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Content of the lecture. Principles of confocal imaging. Different implementations/modes. Primer on multi-photon (MPH) and multi-harmonic (MHG) generation. Dyes Ca2+ sensitive Voltage-Sensitive Photostimulation: Channel Rhodopsin, Caged glutamate FRET Methods of staining

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content of the lecture
Content of the lecture
  • Principles of confocal imaging. Different implementations/modes.
  • Primer on multi-photon (MPH) and multi-harmonic (MHG) generation.
  • Dyes
    • Ca2+ sensitive
    • Voltage-Sensitive
    • Photostimulation: Channel Rhodopsin, Caged glutamate
    • FRET
    • Methods of staining
  • Electron microscopy (EM)
confocal microscope design
Confocal microscope. Design

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

  • New
  • Input pinhole
  • Exit pinhole
  • Scanning head
  • Detector – PMT

www.olympusconfocal.com

confocal microscope vs widefield
Confocal microscope vs. widefield.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

www.olympusconfocal.com

confocal microscope images examples
Confocal microscope. Images examples.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

www.olympusconfocal.com

confocal microscope scanning unit
Confocal microscope. Scanning unit.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

www.olympusconfocal.com

confocal microscope disk scanning
Confocal microscope. Disk-scanning.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Mechanical television in 1920s

confocal microscope vs widefield1
Confocal microscope vs. widefield.

Thin sections (.5-1.5μm)

Max thickness ~50μm

High contrast and definition

Reduced photo-damaged

Scanning

 “slow”

 “-” eyepieces

 digital zooming

Limited number of laser colors

expensive

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Widefield

Confocal

  • The whole picture is taken at once
    • Eyepiece image
    • Potentially fast imaging
    • High photodamage
  • High background noise (secondary fluorescence)
  • “Cheap”
nonlinear techniques
Nonlinear techniques

Nonlinear Techniques

Confocal Imaging

Dyes

EM

Aux

λ

IVR

λ

<10-17s

<10-17s

λ/2

λ/2

λ

λ

absorption

scattering

Two-photon microscopy

Second harmonic generation

Virtual state

nonlinear techniques other schemes
Nonlinear techniques. Other schemes...

Nonlinear Techniques

Confocal Imaging

Dyes

EM

Aux

Multi-photon microscopy

Multi-harmonic generation

Virtual state

λ

λ

IVR

λ

λ

λ/3

λ/3

λ

λ

absorption

scattering

nonlinear techniques other schemes1
Nonlinear techniques. Other schemes.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Coherent Anti-Stock Raman Scattering Microscopy

λs

λp

λas

λp

ν=1

ν=0

light attenuation spectra
Light attenuation spectra.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

two photon microscopy
Two-photon microscopy.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Absorption probability at the focus

Total absorption probability

Svoboda & Yasuda, 2006

two photon microscopy1
Two-photon microscopy.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

confocal vs 2ph non voltage sensitive
Confocal vs. 2PH (non voltage-sensitive)

Less photo-damaging

Deeper penetration

Light pathway/scheme

Femtosecond laser

Requires proper lenses (not a problem for 2PH now)

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Confocal

MPH

  • Usually better resolution
second harmonic generation shg
Second Harmonic Generation (SHG)

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Induced Polarization:

Signal intensity:

second harmonic generation shg1
Second Harmonic Generation (SHG)

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Dombeck et al, 2006

second harmonic generation shg2
Second Harmonic Generation (SHG)

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Sacconi et al, 2006

voltage sensitivity mechanisms
Voltage-sensitivity mechanisms

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

  • Conformational changes in the system dye-molecule – membrane
  • In the case of scattering techniques (SHG, CARS,..) another mechanism – alteration of thealignment degree with voltage
2ph vs shg
2PH vs. SHG

High marker concentration (~N2)

Mostly forward direction

Narrow emission spectrum

Short life-time (<ps)

Excellent membrane contrast

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

2PH

SHG

  • Low marker concentration
  • Forward & backward directions
  • Relatively wide fluorescence spectrum
  • Relatively long lifetime (~ns)
  • Poor membrane contrast
combination of 2ph and shg
Combination of 2PH and SHG

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Nikolenko et al, 2003

Moreaux et al, 2001

electron microscopes
Electron microscopes
  • Louis d’Broyle, 1927: electrons, like photons, can behave like waves. But with very small wavelength.
electron microscopy
Electron microscopy

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

JEM-1400

www.biologie.uni-hamburg.de

electron microscopy1
Electron microscopy

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Briggman, Denk, 2006

electron microscopy examples
Electron microscopy. Examples.

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

DEMO! : rat’s brain EM sections from Kristen Harris

image obtained with sem
Image obtained with SEM

Geological Survey of Canada, Electron Beam Laboratory

em advantages and drawbacks
EM advantages and drawbacks

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

  • Best resolution, available now
  • Large depth of field
  • Distinctive staining techniques (electron-dense (heave metal) + selective for the tissue of interest (neurons, syn)
  • Only slices (postmortem)
  • Expensive
  • Very slow
    • Creation of the stack of images
    • Reconstruction of the volume
end end
END? END!

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

slide29

Lateral resolution

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

  • Defining the resolution using line-gratingobjects – distance between the maximum and the minimum in “Fraunhoffer slit” diffraciton.

? Is it true only for WF illumination?

  • Defining the resolution using point objects – diameter of the first dark disk on the Airy diffraction image.

Rayleigh criterion: the images of two equally bright spots are resolved if d≥ rAiry (assumes that the sources radiate coherently).

slide30

Axial resolution

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

Has a diffraction nature as well as the lateral resolution

Thus z resolution is usually substantially larger than the xy resolution.

Nevertheless don’t forget – inability to resolve objects doesn’t mean that it’s impossible to know there location

slide31

Depth of field

Confocal Imaging

Nonlinear Techniques

Dyes

EM

Aux

The depth of field of a microscope is the depth of the image (measured along the microscope axis translated into distances in the specimen space) that appears to be sharply in focus at one setting of the fine-focus adjustment.

In bright field microscopy the depth of field should be approximately equal to the axial resolution, at least in theory.

In the dark field or conventional fluorescence microscopy because of the out of focus excitation of the specimen, the depth of field is much greater than the axial resolution.

In confocal imaging the depth of field correspond to the axial resolution. Moreover, it was shown using information theory (Ingelstam, 1955), that the lateral resolution becomes better by a factor of sqrt(2), when the depth of field becomes vanishingly small