Download
Skip this Video
Download Presentation
The current state of Confocal Scanning Laser Microscopy
Loading in 2 Seconds...
Download
Download Presentation
1 / 47
The current state of Confocal Scanning Laser Microscopy - PowerPoint PPT Presentation
- 152 Views
- Uploaded on
The current state of Confocal Scanning Laser Microscopy. Hjalmar Brismar Cell Physics, KTH. What are we doing in Cell Physics Confocal microscopy History Present Applications Areas of development Excitation Detection Scanning. Cell Physics.
Download Presentation 
An Image/Link below is provided (as is) to download presentation
The current state of Confocal Scanning Laser Microscopy
An Image/Link below is provided (as is) to download presentation
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.
While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
What are we doing in Cell Physics
- Confocal microscopy
- History
- Present
- Applications
- Areas of development
- Excitation
- Detection
- Scanning
Cell Physics
- Study the biological cell from a physical perspective
- Use tools and concepts from physics on biological problems
- Develop methods and techniques
- Describe biological functions and systems within a physical/mathematical framework
- We focus on:
- Cell volume
- Osmolyte transport
- Water transport
- Cell mass
- Measurement techniques
- Cell cycle/cell mass regulation
- Intracellular signalling
- Frequency modulated Ca2+ signals
Instrumentation
- Microscopy (widefield, confocal, multiphoton)
- Fluorescencent probes
- Fluorescent labels, antibodies
- Genetically engineered, GFP
- Electrophysiology
- Patch clamp
- MEA, multi electrode arrays
Confocal microscopy
- Marvin Minsky, 1955
- Laser (1958)1960
- Affordable computers with memory > 64kB
- CSLM 1986-87
Confocal
Confocal evolution
- 1 st generation CSLM (1987)
- 1 channel fluorescence detection
- 50 Hz line frequency
- 2nd generation (commercial systems ca1990)
- 2-3 channel detection
- >=100 Hz
- 3rd generation (1996)
- 4 channel detection
- 500 Hz
- 4th generation (2001)
- 32 channels
- 2.6 kHz
- AOM, AOBS control
Confocal industry
- Carl Zeiss (physiology, dynamic measurements)
- Leica (spectral sensitivity)
- Biorad (multiphoton)
- (olympus)
- (nikon)
- (EG&G Wallac)
- …
Spectra Physics Millenia X - Tsunami
Spectra Physics 2017UV
Applications - Techniques
- GFP
- FRAP
- FRET
- Multiphoton excitation
GFP- Green Fluorescent Protein
Aequoria Victoria
GFP
- Discovered 1962 as companion to aequorin
- Cloned 1992, expression 1994
- 238 Aminoacids
- 27-30 kDa
- Fluorophore made by 3 aminoacids (65-67) ”protected” in a cylinder
Dynamics
GFP-Tubulin in Drosophila
mobile
Bleach
Protein mobility – bleaching experimentsFRAP – Fluorescence recovery after photbleaching
Interaction - FRET(Fluorescence Resonance Energy Transfer)
Excitation 430-450 nm
Donor
CFP
ProteinA
< 5-10 nm
Emission
>570 nm
YFP
Acceptor
ProteinB
NKA – IP3R
After
Before
Photobleaching ofacceptor removes FRETdetected as increased donor signal
Distance < 12 nm
Ouabain binding to NKAshortens the distance – stronger interaction –increased FRET efficiency15-25%
Donor
GFP-NKA
Donor diff
Acceptor
Cy3-IP3R
20
80 mm
Better penetration (2-400 mm)
Enables measurements from
intact cells in a proper physiological
environment.
Electrophysiology
40
60
80
1-photon
2-photon
CFP – YFP separated by a 6 aminoacid linker
Fluorochrome distance 5 nm
2-photon @ 790 nm
2-photon @ 790 nm
790
790
YFP – Calcyon
No excitation at 790 nm
YFP excited at 880 nm
790
880
2-photon @ 790 nm
1-photon @ 514 nm
Development - Excitation
Currently used lasers
- Ar ion, 458,488,514 nm
- HeNe 543, 633 nm
- Ar ion 351,364 nm
- ArKr 488,568 nm
- HeCd 442 nm
- Diode 405 nm
- HeNe 594 nm
- Multiphoton excitation, TiSa 700-1100
We need affordable, low noise, low power consumption lasers
370-700 nm !
Development - Detection
- Spectral separation
- Optical filters
- Prism or grating
- Detectors
- PMT
- Photon counting diodes
We need higher sensitivity, QE !
Development - Scanning
- Speed
- Flexibility
Ultrafast 3D spline scan
- Biological motivation
- Ca2+ signals
- Measurement approach
- Intracellular ion measurements
- Combined electrophysiology
[Ca2+]
Data from live cell experiments combined with biochemical data is used as input for mathematical modeling-simulationsModels verified by experiments can provide
new information and direct the further investigations
Approach
- High resolution 3D recording of Ca2+
- High speed recording
- Combined CSLM - electrophysiology
- Big cells – hippocampal pyramidal neurons
Arbitrary scan – 2D
(Patwardhan & Åslund 1994)
Design criteria
- Z-axis precision >= optical resolution
- Bidirectional scan (to gain speed)
- Focusing distance 20-50+ um
- >100 Hz
- Nonharmonic
Ideas for ultrafast 3D scan
- Stage scan
- High mass, impossible patch clamp
- Scan objective
- Well defined mass, side effects in specimen ?
- Scan focusing lens inside objective
- Tricky optics ?
