Selection of Imaging Modality

1
Type of Spectrophotometry
Expanded UV-visible region
1021
10-10
200
1019
Ultraviolet
X-ray
10-8
Violet
400
1017
10-6
Blue
Vacuum Ultraviolet
Wavelength (nm)
1015
Green
Wavelength (cm)
Ultraviolet-visible
10-4
Frequency (Hz)
Infrared
1013
Yellow
600
10-2
Orange
1011
Micro
Red
1
800
Wave
109
102
Radio (nmr)
107
104
Parts of the Electromagnetic Spectrum which are
employed for Spectrophotometry
2
Chemical Bond Energy
E hu hc / l h planks 6 x 10-34 J
sec c 3 x 108 m/sec So for green light
(500 hm) E 2 x 10-19 J / photon
ATP - 10 Kcal / mole 4 x 104 joules /
mole Avagadros 6 x 10-23 molecules /
mole Bond Energy of Terminal 1 x 10-19
J Phosphate of ATP
3
Fluorescent Probe Chemistry

• Absorbance (Excitation Wavelength)
• x Extinction Coefficient (A/mole)
• (Beers Law)

Fluorescence X Quantum Efficiency
QE is effected by a range of environmental
factors As well as the efficiency of the
recording device
4
Quantum Efficiency
Fl x Quantum Efficiency
How Much Absorbed Light Gets Converted to
Fluorescence
5
Factors Effecting Quantum Yield
Phenol
FLUORESCENCE INTENSITY
Phenol
FLUORESCENCE INTENSITY
FLUORESCENCE INTENSITY
Analine
Analine
pH
6 8 10 12 14 16 18 20 22 24 26
Concentration
Temperature
6
Calcium Green Fura-2
Calcium Green fluorescence enhancement
FURA-2 absorbance (Excitation) shifts
Ratio Imaging
7
Loading Fura-2 with a Patch Pipette
AM (Acetoxymethylester)- Loading
8
Filter Wheel System for Ca2 Analysis
PC
9
Photobleaching
Fluorescein Actin Texas Red - Tubulin
10
Multi-Spectral Imager
11
Astroglia and Neurons GFAP-Alexa and Propidium
Iodide
PI gt 600 nm
Overlay
488 nm Excitation
Alexa 530
12
Fluorescence Energy Transfer
Fluorophores must be less than 1 nm apart for
FRET to occur
13
Functional FRET Probes
INACTIVE
ACTIVE
Enzymes, Protein Kinases, Ca2 Binding Proteins
calmodulin
FRET Occurs when Fluorophores are less than 100
Angstroms Apart
14
Fluorescence Energy Transfer
Intensity of Absorption/Fluorescence
EX 1
EM 1
EX 2
EM 2
Wavelength
In FRET a Decrease in Fluorescence of the Donor
is Equal to the Increase in Fluorescence of the
Acceptor
15
3D Imaging Point Spread Function
Through-Focus Imaging of a 0.2 mm Fluorescent Bead
16
Confocal Microscopy
- Phototube
17
Confocal Microscopy
Upsides Direct viewing from a non-blurry
section. Works great for thick samples. Downsid
es Laser light is powerful, and can easily
bleach the sample- best for fixed
preparations. For 3D imaging scanning in both x,y
(2d miage) and z-dimensions required- lots of
bleaching. Can be rather slow for a full 2D image
18
Demonstration of Two-Photon Excitation
Cone of Excitation Using Standard Optics
Two-Photon Spot Illumination
Energy in 2 680 nm Photons Energy in 1 340 nm
Photon
19
3D Imaging Point Spread Function
Through-Focus Imaging of a 0.2 mm Fluorescent Bead
20
3D Deconvolution Imaging
Through-Focus Imaging of a 0.2 mm Fluorescent Bead
21
Phospholamdan Distribution in Mouse Aorta
Vascular Smooth Muscle Cell
Original Data
Deconvolved Data
Optical section at basolateral membrane
Cell-Matrix Interface
22
SERCA3 (Red) and SERCA2 (Green) Distribution in
MAEC
N
N
N
N
N
N
N
N
N - Nucleus
23
Smooth Muscle Cell Expressing Golgi-Targeted GFP
and Loaded with Texas Red Dextran by Pinocytosis
GOLGI
N
ENDOSOMES / LYSOSOMES
24
Overlay Images from a SMC Loaded for
Mitochondria (DASPEI Green) Lysosomes
(DND-189, Red)
37oC
Simultaneous Acquisition 470 nm EX 510 and
gt570 nm EM
25
3D Deconvolution Imaging
Upsides Images can be collected rapidly with
little photobleaching imaging a
live cells without photo-damage. Images of dim
samples can be acquired maximized light
throughput. Downsides Since image processing
is required, it takes time to obtain the final
deblurred images. In thick samples, there is too
much information (blurred light) to accurately
process the data to obtain high resolution data
sets.
26
Selection of Imaging Modality

• Temporal Resolution How Fast is the Response?
  Smooth vs Skeletal Muscle
• Spatial Resolution Whole Field of
  View? Individual Cells within a
  Field? Subcellular Compartments?
• Spectral Resolution Single Probe
  Distribution 2-4 Wavelengths Wide-Band
  Multiple Probes

27
Total Internal Reflectance Microscopy
Evanescent Illumination
28
Functional Analysis and Spatial Distribution of
Ion Channels
Ian Parker UC- Irvine
29
Spatial Analysis of Ca2 Channel Activity
Spatial Position Raster Line
Time (msec)
30
The Subcellular Organization of Ca2 Signaling
Acetylcholine
ICRAC / TRP
IP3
Na
Caveolae
Ca2
Ca2
Ca2
31
Principle of Operation for Atomic Force Microscopy
32
Atomic Force Microscopy
Variety of Cantilevers and Tip Profiles Available
SEM image of AFM cantilevers (magnification 45x)
Bead attached to the tip 2 ?m diameter, glass, k
0.06 N/m Magnification 10,000x
SEM images of common tip shapes
33
AFM CONTACT MODE IMAGING
AFM CONTACT MODE IMAGING Vascular Smooth Muscle
Cell Rat Skeletal Muscle Arteriole
34
Dual Fluorescence and AFM Imaging
Blue Vinculin Yellow Mitochondria AFM Contact
Surface
35

• Biomedical Imaging and Spectroscopy (BMIS)
  Graduate Training Program
• 5 yrs. renewable support, Currently 4 Ph.D.
  students/yr
• - Support for 1 yr salary for thesis projects
  using bio-imaging
• Advanced Research Institute for Biomedical
  Imaging (ARIBI)
• HHMI Grant for Training Program in Biomedical
  Imaging
• Curriculum Development
• Faculty Development and Support