Modes of light microscopy Choosing the appropriate system
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1 Modes of light microscopy Choosing the appropriate system Wide-field microscopy Confocal microscopy Multi-photon microscopy
2 Wide-field, inverted fluorescence microscope Nikon MicroscopyU
3 Endosome migration in living cells imaging a flat cell via wide field microscopy and a CCD
4 Some cells can be induced to be flat the cytoskeleton of a fibroblast grown on a solid substrate
5 But most cells are 3-dimensional 3D rendering of E-cadherin and nuclei in polarized epithelial cells
6 Cells are 3-dimensional the need for optical sections Conventional microscope Confocal microscope
7 Imaging 3-dimensional structures conventional epifluorescence Camera face
8 Imaging 3-dimensional structures Confocal microscopy
9 Serial confocal optical sections of the microtubule cytoskeleton of polarized epithelia Base Middle Apex 7.5µ 6.0µ
10 3-D rendering of the organization of microtubules in polarized epithelia Bob Bacallao
11 Resolution of confocal microscopy is only ~ 1.4 times better the big improvement lies in background rejection Conventional microscope Confocal microscope
12 Confocal microscopy builds an image by point scanning Need to acquire one point at a time. This limits acquisition to ~1 frame/sec. Limited by number of photons per pixel.
13 In a 1 second wide-field exposure all pixels are exposed (in parallel) for 1 second. For a 1 second acquisition using a confocal microscope, each pixel is collected for less than 4 µs, requiring 256,000 x higher incident intensity and 256,000 separate measurements. Point imaging is fundamentally different from wide-field imaging and generally requires lasers, sensitive detectors, and fast computers. These key components became available for common use in the 1980's.
14 Multi-color confocal microscopy - the apical recycling endosome is distinct from the trans-golgi network
15 Simultaneous analysis of multiple parameters - microtubules and chromosomes of a dividing epithelial cell Ruben Sandoval
16 Subcellular distributions of internalized transferrin and Rab25 in living MDCK cells
17 Multicolor confocal microscopy Image is built up through a raster scan requiring approximately 1 second figure through the unknowing courtesy of the Integrated Microscopy Resource, Madison, Wisconsin
18 BioRad MRC-1024 Lightpath M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
19 BioRad 488 nm Excitation M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
20 BioRad Fluorescein Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
21 BioRad Fluorescein Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
22 BioRad Fluorescein Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
23 BioRad Fluorescein Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
24 BioRad Fluorescein Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
25 BioRad 568 nm Excitation M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
26 BioRad Rhodamine Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
27 BioRad Rhodamine Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
28 BioRad Rhodamine Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
29 BioRad Rhodamine Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
30 BioRad 647 nm Excitation M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
31 BioRad Cy-5 Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
32 BioRad Cy-5 Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
33 BioRad Cy-5 Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
34 BioRad Cy-5 Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
35 BioRad Cy-5 Emission M4 M2 PMT 2 Iris Aperture Double Dichroic (T1) Neutral Density Filter Wheel Laser Lines Emission Filter Wheel Excitor Filter Wheel PMT 3 560DCLP (T2A) 640DCSP M1 PMT 1 M3
36 Advantages of scanning by Acousto-optical tuneable filters (AOTFs) Modulate individual lines Sequential multicolor Blank retrace Continuous modulation
37 Spectral confocal microscopy Zeiss LSM510-Meta detector system 32 channel Metadetector collects spectrum of each pixel deconvolution permits distinction of multiple, closely spaced fluorophores linear unmixing
38 Zeiss LSM510-Meta detector system
39 Zeiss LSM510-Meta system 20 channels of fluorescence
40
41 Zeiss 510 META linear unmixing of GFP and YFP Original image Image after linear unmixing
42 GFP versus autofluorescence VC4 and VC5 motor neurons (green) + gut autofluorescence (red) Removal of autofluorescence using spectral fitting and unmixing Imaged by David Miller
43 Practical confocal microscopy Image collection settings Objective choice
44 Practical confocal microscopy Image collection settings - prioritizing the pinhole, PMT and laser power
45 Effect of pinhole diameter on image quality Institut Jacques Monod
46 Effect of PMT Voltage on Signal, and Signal-to-Noise Ratio PMT1-mean PMT1-S/N 200 Biorad-PMT PMT1-mean intensity PMT1-signal-to-noise ratio PMT gain
47 Effect of Laser Power on Signal Relative fluorescence % NDF 3% NDF 10% NDF Number of scans 30% NDF
48 Practical confocal microscopy Image collection settings Objective choice
49 Practical confocal microscopy Image collection settings Objective choice Chromatic aberration
50 Axial chromatic aberration
51 Axial chromatic aberration
52 Chromatic aberration - F-Cy5 ratios and the Plan fluor 40 Single section (solid line) percentage Projection (dashed line) R/F ratio
53 Practical confocal microscopy Image collection settings Objective choice Chromatic aberration Spherical aberration
54 Spherical aberration
55 100x, Planapo, Oil immersion objective 0 microns depth 35 microns depth Spherical aberration 60x, Planapo, Water immersion objective 0 microns depth 66 microns depth
56 Collar adjustment of the 60x water immersion objective
57 Collar adjustment of the 60x water immersion objective Ruben Sandoval
58 Objective corrections require multiple lens elements Corrections: Color - Achromat, Apochromat Flat Field - Plan Immersion Media Cover Glass Polarization UV, IR transmission The more correction that a lens uses, the less transmission
59 100X, Oil immersion, NA X, Oil immersion, NA X, Oil immersion, NA X, Water immersion, NA X, Water immersion, NA.75 Light collection efficiency 10 4 x (NA 2 /mag) Corrections (# elements - transmission) Planapochromat 10 66% Plan-fluor 6 79% Planapochromat 10 66% Planapochromat 10 66% Plan-fluor 6 79% Ask yourself Is correction for chromatic aberration important to you?
60 100X, Oil immersion, NA X, Oil immersion, NA X, Oil immersion, NA X, Water immersion, NA X, Water immersion, NA.75 Light collection efficiency 10 4 x (NA 2 /mag) Corrections (# elements - transmission) Planapochromat 10 66% Plan-fluor 6 79% Planapochromat 10 66% Planapochromat 10 66% Plan-fluor 6 79% Ask yourself Is correction for chromatic aberration important to you?
61 Confocal microscopy - 2-dimensional imaging detectors
62 Confocal microscopy builds an image by point scanning and so is slow Need to acquire one point at a time. This limits acquisition to ~1 frame/sec. Limited by number of photons per pixel.
63 Spinning disk confocal microscope Spinning disk scans 1200 spots across a field at 30 Hz 360 Hz frame rate, we ve collected at up to 40 Hz Characterize rapid dynamics Signal to noise better than conventional confocal systems Reduce illumination for reduced phototoxicity and photobleaching Figure courtesy of Florida State Univ.
64 Transferrin labeled endosomes migrating around an MDCK cell Perkin-Elmer/Yokagawa spinning disk video-rate confocal microscope 1100 time points collected at 11 fps over 100 seconds
65 Real-time movement of Rab25 around an MDCK cell Perkin-Elmer/Yokagawa spinning disk confocal microscope 600 time points collected and displayed at 20 fps over 30 seconds
66 Living MDCK cells expressing GFP-tubulin and GFP-Rab7 Perkin-Elmer/Yokagawa spinning disk video-rate confocal microscope
67 Axial resolution of the Ultraview and the Zeiss 510 confocal microscopes 100x objectives 1 Vertical resolution 100x objective Relative intensity microns from focus
68 Axial resolution of the Ultraview and the Zeiss 510 confocal microscopes 60x objectives 1 Vertical resolution 60x objective Relative intensity microns from focus
69 Ultraview system shows large linear range characteristic of CCD detectors corrected mean s/n Perkin-Elmer Integration time test mean signal signal-to-noise ratio integration time (ms)
70 The Ultraview system for imaging living cells Rab25 in MDCK cells Zeiss 510 PerkinElmer Ultraview 200 images collected at 2 frames per second, 0.13 micron pixels, 12 bit pixel depth
71 Frame 1 Frame 200 PerkinElmer Ultraview Zeiss 510
72 1 Fraction of initial fluorescence frame number S/N - initial S/N - final Ultraview Comparison of imaging performance Zeiss 510 versus PerkinElmer Ultraview Images collected at 1.7 frames per second, micron pixels, 12 bit pixel depth Zeiss
73 Why is the Ultraview so good? 1/1200 the intensity less saturation Integrate 8 µs x 360 per second vs. 4 µs 2-fold higher quantum efficiency 360 breaks between illuminations Less digitizer and amplifier noise
74 Field imaging (Ultraview) versus point scanning confocal systems Field imaging detectors CCDs are very clean, with wide dynamic range Images can be collected rapidly Different colors must be collected sequentially, or on different detector arrays Point scanning detectors Multiple colors can be collected simultaneously PMTs are noisier than CCD systems Slow image acquisition and reasonable frame rates require very brief collection per pixel high illumination and few photons
75 Multi-photon microscopy
76 3D imaging of kidney tissue by confocal microscopy
77 3D imaging of kidney tissue by 2-photon microscopy
78 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons
79 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
80 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
81 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
82 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
83 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
84 Multi-photon fluorescence excitation depends upon the simultaneous absorption of multiple photons What is simultaneous? Multiple photons must arrive within the duration of the intermediate virtual state of the electron ~ 1 attosecond (10-18 seconds) What is the relative frequency of such absorptions? Winfried Denk calculated that a molecule of rhodamine B exposed to direct sunlight will experience: A one-photon absorption around once per second. A two photon absorption once every 10,000 years. A three-photon absorption... well actually never in the history of the universe.
85 How to increase the probability of multi-photon absorption for multiphoton microscopy? You could increase illumination 600,000 fold.
86 How is the probability of multi-photon absorption increased in multiphoton microscopy? Photon crowding Dave Piston, Vanderbilt
87 How is the probability of multi-photon absorption increased in multiphoton microscopy? Photon crowding Photon crowding in space - the cross-sectional density of photons is highest at the focal point. Photon crowding in time - laser emissions are pulsed into brief (~100 femtosecond) packets
88 How is the probability of multi-photon absorption increased in multiphoton microscopy? Photon crowding Photon crowding in space - the cross-sectional density of photons is highest at the focal point. Photon crowding in time - laser emissions are pulsed into brief (~100 femtosecond) packets
89 Pulsed laser emissions provide for power sufficient for multiphoton absorption without photo-damage Pulsed laser provides low average power but peak power high enough for 2- photon absorption Sample illuminated for only 8 one million th of the pixel dwell time
90 One and two photon fluorescence excitation One photon absorption is proportional to illumination Fluorescence is stimulated throughout the lightpath Brad Amos - MRC Two-photon absorption is proportional to the squared power of illumination Photon density sufficient to excite fluorescence occurs only at the focal point
91 Fluorescence microscopy time pixel dwell time 4 microseconds 100X fluorescence lifetime 10 nanoseconds 100,000X 800 nm pulse length 100 femtoseconds 10,000X intermediate virtual state 10 attoseconds duration (seconds)
92 Benefits of Multi-Photon Microscopy no photobleaching in out-of-focus planes figure through the unknowing courtesy of the Integrated Microscopy Resource, Madison, Wisconsin
93 Figure courtesy of the National High Magnetic Field Laboratory Florida State University And Dave Piston, Vanderbilt Univ. Multiphoton photobleaching is limited to the focal point
94 Benefits of Multi-Photon Microscopy no photobleaching in out-of-focus planes no emission aperture - less loss to scattering figure through the unknowing courtesy of the Integrated Microscopy Resource, Madison, Wisconsin
95 Multi-photon microscopy is less sensitive to light scatter by tissues attenuation of signal Image courtesy of Istituto Nazionale per la Fisica della Materia - Genoa
96 Benefits of Multi-Photon Microscopy no photobleaching in out-of-focus planes no emission aperture - less loss to scattering IR light penetrates deeper, with less damage figure through the unknowing courtesy of the Integrated Microscopy Resource, Madison, Wisconsin
97 Multi-photon Excitation Allows Deeper Imaging in Intact Tissue Confocal Two-Photon Excitation Dave Piston, Vanderbilt
98 Imaging complex structures Kidney tubules of a newborn mouse kidney Carrie Phillips, Nephrology and Pathology
99 Imaging complex structures Kidney tubules of a newborn mouse kidney Carrie Phillips, Nephrology and Pathology
100 Single optical section of neural network
101 Imaging complex structures Neural network in mouse brain adapted to chronic alcohol Feng Zhou, Anatomy and Cell Biology
102 Imaging complex structures Segmentation of a single neuron Feng Zhou, Anatomy and Cell Biology
103 Imaging complex structures Neural network in mouse brain adapted to chronic alcohol Feng Zhou, Anatomy and Cell Biology
104 Multiphoton imaging of kidney
105 3D imaging of newborn mouse kidney by 2-photon microscopy Carrie Phillips
106 Imaging complex structures Glomerulus of a newborn mouse kidney Carrie Phillips, rendered by Anatomical Travel, Inc.
107 Imaging complex structures branching tubulogenesis in an embryonic mouse kidney Carrie Phillips, Nephrology and Pathology
108 140 micron thick volume of embryonic mouse kidney - branching tubulogenesis Carrie Phillips... and real time rendering on a PC using Voxx software Jeff Clendenon
109 Vital imaging by multiphoton microscopy The extended depth provided by multi-photon microscopy permits high-resolution imaging of the cells of living animals. The ability to image multiple fluorophores supports correlations of multiple proteins and physiological processes.
110 Experimental arrangement for intravital imaging of rat kidney Anesthesia is provided via 1% halothane and low-flow oxygen. Fluorescent probes are administered via tail-vein injection. Blood gases are monitored via femoral artery.
111 Two-photon image of kidney of living rat injected with Hoechst, 500 Kd fluorescein dextran and 3 Kd Texas-Red dextran Ruben Sandoval and Katrina Kelly
112 Multiple functions apparent in images of kidneys of animals injected with large and small dextrans proximal tubule endocytosis glomerular filtration tubular solute concentration capillary blood flow
113 Multiple functions apparent in images of kidneys of animals injected with large and small dextrans proximal tubule endocytosis glomerular filtration tubular solute concentration capillary blood flow
114 2-photon microscopy of glomerular filtration in a living rat Hoechst-labeled nuclei, 500 Kd fluorescein dextran in blood, 3 Kd Texas-Red dextran in lysosomes and tubule lumens Ruben Sandoval and Katrina Kelly
115 Multiple functions apparent in images of kidneys of animals injected with large and small dextrans proximal tubule endocytosis glomerular filtration tubular solute concentration capillary blood flow
116 2-photon microscopy of tubular solute concentration in a living rat Hoechst-labeled nuclei, rhodamine-albumin in blood, 3 Kd fluorescein dextran in lysosomes and tubule lumens Ruben Sandoval and Katrina Kelly
117 Multiple functions apparent in images of kidneys of animals injected with large and small dextrans proximal tubule endocytosis glomerular filtration tubular solute concentration capillary blood flow
118 Imaging capillary blood flow by 2-photon microscopy Rhodamine-albumin, 3K fluorescein dextran and Hoechst
119 Imaging capillary blood flow (and proximal tubule autofluorescence) by 2-photon microscopy 500K fluorescein dextran and Hoechst
120 Practical multiphoton microscopy
121 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
122 2 photon versus confocal microscopy POP go the endosomes One photon image at first, tenth, whatever scan Two photon image Two photon image at fourth scan
123 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
124 Deep tissue imaging by multi-photon microscopy 2 photon image collected 120 microns into kidney section
125 Imaging complex structures Dendritic spines in mouse hippocampal neuron Feng Zhou, Anatomy and Cell Biology
126 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
127 2-photon cross sections are not necessarily predicted by single photon excitation spectra BioRad
128 2-photon fluorescence excitation 2-photon cross sections Xu et al., BioImaging, 1996
129 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
130 Imaging complex structures Comparing multiple probes in kidney tissue Ruben Sandoval, Nephrology
131 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
132 2-photon image volume of kidney of a living rat (before any fluorescence labeling)
133 2-photon image autofluorescence and Texas-Red gentamicin A tuneable laser is a good idea 820 nm excitation 860 nm excitation
134 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options
135 Signal attenuation with depth into fixed tissues - Multiphoton microscopy Top view Side view
136 32% 80% Signal attenuation with depth in multiphoton microscopy 10 µm Live animal 34 µm 19 µm Fixed tissue 50 µm 81 µm 63 µm 2x
137 Multi-photon microscopy is less sensitive to light scatter by tissues attenuation of signal Mouse Imaging Centre The Hospital for Sick Children University of Toronto
138 Sources of signal attenuation at depth Fluorescence emissions scattering refraction spherical aberration Illumination scattering refraction spherical aberration
139 Red emissions Green emissions 12 µm Use Red fluors scattering decreases as the fourth power of wavelength 127 µm 860 nm excitation
140 Two color 2-photon imaging of 13 day old embryonic mouse kidney [ microns ] Red - peanut agglutinin Green - Len culinaris agglutinin
141 Non-descanned detectors collect scattered light more efficiently Descanned PMT Non-descanned PMT
142 Non-descanned detectors collect scattered light more efficiently Descanned PMT Non-descanned PMT
143 Sources of signal attenuation at depth Fluorescence emissions scattering refraction spherical aberration Illumination scattering refraction spherical aberration
144 17ocbleach Attenuation of illumination is reflected by lower photobleaching at depth After 1 scan After 40 scans surface 39 µm Attenuation of illumination photobleaching rates Live rat, 800 nm excitation, Hoechst
145 Illumination attenuation decreases at longer wavelengths light scatter decreases with wavelength nm 740 nm photobleaching decay rate depth
146 Signal attentuates with depth into kidneys, but can be regained with increased illumination
147 Signal attentuates with depth into kidneys, but can be regained with increased illumination And, unlike confocal microscopy, scatter has minimal effect on resolution
148 Practical multiphoton microscopy Photobleaching Resolution Multiphoton fluorescence dyes Imaging multiple colors Laser choices Detector options Objective choice
149 Objectives for Multiphoton Microscopy A new generation of microscope objectives is being developed that is optimized for infrared transmission, and designed for light collection rather than image formation.
150 The importance of high numerical aperture to 3d microscopy - XY planes of a kidney sample 20x, NA.75 60x, NA 1.2
151 The importance of high numerical aperture to 3d microscopy - XY planes of a kidney sample 20x, NA.75 60x, NA 1.2
152 Increase in the use of multiphoton microscopy since number of publications years since 1993
153 Increase in the use of multiphoton microscopy since 1993 total "kidney or renal, but not cultured cells" number of publications years since 1993
154 Summary Which system to choose? Do you need to image? Do you need optical sections? Confocal or image deconvolution? Confocal or multi-photon?
155 Summary Which system to choose? Do you need to image? Do you need optical sections? Confocal or image deconvolution? Confocal or multi-photon?
156 Summary Which system to choose? Do you need to image? Do you need optical sections? Confocal or image deconvolution? Confocal or multi-photon?
157 Summary Which system to choose? Do you need to image? Do you need optical sections? Confocal or image deconvolution? Confocal or multi-photon?
158 pixel dwell time 4 microseconds 100X fluorescence lifetime 10 nanoseconds 100,000X 800 nm pulse length 100 femtoseconds 10,000X intermediate virtual state 10 attoseconds duration (seconds)
Why and How? Daniel Gitler Dept. of Physiology Ben-Gurion University of the Negev. Microscopy course, Michmoret Dec 2005
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