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 Magnification makes objects bigger

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

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

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

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

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

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

10. WHAT DETERMES RESOLUTION? 1.Contrast is necessary to detect detail (edges) from background 2.Diffraction fundamentally limits resolution diffraction occurs at the objective lens aperture

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

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

13. Central maximum of one peak overlies 1 st minimum of neighboring peak Just resolved RAYLEIGH CRITERION Generally accepted criterion of resolution Intensity Just resolvedWel resolved Single point sourcce

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

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

16. d 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. To reduce d, and therefore achieve better resolution: 1.  wavelength 2.  N.A. Useful magnification is limited to 500-1000 X N.A., so about 1,000 X Resolution therefore is given by:

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

18. Airy Disk

19. AIRY DISK

20. INTENSITY Z-POSITION 255 0

21. AIRY DISK INTENSITY Z-POSITION 255 0

22. AIRY DISK INTENSITY Z-POSITION 255 0

23. PSF Z

24. Z psf

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

26. OBJECTIVE LENS Resolution  Intensity  > corrections Intensity NA NA 4 mag 2 (For epiflourescence; for transmission it is NA 2 of objective time NA 2 of condenser)

27. OBJECTIVE LENS For thick specimens, spherical aberrations from the mismatch in refractive index between glass and water lead to large distortions as you move away from the coverslip. Spherical aberrations not only create geometric distortions but also significantly reduce the amount of light collected The c-apochromat lenses use water instead of oil as the immersion medium and use a correction collar to correct for the precise amount of glass (coverglass) between the immersion medium and the aqueous sample. This allows the lens to properly correct for spherical aberration at different sample depths. The thickness of the coverglass must be measured to properly set the correction collar. Spherical Aberration an important issue for thick specimens

28. 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

29. 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. How CCD cameras Make an image

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

31. 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” VOXELS ARE 3D PIXELS

32. + = 2 Frame averaging (time averaging on CCD) (manipulating arrays of numbers in meaningful ways) DIGITAL IMAGE MANIPUTATIONS 05155 210255 27164 05157 35160 011271 05145 27180 15152 111263 16153 16163

33. (manipulating arrays of numbers in meaningful ways) DIGITAL IMAGE MANIPUTATIONS look up table (LUT) manipulations e.g. contrast stretching Input value Output value LUT 05155 210255 27164 05157 00500 002550 00600 00500

34. (manipulating arrays of numbers in meaningful ways) DIGITAL IMAGE MANIPUTATIONS = image math e.g. ratio imaging 255075100 25100150100 25100150100 25100150100 5555 510 5 5 5 5555 5 1520 5101520 5101520 5101520

35. + = 2 Input value Output value = Frame averaging (time averaging on CCD) look up table (LUT) manipulations e.g. contrast stretching image math e.g. ratio imaging (manipulating arrays of numbers in meaningful ways) LUT DIGITAL IMAGE MANIPUTATIONS 05155 210255 27164 05157 35160 011271 05145 27180 15152 111263 16153 16163 05155 210255 27164 05157 00500 002550 00600 05157 255075100 25100150100 25100150100 25100150100 5555 510 5 5 5 5555 5 1520 5101520 5101520 5101520

36. Image enhancement

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

38. FLUORESCENCE MICROSCOPY

39. FLOURESCENCE Ground State Excited Energy States E t lifetime

40. Stokes Shift

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

42. 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 1.Must be within linear range of detector (no 0’s, not above maximum level) 2.Must subtract background (generally cell-free area) 3.ALL conditions in microscope must be identical

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

44. Calcium-dependent Excitation Spectra of FURA-2

46. Image Math = 285279105 26102156112 28104152104 275879112 3245 12612 3424 284 6152126 7131928 9131825 10121728 1566 2348 4335 5228 255075100 25100150100 25100150100 255075100 _ 5101520 5101520 5101520 5101520 _ = Cell with 340ex Bkgd with 340ex Bkgd corrected image 340ex Cell with 360exBkgd with 360exBkgd corrected image 360ex

47. Image Math 255075100 25100150100 25100150100 255075100 5101520 5101520 5101520 5101520 Bkgd corrected image 340ex Bkgd corrected image 360ex 5555 510 5 5 5 5555 Ratio image (340/360)

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

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

51. Courtesy of Billy Tedford and John Carson

52. TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF)

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

54. CONFOCAL MICROSCOPY

55. Diffracted light is spread out in Z as well as x and y. x,y plane x,z plane

56. coverslip specimen slide coverslip specimen slide Conventional illumination Point scanning illumination

57. focal plane objective lens Out-of-focus rays in-focus rays illuminating aperture dichroic Imaging aperture photomultiplier CONFOCAL MICROSCOPY

58. Photomultipliers convert photons into a proportional current

59. Widefield Fluorescence Confocal White et al. 1987. J. Cell Biol. 105: 41-48

60. Scan Time Issues Typical scan rate 1s /scan 512X512 t = 1 sec X = 512 Y = 512 t = 0 X = 128 Y = 128 t = 0 t = 0.25 sec

61. Scan Time Issues Two scan types: 1. 2. Bidirectional Unidirectional Bidirectional scanning can have speed limitations and alignment requirements

62. Digital Zoom How close together can we scan? 10 X 8 = 80 points

63. Sampling Theory The Nyquist Theorem describes the sampling frequency (f) required to represent the true identity of the sample. –i.e., how close together should you sample an image to know that your sample truly represents the image? To capture the periodic components of frequency f in a signal we need to sample at least 2f times in essence you must sample at 2 times the highest frequency.

64. Sampling Theory Sample at = frequency of image resolution Sample at ½ frequency of image resolution

65. Sampling Theory Using 1.4 N.A. lens, max resolution is 0.2 um To get 0.2 um resolution in the final image, you must sample at 0.2/2 =.1 um/pixel. Over sampling (< 0.1 um/pixel) causes more bleaching and phototoxicty with no increase in resolution. It can also cause problems in quantifying fluorescence images. Sampling in Z works by the same principle. Sample at 1/2 x the z resolution defined by the lens and confocal aperture size.

66. ANALYZING DYNAMIC EVENTS WITH FLUORESCENCE MICROSCOPY (THE "F" TECHNIQUES)

67. FLUORESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING (FRAP)

68. BLEACH DIFFUSION INTENSITY POSITION INTENSITY POSITION INTENSITY POSITION NO DIFFUSION INTENSITY INITIAL FLUORESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING

69. Fluorescence Redistribution

71. BLEACH INTENSITY POSITION INTENSITY POSITION INITIAL POSITION DIFFUSION INTENSITY FLUORESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING NO DIFFUSION INTENSITY

72. Initial slope = Diffusion coefficient % recovery = fraction of molecules diffusing FRAP ANALYSIS

73. Photobleaching of cytoplasmic components Images are collected every 0.345 s

74. Photobleaching of cytoplasmic components Methods for analyzing the data start with an appropriate model of the biology

75. “FLIP” Method: Repetitive bleach and redistribution cycles, where movement of fluorescent probe out of unbleached region is analyzed. Uses: 1.Best method to analyze binding rates, has been used to measure off rates of membrane binding proteins such as rac. 2.Used also to measure continuity within/between cellular compartments

76. Fluorescence Loss After Photobleaching

77. Photoactivatable GFP GFP exists as a neutral phenolic form that absorbs at 413 nm, and an anionic phenolate form that absorbs at 488 nm. Wild-type GFP, as well as the photoactivatable GFP ("PA- GFP"), can be excited at ~400 nm and ~488 nm. Absorption of 413 nm light converts the neutral to the anionic form, producing the observed photoactivation PA-GFP contains a T203H mutation that biases the equilibrium toward the neutral form. Wild-type GFP shows a 2-3 fold increase in fluorescence after activation, PA-GFP fluorescence increases 50-100 fold. photoactivated for ~1 s at 413 nm

78. Photoactivatable GFP photoactivated for ~1 s at 413 nm

79. FLUORESCENCE CORRELATION SPECTROSCOPY

80. Fluorescence Fluctuations time Intensity

81. Analysis of fluorescence fluctuations Intensity = # molecules time 1 molecule Related to D 2 molecules 1 molecule Related to concentration amplitude: number of molecules Decay time: diffusion time G(t) Time Correlation function

82. Fluctuation trace