Multi-photon Fluorescence Microscopy - PowerPoint PPT Presentation

About This Presentation
Title:

Multi-photon Fluorescence Microscopy

Description:

Multi-photon Fluorescence Microscopy Topics Basic Principles of multi-photon imaging Laser systems Multi-photon instrumentation Fluorescence probes Applications ... – PowerPoint PPT presentation

Number of Views:315
Avg rating:3.0/5.0
Slides: 47
Provided by: neta75
Category:

less

Transcript and Presenter's Notes

Title: Multi-photon Fluorescence Microscopy


1
Multi-photon Fluorescence Microscopy
2
Topics
  • Basic Principles of multi-photon imaging
  • Laser systems
  • Multi-photon instrumentation
  • Fluorescence probes
  • Applications
  • Future developments

3
Multi-photon ExcitationA non-linear process
  • Excitation caused by 2 or more photons
    interacting simultaneously
  • Fluorescence intensity proportional to
  • (laser intensity)n , n number of photons
  • fluorescence localised to focus region

4
History - Multi-photon
  • Originally proposed by Maria Goeppert-Mayer in
    1931
  • First applications in molecular spectroscopy
    (1970s)
  • Multi-photon microscopy first demonstrated by
    Denk, Strickler and Webb in 1989 (Cornell
    University, USA)
  • With Cornell, Bio-Rad is the first to commercial
    develop the technology in 1996

5
Multi-photon microscopy
  • The only contrast mode is fluorescence ( IR
    transmission/DIC is possible)
  • Lateral and axial resolution are determined by
    the excitation process
  • Red or far red laser illumination is used to
    excite UV and visible wavelength probes
  • (e.g.. 700nm for DAPI)

6
Multi-Photon Excitation Physical Principles
7
Consequence of multi photon excitation
  • 1-Photon 2-Photon
  • Excitation occurs everywhere Excitation
    localised
  • that the laser beam interacts
  • with samples Excitation efficiency
    proportional
    the square of laser intensity
  • Excitation efficiency
  • proportional to the intensity
    Emission highest in focal region
    where intensity is highest

8
Classical and confocal fluorescence
Multi-photon fluorescence
9
Key points for multi photon excitation
  • Wavelength of light used is approximately 2 x
    that used in a conventional system. (i.e. red
    light can excite UV probes)
  • Excitation process depends on 2-Photons arriving
    in a very short space of time (i.e. 10
    seconds)
  • Special kind of laser required

-16
10
Lasers for MP
  • Mode-locked femto-second lasers

11
CW and Pulsed Lasers
CW
Pulsed
Short Pulse Advantage Fluorescence
proportional to 1/pulse width x repetition rate
12
  • Laser Options
  • Coherent, Verdi-Mira (MiraX-BIO) X-Wave Optics,
    good
  • beam pointing, beam reducer needed
  • Spectra Physics, Millennia/Tsunami Established
    system,
  • extended tuning optics, good beam diameter
  • Coherent Vitesse NdYlf Turn-key, fixed
    wavelength
  • lasers, small footprint
  • Coherent Vitesse XT and Spectra physics Mai Tai
    - small
  • footprint, limited tuning TiS ( 100 nm range)
    computer
  • controlled

13
General Laser Specifications for MP Microscopy
  • Pulse Width lt250 fsecs
  • Repetition Rate gt75 MHz
  • Average Power gt250 mW

14
Comparison of Lasers Available ForMulti-Photon
Microscopy
15
Why Femto-second?
  • High output powers needed in deep imaging -
  • higher average power generated by pico-second
  • pulses may generate heating and tweezing
    effects
  • 3P excitation of dyes (DAPI, Indo-1) with
    pico-second
  • pulses practically impossible
  • Femto-second pulses may cause 3P excitation of
  • endogenous cellular compounds - however
  • no evidence that this causes cell toxicity

16
Relationship between Average Power and Pulse Width
17
Ratio of 3P excitation to 2P excitation as a
Function of Pulse Width
18
What about Fibre-delivery of Pulsed Lasers
  • Advantage - alignment and system footprint
  • Problem - average power output combined with
  • short pulses for a tuneable laser suffer
    considerable
  • power loss, and realignemnt of laser with each
  • wavelength change ( repointing)
  • problem less with fixed wavelength. ie NdYlf
    uses p-sec pulses
  • which are then compressed by fibre

19
Instrument Design
20
MP Optics Instrument design
Detector
Detector
Confocal Aperture
Laser
Laser
Objective Lens
Objective Lens
C
C
C
C
Excitation
Emission
21
Choice of Microscope, upright or inverted or both
Fentosecond TiS laser
Beam Control and Monitoring Unit ( Optics Box)
Radiance2000MP
2 or 4 External detector unit
Scan head convertible from upright to inverted (
MP ONLY option also available)
22
Key specifications
  • Adaptable to a wide range of microscopes - Nikon,
    Olympus and Zeiss
  • Compatible with six femtosecond pulsed lasers
  • Beam conditioning units range from basic
    functionality to flexible fully featured units
  • Beam delivery systems for single scopes and to
    switch between scopes
  • Non-descanned and descanned detector options
  • Reduced system footprints
  • Multi-Photon ONLY scan head version available

23
Why all this trouble?
  • Conventional confocal has many limitations
  • limited depth penetration
  • short life times for cell observation
  • problems with light scatter especially in dense
    cells
  • limitations with live cell work

24
Is not UV confocal the solution?
  • No - its the problem for many of these
    applications

25
Why has UV confocal seen such little popularity
worldwide
  • Despite being available for nearly 10 years, only
    a small number of systems have been installed
  • Chromatic errors
  • High Toxicity to cells and tissues
  • Poor penetration
  • Enhances autofluorescence
  • Almost unusable in plant sciences
  • High scattering
  • User safety
  • Limited options with lenses
  • In two years the installed base of MP systems
    have doubled over all UV systems world wide.

26
Strengths of Multi-PhotonMicroscopy
  • Deeper sectioning - thick, scattering sections
    can be imaged to depths not possible in standard
    confocal
  • Live cell work - ion measurement (i.e. Ca2),
    GFP, developmental biology - reduced toxicity
    from reduced full volume bleaching allows longer
    observation
  • Autofluorescence - NADH, seratonin, connective
    tissue, skin and deep UV excitation

27
(No Transcript)
28
Deep Imaging improved by..
29
Scattered Light Collection
30
Reduction of EmittedFluorescence due to
Scattering Events
31
Relationship between theNumber of Scattering
Events and Depth into Aortic Tissue
350nm
500nm
700nm
32
Scatter light detection improved by External
light Detector
From Vickie Centonze Frohlich IMR, Madison, WI
33
Reduced Photo bleaching...
34
(No Transcript)
35
MP Fluorochromes and Applications
36
Key issues
  • Most commonly used probes can be imaged
  • MP is effectively exciting at UV/blue wavelengths
  • Excitation spectra are broader than for 1-photon
  • Emission spectra are the same as in 1-photon
    excitation
  • All probes are excited simultaneously at the same
    wavelength
  • Probe combinations must be chosen so that they
    are separated by emission spectra
  • Co-localization is exact even between UV and
    visible probes
  • Can use objective lenses which are not full
    achromats (e.g. z focus shift)

37
Fluorescent Probes for MP Imaging
38
(No Transcript)
39
Efficient SimultaneousDetection of Multiple
Labels
40
Following Dynamic Ca2 Changes using MP Excitation
41
Sources of Tissue Autofluorescence
42
Serotonin Distribution in Living Cells
43
Imaging of Serotonin Containing Granules
Undergoing Secretion
44
MP Imaging ofDrug Localisationand Metabolism
45
Non Imaging Possibilities
  • FRAP (Fluorescence recovery after photobleaching)
  • Photoactivation
  • Knock out experiments
  • FCS (Fluorescence correlation spectroscopy)

46
MP in a nutshell
  • Multi-Photon microscopy allows optical section
    imaging deeper into samples than other methods,
    even in the presence of strong light scattering
  • Multi-Photon microscopy allows the study of live
    samples for longer periods of time than other
    methods, reducing cytotoxic damage
Write a Comment
User Comments (0)
About PowerShow.com