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 Magnification makes objects bigger

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

Resolution RESOLUTION means objects can be seen as separate objects

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. 

Image plane How to get better resolution? Objective lens specimen

Objective lens Image plane How to get better resolution? specimen

Objective lens Image plane How to get better resolution? specimen

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

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

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

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

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

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

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 X N.A., so about 1,000 X Resolution therefore is given by:

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

Airy Disk

AIRY DISK

INTENSITY Z-POSITION 255 0

AIRY DISK INTENSITY Z-POSITION 255 0

AIRY DISK INTENSITY Z-POSITION 255 0

PSF Z

Z psf

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

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)

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

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 (for 8 bit image) or 0-4,126 for 12 bit image. Each point in the array is a pixel

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

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

2-D Image becomes array of intensity values (grey levels) from (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

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

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

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

+ = 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

Image enhancement

Original image enhanced image background image enhanced - background image frame averaged enhanced - background

FLUORESCENCE MICROSCOPY

FLOURESCENCE Ground State Excited Energy States E t lifetime

Stokes Shift

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

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

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

Calcium-dependent Excitation Spectra of FURA-2

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

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

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

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

Courtesy of Billy Tedford and John Carson

TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF)

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

CONFOCAL MICROSCOPY

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

coverslip specimen slide coverslip specimen slide Conventional illumination Point scanning illumination

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

Photomultipliers convert photons into a proportional current

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

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

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

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

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.

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

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.

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

FLUORESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING (FRAP)

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

Fluorescence Redistribution

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

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

Photobleaching of cytoplasmic components Images are collected every s

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

“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

Fluorescence Loss After Photobleaching

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 fold. photoactivated for ~1 s at 413 nm

Photoactivatable GFP photoactivated for ~1 s at 413 nm

FLUORESCENCE CORRELATION SPECTROSCOPY

Fluorescence Fluctuations time Intensity

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

Fluctuation trace