2X fluorescence = 2X amt of fluorophore • Because light in the microscope is spread out by diffraction, conditions within and between images are not always identical. • As with any measurement, need to be careful with measurements • Must be within linear range of detector (no 0’s, not above maximum level) • Must subtract background (generally cell-free area) • ALL conditions in microscope must be identical

  40. Fluorescent Ion Indicators Fluorescence properties change when specific ion is bound. For example: fura-2 in low Ca2+ excitation maximum at 360nm fura-2 in high Ca2+ excitation maximum at 340nm ratio of fluorescence intensity at the two wavelengths is a measure of the concentration of Ca2+.

  41. Calcium-dependent Excitation Spectra of FURA-2

  42. Image Math Bkgd corrected image 340ex Cell with 340ex Bkgd with 340ex _ = Cell with 360ex Bkgd with 360ex Bkgd corrected image 360ex _ =

  43. Image Math Bkgd corrected image 340ex Ratio image (340/360) Bkgd corrected image 360ex

  44. Dual Wavelength Ratios are Independent of the Amount of Fluorescent Indicator Ratioing helps eliminate bleaching and dye leakage artifacts and thus are sensitive only to the concentration of analyte

  45. Dual Wavelength Ratios Normalize for Variable Thickness within a Sample (e.g. a cell under a microscope)

  46. Courtesy of Billy Tedford and John Carson

  47. TOTAL INTERNAL REFLECTION FLUORESCENCE(TIRF)

  48. TIRF excites fluorescence only within a narrow region next to the substrate

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