Imaging Introduction. September 24, 2010
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1 Imaging Introduction September 24, 2010
2 What is a microscope? Merriam-Webster: an optical instrument consisting of a lens or combination of lenses for making enlarged images of minute objects; especially: compound microscope (a microscope consisting of an objective and an eyepiece mounted in a drawtube)
3 Why is it important to know how a microscope works?
4 Why is it important to know how a microscope works? Interpreting your images correctly
5 Why is it important to know how a microscope works? Interpreting your images correctly Take the best images possible
6 Why is it important to know how a microscope works? Interpreting your images correctly Take the best images possible Work independently
7 Why is it important to know how a microscope works? Interpreting your images correctly Take the best images possible Work independently Properly use it and not damage it ($35,000 - $70,000)
8 Why is it important to know how a microscope works? Interpreting your images correctly Take the best images possible Work independently Properly use it and not damage it ($35,000 - $70,000) Push the limits of the microscope and use it for new applications
9 Components of microscope
10 Components of microscope
11 Why do we care about this? 20x Objective on Upright Zeiss Not Aligned Aligned w/ Smallest Aperture Aligned w/ Medium Aperture Aligned w/ Largest aperture
12 63x Objective Not Aligned Aligned w/ Smallest Aperture Aligned w/ Medium Aperture Aligned w/ Largest aperture
13
14 Elements of transmitted light path Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
15 From Jeff Lichtman
16 Illumination Cone Diagram
17 Aperature Diaphram applet What would happen if the field diaphram was vared?
18 Benefits of Varying Field and Aperture Diaphragms Field Diaphragm Affects the field of view Stopping it down limits the area of view Stopping it down useful when trying to prevent loss of contrast due to scattered light-the field diaphragm thus can improve image contrast Does not affect the intensity of light in the illuminated region Aperture Diaphram Decreasing can increase contrast Decreasing increases depth of focus Increasing, increases resolution
19 Transmitted Light Methods Brightfield Pros: Does not require high intensity light Ideal for measurements because no distortion Cons: Contrast at the expense of resolution Our eyes see only changes in intensity, they cannot see changes in the phase of light, and light passing through unstained tissue does not change intensity very much because of only slight changes in the index of refraction. Ways to enhance contrast: Stain Tissue Phase Contrast DIC Bright field Phase DIC From FSU Microscopy
20 Phase Contrast Pros Can get contrast without losing resolution On living tissue, dead cells are dark, live cells are bright Cons Phase images are usually surrounded by halos around the outlines of details Requires special objective Phase annuli do limit the working numerical aperture thus resolution Not good with thick specimens Requires much more light than brightfield From FSU Microscopy
21 Differential Interference Contrast (DIC) Pros Can get contrast without losing resolution Prism does not need to be in the back focal plane of the objective Psuedo-3D shadow casting effect Good for thicker specimens because of optical sectioning Smaller specimen features are not obscured by adjoining regions having large optical gradients Cons Cost Sensitive only to gradients in a specific direction, so the image of a given object can change as it is rotated. Analyzer must be removed in fluorescence imaging Requires much more light than brightfield From FSU Microscopy
22 Next Up: Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
23 Next Up: Objective Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
24 How do you choose an objective? From Zeiss Brochure
25 Objective Innerds Why so many lenses? From Zeiss Brochure
26 We don t live in an ideal world Spherical Aberrations Field Curvature Chromatic Aberrations From Zeiss Brochure
27 Main Classes of Objectives Achromats 2 λ color & 1 λ spherical corrections Fluorite 3 λ color & spherical corrections Apochromats 4 λ color and spherical correction Plan Flat field corrected Sluder & Nordberg 2007
28 Main Classes of Objectives Achromats 2 λ color & 1 λ spherical corrections Fluorite 3 λ color & spherical corrections Apochromats 4 λ color and spherical correction Plan Flat field corrected IR and UV is always special and need to pay particular attention Sluder & Nordberg 2007
29 What do the corrections depend on? From Zeiss Brochure
30 Why do corrections matter? Co-localization Beads Fission Yeast North 2006
31 Image Sharpness Improper Coverslip thickness North 2006; FSU Microscopy
32 Image Sharpness Improper Coverslip thickness North 2006; FSU Microscopy
33 Numerical Aperature From Zeiss Brochure
34 Numerical Aperture Effects
35 Resolution: 1.22 λ / (NA obj + Na condenser ) More Next Week! From Jeff Lichtman
36
37 Tube Length From Zeiss Brochure
38 Ray Optics
39 From Jeff Lichtman
40 Infinity Corrected Microscope Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
41 Infinity Corrected Microscope Tube Lens Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
42 Benefits of Infinity Corrected Microscopes Flexibility: Inserting pieces of glass in Infinity space do not cause a lot of chromatic aberrations More correction lenses can go in the objective, so less aberrations
43 Elements of reflected light path Tube Lens Objective Condenser Aperture Diaphragm Collector Lens Field diaphragm Adapted from Sluder & Nordberg 2007
44 Elements of reflected light path Tube Lens Aperture Diaphragm Collector Lens Objective Condenser Field diaphragm Adapted from Sluder & Nordberg 2007
45 Elements of reflected light path Light manipulation are the same just comes from above! Field diaphragm in the incident light path-really useful to remove background!!! Filter Cube Tube Lens Aperture Diaphragm Collector Lens Objective Condenser Field diaphragm Adapted from Sluder & Nordberg 2007
46 Reflected Köhler Illumination
47 Reflected Köhler Illumination Why can we come from above in Fluorescence?
48 Spectral Separation Alexa 488 Excitation Emission This spectral separation of the excitation wavelengths and emission wavelengths allows us to use the same light path through the objective and separate emission from excitation when needed.
49 Why is there spectral separation? Jablonski Diagram
50 Where does the spectral spread come from?
51 The Filter Cube: How we separate light paths To Detector Emission Filter Excitation Filter Dichroic Mirror From Illuminator From FSU Microscopy
52 Dichroic Mirror Normal Mirror Designed to pass light above a certain wavelength and reflect light below it DM 565
53 Dichroic Mirror Reality % Transmittance Ideal Reality
54 Filter Cube Specs Excitation Dichroic Emission % Transmittance
55 2-photon considerations
56 How the Cube works To Detector Light Source Fluorecence Excitation light Fluorescent Specimen
57 Filter Design
58 Fluorescent Lamps
59 4 Major Fluorescent Lamp Sources Mercury Metal halide Xenon LED
60 4 Major Fluorescent Lamp Sources Mercury Metal halide Xenon LED
61
62 Binocular/Trinocular head From FSU Microscopy
63 Function of Eyepieces Image formation
64 Eyepieces Ever get your specimen into focus using your eyes and then you take the image with the computer and it is blurry or you see nothing (for confocal)? Sluder & Nordberg 2007
65 Eyepieces Ever get your specimen into focus using your eyes and then you take the image with the computer and it is blurry or you see nothing (for confocal)? Parfocality! Sluder & Nordberg 2007
66 Eyepieces From FSU Microscopy
67 Questions? Next Time Confocal Microscopy PSF 2-photon Microscopy
68 References Sluder, Greenfield, and Joshua J Nordberg Microscope basics. Methods in cell biology 81, no. 06: doi: /s x(06) North, Alison J Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. The Journal of cell biology 172, no. 1: doi: /jcb artid= &tool=pmcentrez&rendertype=abstract.
69 Imaging Path Illumination Path From Jeff Lichtman
70
71 Numerical Aperature 1.4NA Oil Lens 1.4 = (1.5) sin(u) u = 69 degrees
72
73
74
75
76
77 Inverted Microscope
78 From Jeff Lichtman
79 What is the main difference between these two? Barska Compound Microscope $300 Olympus BX51 $35,000
80 What is the main difference between these two? Barska Compound Microscope $300 Olympus BX51 $35,000 Infinity Correction!
81 Locations Alternate field stop location on other microscopes Field stop Neutral Density Filters
82 Aperature Diaphragm Condenser Turret Polarizer
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