Quantitative Imaging

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Amplification in the magnifying lens is not impeccable; the amplified picture is obscured ... The determination of a magnifying lens is the most brief separation two focuses ...

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Quantitative Imaging Using imaging to investigate atomic occasions in living cells Ann Cowan

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FUNCTION OF MICROSCOPY Function of any microscopy is NOT just to amplify! Capacity of the magnifying instrument is to RESOLVE fine detail.

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Magnification makes objects greater Magnification

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Magnification in the magnifying instrument is not impeccable; the amplified picture is obscured by diffraction Magnification

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RESOLUTION implies items can be viewed as independent articles Resolution

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RESOLUTION l  d N.A. The determination of a magnifying instrument is the most limited separation two focuses can be isolated and still be seen as 2 focuses. Not determined simply determined Well determined MORE IMPORTANT THAN MAGNIFICATION !!

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How to show signs of improvement determination? Picture plane Objective focal point example

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example How to show signs of improvement determination? Picture plane Objective focal point

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example How to show signs of improvement determination? Picture plane Objective focal point

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WHAT DETERMES RESOLUTION? Differentiation is important to distinguish detail (edges) from foundation Diffraction on a very basic level breaking points determination diffraction happens at the target focal point gap

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IMAGE OF A SELF-LUMINOUS POINT IN THE MICROSCOPE greatest First least Light from every purpose of the question is spread out in the magnifying instrument since light diffracts at the edges of the focal point = Airy Disk Objective focal point

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RAYLEIGH CRITERION Generally acknowledged basis of determination Single point sourcce Just determined Just determined Wel determined Intensity Central most extreme of one pinnacle overlies 1 st least of neighboring pinnacle

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What decides the separation between Peaks? Objective θ example The most extreme edge of light gathered by the goal focal point. Bigger edge of accumulation = Better determination

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Maximum edge of light gathered from a point decides width of Airy Disk q example Objective focal point Image plane Min remove between focuses: wavelength refractive list λ  d sin q n Numerical Aperture (N.A.) = n sin q

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Resolution accordingly is given by: l  d N.A. To lessen d , and in this way accomplish better determination:  wavelength  N.A. Light magnifying lens: greatest N.A. is 1.4, for noticeable (e.g. green light),  = 500 nm hence best determination is 0.2 um. Valuable amplification is constrained to 500-1000 X N.A., so around 1,000 X

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Contrast is required to see objects Increasing Contrast light from a protest should either be diverse in power or shading (= wavelength) from the foundation light

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Airy Disk

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AIRY DISK

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AIRY DISK 255 INTENSITY 0 Z-POSITION

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AIRY DISK 255 INTENSITY 0 Z-POSITION

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AIRY DISK 255 INTENSITY 0 Z-POSITION

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PSF Z

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Z psf

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FWHM Z-POSITION INTENSITY Z determination Z Resolution characterized as FWHM = the full width at half maximal force of a z line of a point hotspot For 1.4 N.A. focal point, Z determination ~ .5 um By Nyquist hypothesis, need to gather at 0.25 um Z steps

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 NA 4 mag 2 OBJECTIVE LENS Resolution  Intensity  > remedies Intensity (For epiflourescence; for transmission it is NA 2 of target time NA 2 of condenser)

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Digital Images Are Arrays of Numbers Value at every point is the measure of light gather from every point in a picture 2-D Image gets to be exhibit of force qualities (dark levels) from 0 - 255 (for 8 bit picture) or 0-4,126 for 12 bit picture. Every point in the exhibit is a pixel

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How CCD cameras Make a picture Figure 1. The pixels of a CCD gather light and change over it into parcels of electrical charge Figure 2. The charges are immediately moved over the chip. Figure 3. The charges are then cleared off the CCD and changed over to simple electrical driving forces, which are then measured as computerized numerical qualities.

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

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VOXELS ARE 3D PIXELS 2-D Image gets to be cluster of power qualities (dim levels) from 0 - 255 (for 8 bit picture) or 0-4,126 for 12 bit picture. Every point in the cluster is a pixel For progressive Z segment, 2D exhibits are stacked into 3D varieties of qualities, every component is known as a "voxel"

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DIGITAL IMAGE MANIPUTATIONS (controlling varieties of numbers in important ways) Frame averaging (time averaging on CCD) 2 + =

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DIGITAL IMAGE MANIPUTATIONS Output esteem Input esteem LUT (controlling varieties of numbers in significant routes) look into table (LUT) controls e.g. differentiate extending

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DIGITAL IMAGE MANIPUTATIONS (controlling varieties of numbers in significant ways) picture math e.g. proportion imaging =

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Image upgrade

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Original picture improved picture foundation picture upgraded - foundation picture outline arrived at the midpoint of upgraded - foundation

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FLUORESCENCE MICROSCOPY

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FLOURESCENCE Excited Energy States E Ground State lifetime t

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Stokes Shift

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EPIFLUORESCENCE First boundary channel Second hindrance channel dichroic reflect target focal point example

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Flourescence identification is straight and can be utilized to measure relative or supreme measures of particles If conditions are indistinguishable, 2X fluorescence = 2X amt of fluorophore Because light in the magnifying lens is spread out by diffraction, conditions inside and between pictures are not generally indistinguishable. Similarly as with any estimation, should be cautious with estimations Must be inside direct scope of finder (no 0's, not above most extreme level) Must subtract foundation (by and large sans cell range) ALL conditions in magnifying lens must be indistinguishable

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Fluorescent Ion Indicators Fluorescence properties change when particular particle is bound. For instance: fura-2 in low Ca 2+ excitation greatest at 360nm fura-2 in high Ca 2+ excitation most extreme at 340nm proportion of fluorescence power at the two wavelengths is a measure of the convergence of Ca 2+.

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Calcium-subordinate Excitation Spectra of FURA-2

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Image Math Bkgd redressed picture 340ex Cell with 340ex Bkgd with 340ex _ = Cell with 360ex Bkgd with 360ex Bkgd revised picture 360ex _ =

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Image Math Bkgd adjusted picture 340ex Ratio picture (340/360) Bkgd amended picture 360ex

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Dual Wavelength Ratios are Independent of the Amount of Fluorescent Indicator Ratioing takes out fading and color spillage antiques and along these lines are delicate just to the centralization of analyte

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Dual Wavelength Ratios Normalize for Variable Thickness inside a Sample (e.g. a cell under a magnifying instrument)

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Courtesy of Billy Tedford and John Carson

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TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF)

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TIRF energizes fluorescence just inside a restricted locale beside the substrate

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CONFOCAL MICROSCOPY

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

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coverslip example slide Conventional enlightenment Point checking light

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CONFOCAL MICROSCOPY photomultiplier Imaging opening lighting up gap dichroic in-center beams Out-of-center beams target focal point central plane

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Photomultipliers change over photons into a corresponding current

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

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X = 128 t = 0 Y = 128 t = 0.25 sec Scan Time Issues Typical output rate 1s/filter 512X512 t = 0 X = 512 Y = 512 t = 1 sec

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Scan Time Issues Two sweep sorts: 1. Unidirectional Bidirectional 2. Bidirectional checking can have speed restrictions and arrangement prerequisites

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Digital Zoom 10 X 8 = 80 focuses How near one another would we be able to filter?

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Sampling Theory The Nyquist Theorem portrays the examining recurrence ( f ) required to speak to the genuine character of the example. i.e., how near one another would it be a good idea for you to test a picture to realize that your example genuinely speaks to the picture? To catch the intermittent parts of recurrence f in a flag we have to test no less than 2 f times basically you should test at 2 times the most elevated recurrence .

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Sampling Theory Sample at = recurrence of picture determination Sample at ½ recurrence of picture determination

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Sampling Theory Using 1.4 N.A. focal point, max determination is 0.2 um To get 0.2 um determination in the last picture, you should test at 0.2/2 = .1 um/pixel. Over examining (< 0.1 um/pixel) causes all the more fading and phototoxicty with no expansion in determination. It can likewise bring about issues in measuring fluorescence pictures. Examining in Z works by a similar standard. Test at 1/2 x the z determination characterized by the focal point and confocal opening size.

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

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FLUORESCENCE REDISTRIBUTION AFTER PHOTOBLEACHING (FRAP)

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Fluorescence Redistribution

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Fluorescence Redistribution

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

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Photobleaching of cytoplasmic segments Images are gathered each 0.345 s

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Photobleaching of cytoplasmic parts Methods for breaking down the information begin with a proper model of the science

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"FLIP" Method: Repetitive dye and redistribution cycles, where development of fluorescent test out of unbleached area is investigated. Utilizes: Best strategy to dissect restricting rates, has been utilized to mark off rates of layer restricting proteins, for example, rac. Utilized additionally to gauge congruity inside/between cell compartments

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Fluorescence Loss After Photobleaching

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Photoactivatable GFP

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