MICROSCOPY PRIMER
„Learn to live with Uncertainty“
Josef Gotzmann
Head of Bio-Optics Facility
Max F. Perutz Laboratories
Vienna Biocenter
Josef.gotzmann@meduniwien.ac.at
Download Lecture at the homepage:
https://www.mfpl.ac.at/research/scientificfacilities/biooptics-light-microscopy.html
--------------------------------------------------
© Josef Gotzmann
Download, Read AND Understand
Administrative Rules
at:
the homepage
“download section”
Read them thoroughly BEFORE you
come for any training session!!
© Josef Gotzmann
“INDIVIDUALIZED”
TRAINING STRATEGY
LEGIBILITY FOR TRAINEES
(valid for central facility!)
Potential trainees MUST
• provide an organized experimental strategy to
discuss with the facility staff
• already have own samples for a specialized
training session
© Josef Gotzmann
NEW “INDIVIDUALIZED”
TRAINING STRATEGY
Administrative Rules
1.
Fill in the “Training application form” and meet the facility staff to
discuss most forward strategies and find the proper microscope(s) to be
trained on
2.
Attend the Introductory Lecture [1.) and 2.) may be switched]
3.
Fill in the confirmation on usage of microscopes/user fee regulations
(trainee AND group leader)
4.
Organize a training unit with the facility staff – training units will be split
into
“how to do” and “optimize my own sample”
sessions (on two separate days)
3a. Optional: facility personnel evaluates potential applicability if selection of
proper microscope system remains unclear
© Josef Gotzmann
Cancelation policy:
As user cancellations are being recorded, the following penalty rules will be applied:
• One cancelation per user and month and microscope system
is tolerated (not included cancelations of running slots, if 1/2
of the booked time are used up; or if people come up with a
RELIABLE excuse [e.g. sickness])
• Violation of rule 1 (second cancelation) leads to a warning by
the facility personnel, which is cc’d to the respective group
leader. It is common sense that rule 1 is skipped then for the
coming month for the user concerned.
• Another cancelation violation will lead to charging the group
leader the canceled booking time
• Repetitive infringement of the policy will lead to exclusion of
user registration for a period of 1-6 months (imposed by the
head of facility).
Lecture Contents
• General (physical) principles – nearly formula- free
• Microscope hardware considerations (Illumination,
objectives, detection)
• Contrasting techniques in light microscopy
• Fluorescence and Tools
• Live Imaging – Practical hints
• Confocal Microscopy
• Confocal Microscopy Applications – The F- techniques
• Advanced microscopic methods and what they are
good for
• Laser Safety Instructions
© Josef Gotzmann
Light
Energy
Light is an electromagnetic wave:
radiation (direction and speed)
and
wave properties (intensity and
wavelength)
Geometric Optics
And
Wave Optics
© Josef Gotzmann
Refraction
(Brechung) and
Reflection
Interference,
Diffraction
(Beugung),
Polarisation,
Types of Light
• Monochromatic
• Polychromatic
• Linearly polarized
• Nonpolarized
E-vector
• Collimated (coaxial paths of propagation
• Non-collimated =
Divergent
• Coherent – same phase
• Non-coherent
through space – indep. of l, phase or polarization)
© Josef Gotzmann
Refraction
Refraction and Refractive Index
(measure for optical density):
Air: 1,0003
Water: 1,3333
Silica glas: 1,459
Immersion-oil: appr. 1,52
Diamond: 2,417
less dense
more dense
Refraction varies by frequency
Lenses, Focus and Aberrations
Correction
Reason: lens failures-glass inconsistencies, partial reflection (sample thickness!),
Optical solution: aspheric lenses (cheaper is apertures)
Can be
longitudinal (as
shown) and
lateral
(perpendicular to
focal point)
http://www.funsci.com/f
un3_en/ucomp1/
„plan“-lenses:
Correction for
field curvature
Reason: prism-effect at lens edges
Optical solution: achromatic or apochromatic lenses (2 types of glass) – Fluorescence !!
Other aberrations include: Distortion (fisheye) / astigmatism
Essential Wave Properties
• Wavelength (nm):
• Amplitude – Intensity:
• Phases
l / 2-shift
Constructive Interference Destructive
• Diffraction
Diffraction
Pattern with
main and
side maxima
http://www.sgha.net/articles
/diffraction.jpg
http://www.a-levelphysicstutor.com/wav-light-diffr.php
The Microscope
3
Detection
1
Illumination
© Josef Gotzmann
http://micro.magnet.fsu.edu/primer/anatomy/bh2cutaway.html
Simple Geometry of a Microscope
Magnifying glas)
© Josef Gotzmann
Types of microscopes and
Illumination
UPRIGHT
Reflected or Incident Light
(Auflicht)
Transmitted Light
(Durchlicht)
INVERSE
Also for thick and intransparent specimen
© Josef Gotzmann
elearningcenter.univie.ac.at/fileadmin/.../Friday_Lecture_Volgger.ppt
Light Sources / 1
Halogen Lamps
Mercury Arc Lamps
XenonLamps
LEDs
www.olympus.de
-High power
-200-500h
-Peak intensities
-Needs alignment
http://www.olympusmicro.com/
- < power than mercury
-1500-3000 h
-Uniform spectrum
-Needs no alignment
© Josef Gotzmann
- ultralong lifetime
-cheap
-Narrow spectra (lack betw.
530 and 580nm)
-Individual bulbs – user
specs.
-Weaker emission
intensities
Light Sources / 2
Laser
• Light amplification by stimulated emission of radiation (energy (from
pumping) absorption -> photon -> stimulation of absorption -> photon,
photon…. -> resonator for long ways)
• Medium for amplification can be gas (HeNe, Ar, Kryptone…) or solid state
(Al2O3-rubene, corunde, titan-sapphire…)
• Can be continuous wave (cw) or pulsed (photonic packages down to fs)
PROPERTIES
• COHERENT Light: means waves maintain the same phase relationship
while traveling
• Laser light is also monochromatic (one wavelength) and polarized (Evectorial propagation in parallel planes)
© Josef Gotzmann
Objectives
Dipping objectives-physiology
© Josef Gotzmann
D.B.Murphy, „Fundamentals of Light Microscopy and Electronic Imaging“; Wiley-Liss, 2001; http://zeiss-campus.magnet.fsu.edu/tutorials/basics/objectivecolorcoding/index.html
Coverslips / Tools
• #0:
• # 1:
0.08 – 0.13mm
0.13 - 0.16 mm
• # 1.5: 0.16 - 0.19 mm
• # 2:
0.19 – 0.25 mm
Usually glass, permanox plastics can also be used. Conventional TC
plastics not useful for fluorescence applications (absorption!) For live
imaging use glass-bottom dishes (expensive) or chamber slide (cave:
working distance)
© Josef Gotzmann
http://www.ibidi.de/products/p_disposables.html // http://www.glass-bottom-dishes.com/
Resolution
• Definition: the smallest distance between two points that can be displayed
d=
0.61l
NA
Resolution thus depends on:
1. The wavelength of light that reaches the objective
2. Numerical Aperture (NA) ---> Property of the objective
3. Immersion medium (part of NA calculation)
Numerical Aperture
objective
µ
u
n = 1,0
n = 1,5
dry
immersion
Numerical Aperture (NA) = n(sin µ)
Material
Refractive Index
Air
1.0003
Water
1.333
Glycerin
1.4695
Paraffin oil
1.480
Cedarwood oil
1.515
Synthetic oil
1.515
Anisole
1.5178
Bromonaphthalene
Methylene iodide
1.6585
1.740
http://micro.magnet.fsu.edu/index.html
Resolution
!!!!!Magnification identical !!!!!!
Low Aperture
High Aperture
© Josef Gotzmann
Count as
„resolved“
Counts for transmitted and reflected©light
microscopy
Josef Gotzmann
Depth of Field
The axial range, through which an objective can be focused without any appreciable
change in image sharpness, is referred to as the objective depth of field = thickness along
the z-axis where an object in the specimen appears focused !! -> almost only dependent
on NA !
Depth of Focus = the thickness of the image plane itself. Largely dependent on
Magnification !
Numeri
Depth
Magnifi
cal
of Field
cation Apertu
(mm)
re
Image
Depth
(mm)
4x
0.10
15.5
0.13
10x
0.25
8.5
0.80
20x
0.40
5.8
3.8
40x
0.65
1.0
12.8
60x
0.85
0.40
29.8
100x
0.95
0.19
80.0
© Josef Gotzmann
http://www.olympusmicro.com/primer/anatomy/objectives.html
Axial Resolution
• Axial Resolution is worse than lateral:
minimum distance two diffraction images of “points” can approach each other
along the z-axis
z
distance
=
2ln
(NA)2
• Z shrinks inversely proportional to
the 2nd power of the NA
http://zeiss-campus.magnet.fsu.edu/
© Josef Gotzmann
Detection
Digital Cameras: Photons elicit electron hole pairs (photoelectric effect) –
charge converted to voltage – this analogue signal is amplified and
converted into a binary image (AD-conversion)
Digital Coding/Dynamic Range:
Data Depth = levels of grey
1 bit: 0,1
2 bit 00, 01, 10, 11
© Josef Gotzmann
Nyquist-Sampling Theorem – or how many pixel
do I need for a resolution representative image ?
NYQUIST
SAMPLING
CRITERION: the rate of sampling
must be at least 2-fold the sample
frequency
to
be
able
to
reconstruct the analog signal from
a digitalized one.
sampling frequency is limited by
the pixel size of the chip!
Calculation – the resolution at
550nm with an objective 100x,
NA=1,4 calculates to 230nm ->
magnified by a factor of 100 =
23µm ->on the chip the image
must be large enough to cover 2
pixels -> required pixel size is 11,5
µm !
RxM=
2 x pixel size
½ inch chip is usually 6,4mm x 4,8mm: minimum # of pixels horizontally =
6400 / 11,5 calculates to 557 pixels
Lower mag objectives usually need more pixels for optimal resolution on
the CCD-chip (high resolution microscopes usually equipped with no more
than 1,3 Megapixel cameras) © Josef Gotzmann
Detection – Photo-Multiplier-Tube
(PMT)
• Photon excites catodic plate to create electrons -> these electrons are
mirrored on dynodes (parallel electrodes) and create more secondary
electrons -> next dynode…… -> last dynode is the anode -> between
cathode and anode the voltage defines the amplification capacity –
amplification factor between 104-107 – voltage output correlates with
incoming light intensity
© Josef Gotzmann
http://de.academic.ru/pictures/dewiki/80/Photomultiplier_schema_de.png
Fluorescence
Stokes (1852) – Jablonski (1935)
1)
2)
3)
4)
Molecule absorbs Light = Energy
Excitation of electrons
Relaxation of energized electrons
Emission of fluorescent Light of higher
wavelength than exciting light
© Josef Gotzmann
Fluorescence
lEm > lExc
© Josef Gotzmann
Principle of Fluorescence
•
Molecules capable to fluoresce =
Fluorophores
•
•
•
Excitation with light of proper
wavelength lifts electrons from
basal (S0) to excited levels S1 ; each
of these energy levels is itself
divided into several possible
vibrational states of the molecule
Emission free conversion to lowest
state energy level (IC = Internal
Conversion)
Two possible ways:
–
–
Intersystem Crossing (ICS):
Conversion to T1 triplet state
without any radiative emission and
return to S0
Return to energy state S0 by
emission of a photon (Energy
difference = l) 
Fluorescence
© Josef Gotzmann
Quantum Efficiency
• Only emitted light is relevant for fluorescence detection in microscopy –
intersystem conversion processes equals to loss of fluorescence efficiency
• Quantenausbeute (quantum yield or quantum efficiency [QE]) in steady
state:
Quantu
Conditio
QE =
Number of emitted photons
-------------------------------------Number of absorbed photons
• QE is the essential for a fluorophore to qualify
for optimal use in microscopy
© Josef Gotzmann
m Yield
[Q.Y.]
Q.Y. [%]
Standard
s
ns for
Q.Y.
Measure
ments
Cy3
4
Cy5
27
Cresyl
Violet
Excitatio
n [nm]
Ref.
PBS
540
2
PBS
620
2
53
Methano
580
l
3
Fluoresc
ein
95
0.1 M
NaOH,
22oC
496
3
POPOP
97
Cyclohex
ane
300
3
Quinine
sulfate
58
0.1 M
H2SO4,
22oC
350
3
Rhodami
ne 101
100
Ethanol
450
4
Rhodami
ne 6G
95
Water
488
4
Rhodami
ne B
31
Water
514
4
Tryptoph
13
an
Water,
20oC
280
3
LTyrosine
Water
275
3
14
Factors affecting QE
– Quenching by collision with other molecules
– Static Quenching: when a complex is formed between the
fluorophore and a quenching molecule
– Fluorescent resonance energy Transfer (FRET): radiation-free transfer
of energy from an excited donor molecule on to an acceptor molecule
(can also be used for dynamic association studies- see later). Occurs
preferentially in multi-colour applications – cave: keep fluorophore
concentrations as low as possible.
• Works only over a limited narrow spatial neighborhood in the
range of 20 – 70 Angström
• Emission spectra of Donor and Excitation spectra of Acceptor
molecules must overlap significantly
– Photobleaching: Interaction with light– ROS – can lead to
photochemical changes in molecule structure and in worst case to
loss of fluorescent properties -> is being used for dynamic analyses
– Power saturation-Damage: power limit at specimen over which
fluorophores become destroyed (1mW – confocal / 50 mW for 2photon)
– Red shifting by solvent relaxation: Interaction with solvent dipoles
reduces the energy of emitted photons
© Josef Gotzmann
FLUORESCENCE MICROSCOPY
© Josef Gotzmann
Types of Filters
• Longpass-filter
• Shortpass-filter
• Bandpass-filter
http://www.semrock.com/
Longpassfilter
e.g. Longpass 420:
Number defines cut-off wavelength. This number is selected at
the „cut-on point“ and will always be specified at 50% of
transmission
cut-on point
© Josef Gotzmann
Shortpassfilter
e.g. Shortpass 500:
Number defines wavelength up to which transmission occurs. It
defines the „cut-off point“) at 50& transmission
cut-off point
© Josef Gotzmann
Bandpassfilter
e.g.: Bandpass 465/70 (alternatively: 430-500)
70 = Bandwith: defines broadness of the peak at 50% of
transmission
465 = median wavelength – arithmetic average of cut-of
wavelengthes (Cave: often not identical with peak maximum)
median wavelength
Bandwith
© Josef Gotzmann
Full Cube assembly
31001 (Chroma)
Excitationfilter
Dichroic mirror
Emissionfilter
© Josef Gotzmann
http://www.chroma.com/
400
500
600
700
Problem 1: cross-excitation
a fluorophore is not just excited by wavelength at its peak value, but also by
wavelength at certain range around the peak, which can extends into the area
used by other fluorophores.
FITC TRITC excitation peaks
© Josef Gotzmann
Problem 2: cross emission (emission bleeding
through)
When emission spectra of two fluorophores overlaps, emission from one channel will
extend to another channel.
DAPI-FITC emission peaks
FITC TRITC emission peaks
Filter set for simultaneous detection of triple
fluorescence
82000v2 Filtersatz
von Chroma
Für DAPI, FITC, TRITC
Excitationfilter
Dichroic mirror
Emissionfilter
© Josef Gotzmann
http://www.chroma.com/
400
500
600
700
Organelle Lights ™
ORGANELLES
Nuclei (Syto, Yopro,
Topo, histone-FP)
Mitochondrien(rot)
+Lysosomen (grün)
(MitoTracker – Lysotracker)
Golgi
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-andTissue-Analysis/Cell-Structure/CS-Misc/Organelle-Lights-reagents.html
Endosomes
ER (membrane stains like
DiOC6, ConA)
Cytoskeleton (Phalloidin, Taxol)
© Josef Gotzmann
http://celldynamics.org/celldynamics/
FUNCTIONAL ANALYSES
Apoptosis TdT(terminal deoxy transferase)-mediated dUTP-X nick end labeling
Annexin 5 (detects phosphatidylserin on surface / Live-Dead Kits from Molecular Probes)
Cell cycle (BrdU, Fucci Cell Cycle Sensor)
Fluorosensors
(cAMP)
Ionic and pH Indicators
(FURA, Indo, pHrhodo)
© Josef Gotzmann
Enzyme Activities
(alk-Phosphatase)
Special Fluorophores
• Photo-Activation
• Photo-Conversion
© Josef Gotzmann
http://www.nature.com/focus/cellbioimaging/content/images/ncb1032-f3.jpg
The Point-Spread
Function
For this reversal the „point-spread-function“
(PSF) is either calculated or experimentally
determined and the PSF is the basis for this
mathematic reversal approach.
The point spread function is the image of a
point source of light from the specimen
projected by the microscope objective onto
the intermediate image plane, i.e. the point
spread function is represented by the Airy
disk pattern (see resolution). Due to
diffraction, the smallest point to which one
can focus a beam of light using a lens is the
size of the Airy disk. PSF of a system is the
three dimensional diffraction pattern
generated by an ideal point source of light.
The PSF is a measure for the ability of a
system to create contrast for a given
resolution in the intermediate image plane.
PSF of an individual objective or a lens
system depends on numerical aperture,
objective design, illumination wavelength,
and the contrast mode.
© Josef Gotzmann
http://www.biomedical-engineering-online.com/content/5/1/36
Deconvolution
Limitations to the resolution in an optical system stems from „convolution“: glare,
distortion and blurriness from stray light from out-of-focus areas, especially in fluorescence
microscopy cause acquisition „artefacts“. Also in confocal microscopy these artefacts may
occur from optical inconsistencies in the specimen, glass, or from optical defects (inproper
corrections) in objectives.
Highly sophisticated software calculations can be applied to „reverse“ these artefacts and
create crispy images for better evaluation.
Why do we do it:
•Enhanced resolution in all 3 dimensions x, y, and z
•Reduction of Noise to improve S/N ratio
•Reversal or optimization of optics-based aberrations
Confocal Microscopy
Marvin Minsky, Harvard, 1957
© Josef Gotzmann
http://www.olympusmicro.com/primer/techniques/confocal/index.html
Confocal: Overview
•Laser Illumination
•Illumination pinhole
•Scanning mechanism
•Detector pinhole
•Photomultiplier tube
•
•
•
•
A point light source for illumination
A point light focus within the specimen
A pinhole at the image detecting plane
These three points are optically conjugated
together and aligned accurately to each other
in the light path of image formation, this is
confocal.
• Confocal effects result in supression of out-offocal-plane light, supression of stray light in
© Josef Gotzmann
the final image
Confocal - Pinhole
"optical sectioning“
© Josef Gotzmann
Tight junctions
Adherens junctions
OPTICAL
SECTIONING
Desmosomes
E-cadherin
© Andreas Eger
ß-catenin
© Josef Gotzmann
Desmoplakin
•
•
•
•
After z-stack acquisition you can project the
data to a 3D-image
You can render the image based on volume or
surface characteristics
You can quantitate several features: e.g. colocalization
Basis for these calculations is the 3-dimensional
counterpart of a pixel, the VOXEL
© Josef Gotzmann
3D-rendering
VOXEL
PIXEL
Due to axial resolution being ~2x
lateral resolution fluorescent spots
have elongated shapes
Confocal images: features
• no interference from lateral stray
light: higher contrast.
• no out-of-focal-plane signal: less
blur, sharper image.
• images can be derived from
optically sectioned slices (depth
discrimination)
• Improved resolution (theoretically)
due to better wave-optical
performance.
© Josef Gotzmann
Laser work as point-scanners
Area and ROI scanning
© Josef Gotzmann
Confocal - Illumination
• Laser Modulation -> AOTFs
• No filters, filter wheels, tunable intensities,
wavelenghtes and selection of ROIs
• Ultrasound waves work
as grating and deflect
specific l / intensity of
waves regulates intensity
of light
© Josef Gotzmann
Filters / Sliders guide the light
LSM 710
LSM 510 [Meta]
© Josef Gotzmann
old software layout
Alternatives to select light before detection
Grating / Prism Spectrometer
Grating spectrometer:
Advantages:
• Large splitting
• Linear splitting
Prism spectrometer
• Much higher transparency
• For all wavelengths and polarizations
© Josef Gotzmann
Variable Dichroic –
LSM700 Speciality
Confocal Detection
• PMTs (see above)
• Zeiss Meta Detector – first for spectral detection
(10nm steps) – array of 32 PMTs – less sensitive – can
be used for spectra recording (l-scan -> LSMMeta,
710) and unmixing strategies
© Josef Gotzmann
Emission Fingerprinting
l-scan
unmix
Define fluorophores and spectra
© Josef Gotzmann
www.zeiss.de
Zeiss AIRY-SCAN Superresolution
TECHNICAL FEATURES
LSM710-”Airy”
APPLICABILITY
• Superresolution (x1,7)
• Virtual Pinhole
• Confocal -GaAsP
Virtual Pinhole
• 32 GaAsP detectors
• Chromatically corrected
pinhole
Superresolution
GaAsP
Virtual Pinhole
Mode
3.13 AU
3.0 AU
1.3 AU
1.0 AU
PMT
vs.
GaAsP
Confocal Mode; 1.0 A.U.
SENSITIVITY OF GaAsP Detector
Superresolution
Spinning Disc Microscopy
single Airy pattern unit in diameter
with reference to the focal plane
Loss of light
The spinning disk confocal microscope
collects multiple points simultaneously
rather than scanning a single point at a time
© Science vol 300 David j. Stephens and Victoria J. Allen
© Josef Gotzmann
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/spinningdiskfundamentals/index.html
Spinning Disc
• Advantages:
 Fast acquisition with minimized energy input to the specimen
 Cameras instead of PMTs – no scanning units necessary
 Variable illumination sources: besides lasers, use of solid state
illumination, LEDs, metal halide lamps or even HBO
 Preferential for live imaging
• Disadvantages:
 No selectable pinholes – semi-confocal – pinhole size and
spacing may lead to trade-off in terms of resolution!
 Camera adaptation acc to needs (expensive)
© Josef Gotzmann
Facility Portfolio/1
• 3 Confocal Microscope Sytems
– Zeiss LSM700 (inverse, 2014)
– Zeiss LSM510-Meta (upright, 2005)
– Zeiss LSM710 (inverse, 2012)
Applications:
Routine high resolution imaging, z-sectioning, FRAP,
FRET; optical highlighters; 4D and 5D-imaging,
short- and long-term live imaging possible;
AIRY SCAN technology at LSM710
© Josef Gotzmann
Facility Portfolio/2
• Live Imaging Unit Olympus Cell-Sense (2015):
– Live imaging (T- and CO2-control); Microfluidics
– Destined at 30oC (yeast); sCMOS + colour camera;
multiple positions, multiple wavelengths including
brightfield (DIC), z-Stacks, time-lapse; count &
measure, microfluidics
• Laser Capture Microdissection-Ablation Unit (2009)
- dissect live cells, single cells, and specific cell
clusters; ablate macromolecular
structures;
© Josef Gotzmann
Facility Portfolio/3
• Deconvolution Microscope “Deltavision” (2010)
– Widefield fluorescence, multi-positioning, 5D
imaging, no environmental control, “online”deconvolution
• Spinning Disc Microscope (2012)
- dedicated for short-term live confocal imaging;
- only 63x oil;
- FRAP/PA/PC (405nm);
- No red laser / only CCD-camera
© Josef Gotzmann
Facility Portfolio/4
• Spinning Disc Microscope II (2014)
-
dedicated for long-term live confocal imaging
63x and 100x oil;
EM-CCD & sCMOS cameras (sensitivity & speed)
Versatile manipulation unit FRAP/PA/PC (all cw
lasers – 405/488/561/635 nm);
- 355nm pulsed -> DNA repair induction,
“nanodissection” (actin cables)
- WF option with sCMOS camera
© Josef Gotzmann
- WF: split imaging for ratiometric
data
Facility Portfolio/5
Home-built High Content Imaging (HCM)
TECHNICAL FEATURES
Available
not
before
2016
APPLICABILITY
• Fully automatized
• SCREENING
inverse stand
• STANDARD IMAGING
• Long distance (dry)
objectives
Let us know what you need !!!
• Phase Contrast
• CCD detection (Orca-ER)
• µManager-driven
• Pinkel setup
FACILITY HOMEPAGE:
Irmi will publish her own administrative rules for the in-house microscopes and details on
organization. Training sessions can already be organized with her .
CSF / 1
• FLIM / FCS miroscope: Fluorescent Lifetime
Microscopy and Fluorescent Correlation
Spectroscopy (Picoquant MicroTime200)
– 355, 440, 485, 510, 561, 635 nm pulsed/cw
– FLIM for quantitative live/fixed FRET-based
protein interaction measurements (concentrationindependent)
– FCS for determining e.g. protein diffusion, binding
and oligomerization state
© Josef Gotzmann
Details at: http://www.csf.ac.at/facilities/advanced-microscopy/
FLIM, or fluorescence lifetime
FLIM-FRET
microscopy, is a method that
measures how long it takes
for any fluorophore to go from
it’s excited state to its ground
state, while releasing a photon.
Thus, FLIM- FRET is perfect for two
molecule interaction studies in vivo.
This time is affected by FRET, and
results in decreased time as the
energy resonates to the acceptor
fluorophore.
By using picosecond pulsed lasers
and gated photon counters, the
precise lifetime of a fluorophore can
be measured, and this relies on
nano-environmental conditions
such as hydrophobicity, pH, and
FRET.
What is critical, is that FLIM can
detect FRET quantitatively
independent of fluorophore
concentration.
© Josef Gotzmann
CSF / 2
• SIM: Structured Illumination Microscopy
(OMX Blaze)
– Superresolution microscope based on
reconstruction of signal frequencies
– xyz-resolution improvement: 2-fold each -> 120 x
120 x 250nm
– 405, 488, 568, 635nm lasers
Details at: http://www.csf.ac.at/facilities/advanced-microscopy/
© Josef Gotzmann
Superresolution-S
I
tructured llumination
M
icroscopy
(Sedat, Agard &Gustaffson, UCSF)
Links
http://www.youtube.com/watch?v=0SLmE3-Zg94&feature=related
http://www.youtube.com/watch?v=6I0SF0dXoZg
http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/hrsim/index.html
Gustaffson, PNAS, 2005
The main concept of SI is to illuminate a
sample with patterned light and increase the
resolution by measuring the fringes in the
Moiré pattern (from the interference of the
illumination pattern and the sample).
"Otherwise-unobservable sample information
can be deduced from the fringes and
computationally restored.
http://en.wikipedia.org/wiki/File:3D-SIM-1_NPC_Confocal_vs_3D-SIM.jpg
http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/ssimconcept/index.html
© Josef Gotzmann
Literature
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•
•
•
•
•
•
J.Pawley, Handbook of Biological Confocal
Microscopy, Springer, 2006
D.B.Murphy, Fundamentals of Light Microscopy
and Electronic Imaging, Wiley, 2001 / 2009
E.M.Goldys, Flurescence Applications in
Biotechnology and the Life Sciences, Wiley,
2009
Kevin F. Sullivan, Fluorescent Proteins
(Methods in Cell Biology) , Academic Press,
2008
Molecular Biology of the Cell, Alberts, Garland
Sciences,
Review series on „Imaging in Cell Biology“ in
Nature Cell Biology Vol 5 (2003) Supplement
Review series on „Biological Imaging“ in
Science Vol 300 (2003), 82-99
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
http://www.probes.com/
http://micro.magnet.fsu.edu/primer/techniques/confoc
al/index.html
http://www.zeiss.com/
http://www.leica-microsystems.com/
http://www.microscopy.olympus.eu/microscopes/
http://www.chroma.com/
http://www.microscopyu.com/
http://zeiss-campus.magnet.fsu.edu/index.html
http://www.microscopy-uk.org.uk/
http://www.olympusmicro.com/
http://www.visitron.de/
http://www.sales.hamamatsu.com/en/home
http://www.evidenttech.com/
https://www.omegafilters.com/index.php
http://www.coolled.com/default.htm
http://rsb.info.nih.gov/ij/
http://www.embl.de/almf/ALMF/Welcome.html
http://www.mshri.on.ca/nagy/
http://www.svi.nl/
© Josef Gotzmann
Thank you for your attention!
To be continued:
Laser Safety Instructions
LASER SAFETY
•
•
•
•
Laser Characteristics
Important Parameters
Potential Hazards
Maximum Permissible Exposure (MPE) and
NOHD (Nominal-Optical Hazard Distance)
• Laser Classification
• Laser Safety Glasses
• Laser Safety Rules
Download at the facility homepage
© Josef Gotzmann
Laser Characteristics
• Monochromatic: compared to other sources of light, lasers only
have a single wavelength (“color” in the visible spectrum)
• Coherent: all waves are “in phase”
• Collimated: all waves propagate quasi-parallel (coaxial) – this is the
major reason, why lasers can be focused perfectly (and makes them so
dangerous)
• Linearly Polarized
© Josef Gotzmann
Important Parameters to know
• Type of laser (see slide before)
• Power / Energy
• Mode of Operation
• Beam Quality
© Josef Gotzmann
Power / Energy
• Energy: in Joule (J)
• Power : Energy/Time = J/sec = Watt (W)
• Irradiance: Power
per Area (W/m2)
• Irradiant dose: Energy per Area (J/m2)
most important parameters to
calculate laser hazard
© Josef Gotzmann
Mode of Operation
Energy
Energy
• Continuous Wave: a continuous beam of laser light is
emitted
• Pulsed: the laser light is emitted in pulses at defined
frequencies (down to fs); in addition energy, power and
pulse duration can be variable
cw
Time
•
pulsed
Time
In the Facility all lasers are “cw”, except two 355nm Q-switched DPSS-lasers at the
microdissection unit and the live spinning disc, respectively (running constantly at 80/120Hz)
© Josef Gotzmann
Beam Quality
• Power distribution profile within the beam;
classified according to grade of deviation from
an optimal Gaussian profile; TEM-modes
(Transverse ElectroMagnetic; TEM 00=Gauss);
• “the more equivalent to gaussian, the better
to be focused!”
© Josef Gotzmann
https://en.wikipedia.org/wiki/Transverse_mode#/media/File:Laguerre-gaussian.png
Potential Hazards
• Skin & Eye
• Thermal hazards (skin burns) from high level of optical
radiation – mainly IR
• Photochemical hazards due to high energy (ultraviolet)
radiation
Severity of light-tissue interactions depends on:
1. Spectral coefficient of absorption
2. Energy
3. TIME !!!!
© Josef Gotzmann
Absorption of Light by the Eye
Lens
Cornea
Retina
Mid and Far
IR(1400 nm-1
mm)
Mid UV
(180 nm-315 nm)
Laser Wavelength Region
IR-C = 1 mm to 1400 nm
IR-B = 3000 nm to 1400 nm
Near UV
(315 nm-400 nm)
IR-A = 1400 nm to 700 nm
Visible light = 700 nm to 400 nm
UV-A = 400 nm to 315 nm
UV-B = 315 nm to 280 nm
UV-C = 280 nm to 100 nm
Visible and
Near IR
(400 nm-1400
nm)
© Josef Gotzmann
700-1400nm
most
dangerous
since no
natural
defense
mechanisms!!!
Determining Potential Laser Hazards
MAXIMUM PERMISSIBLE EXPOSURE (MPE)
•
Maximum Permissible Exposure (MPE) limits indicate the highest exposure that
most people can tolerate without sustaining injury. MPE depends on:
•
Wavelength
•
Output Energy and Power
•
Size of the Irradiated Area
• Duration of Exposure
•
Pulse Repetition Rate
• MPE is usually expressed in terms of the allowable exposure time (in
seconds) for a given irradiance (in watts/cm2) at a particular wavelength.
•
MPE’s are useful for determining safety measures (eyewear, filters or windows).
© Josef Gotzmann
Important MPE-based
Power / Exposure Time Limits
visible light, cw:
• For long-term irradiation: 400µW (red) – 40µw
(blue light)
• For incident irradiation(0.25s)*:
power limit:
1mW
• Values lower for pulsed light sources !!
• INVISIBLE IR-light: the exposure time is set to 10s
* (reaction time to avert from irradiation source)
© Josef Gotzmann
Nominal Ocular Hazard Distance
(NOHD)
• Calculated distance where it is safe for
individuals: depends on
– Laser power
– MPE-values
– Divergence (beam width)
Rad = 360o/2P
e.g.: 25W/m2 laser (raw beam) for 0.25s exposure the NOHD is: 610m
Using a 2’’ focusing length -> larger divergence the NOHD is reduced to
31m !
© Josef Gotzmann
Laser Classification / 1
•
Class 1-Exempt Lasers
–
–
Class 1 laser cannot, under normal operating conditions, produce damaging radiation levels (40-400µW
visible). These lasers must be labeled, but are exempt from the requirements of the Laser Safety Program. e.g.: laser printer.
Class 1M lasers cannot, under normal operating conditions, produce damaging radiation levels unless the
beam is viewed with an optical instrument such as an eye-loupe (diverging beam) or a telescope (collimated
beam). This may be due to a large beam diameter or divergence of the beam. Such lasers must be labeled, but are exempt
from the requirements of the Laser Safety Program other than to prevent potentially hazardous optically aided viewing.
•
Class 2-Low Power Visible Lasers
–
Class 2 lasers are low power lasers not exceeding 1 mW in the visible range (400 - 700 nm wavelength) that may
be viewed directly under carefully controlled exposure conditions. Because of the normal human aversion responses
(0.25sec), these lasers do not normally present a hazard, but may present some potential for hazard if
viewed directly for long periods of time.
Class 2M lasers are low power lasers not exceeding 1 mW in the visible range (400 - 700 nm wavelength) that
may be viewed directly under carefully controlled exposure conditions. Because of the normal human aversion
responses, these lasers do not normally present a hazard, but may present some potential for hazard if
•
viewed with certain optical aids.
Class 3-Medium Power Lasers and Laser Systems
–
Class 3 lasers are medium power lasers or laser systems that require control measures to prevent viewing of the direct
beam. Control measures emphasize preventing exposure of the eye to the primary or specularly reflected beam.
–
Class 3R denotes lasers up to 5mW or laser systems potentially hazardous under some direct and specular
reflection viewing condition if the eye is appropriately focused and stable, but the probability of an actual injury
is small. This laser will not pose either a fire hazard or diffuse-reflection hazard. They may present a hazard if
viewed using collecting optics. Visible CW HeNe lasers not exceeding 5 mW radiant power, are examples of this class.
© Josef Gotzmann
Laser Classification / 2
• Class 3B denotes lasers or laser systems that can produce a
hazard if viewed directly. This includes intrabeam viewing
or specular reflections. Except for the higher power Class
3b lasers, this class laser will not produce diffuse
reflections. Visible cw HeNe lasers above 5 mW, but not
exceeding 500 mW radiant power, are examples of this
class.
• Class 4-High Power Lasers and Laser Systems
A high power laser (>0.5W) or laser system that can
produce a hazard not only from direct or specular
reflections, but also from a diffuse reflection. In addition,
such lasers may produce fire and skin hazards. Class 4
lasers include all lasers in excess of Class 3 limitations.
© Josef Gotzmann
Laser Classification / 3
Classifica P maximal Eye Risk
(mWatts)
(long
tion
Skin Risk
term)
Eye Risk
(short
term)
Diffuse
Reflection
- Eye
Diffuse
Reflection
- Skin
1
40µW (blue)400µW (red)
safe
safe
safe
safe
Safe
1M
40µW (blue)400µW (red)
safe
Safe
Safe
Safe
Safe
2
Max. 1mW
Safe
Safe
Safe
Safe
2M
Max. 1mW
Safe
Safe
Safe
Safe
3R
Max. 5mW
Low risk
Safe
Safe
Safe
3B
Max. 500mW
Low risk
Low risk
Safe
4
> 500 mW
© Josef Gotzmann
Lasers available in the facility
• 355nm pulsed TEM00; 50µJ
80Hz, pulselength 1ns
• 405nm, max 120mW
• 458nm, max 20mW
• 477nm, max 20mW
• 488nm, max 120mW
• 514nm, max 15mW
• 543nm, max 10mW
• 561nm, max 100mW
• 633nm, max 100mW
• 635nm, max 100mW
© Josef Gotzmann
Class 3B
Laser Classification / 4
• Note : on conventional laser scanning microscopes
the classification is on the laser’s raw beam power –
usually at least 90% of laser energy is lost until
the laser beam reaches the specimen – so you
will never encounter more than a max of 10mW
when you’d put your eye directly on the objective
lens!!
• Note: “embedded” lasers must be equipped with
interlocks (shut off lasers when opened) and make
embedded lasers Class 1 equipment
© Josef Gotzmann
Anyway -> Laser Safety Glasses
•
•
categorized by OD-values
T = 10 -OD (means OD=3 T=1/1000 of original)
Important to use goggles classified to protect for correct wavelength (bands)
Fassung
OD
unfilter
ed
SKYLINE
Artikelnummer
D..”Dauerstrich”-cw
I..”Impulse Laser”
R..”Riesenimpulslaser”
M..”mode-coupled”
Filtered
F04.P1H01
770 - 800
4+
DIR LB4
800 - 980
6+
DIR LB6
> 980 - 1.065
8+
D LB6 + I LB8 + R
LB7
>1.065 - 1.100
6+
D LB6 + IR LB5
650 - 680
1-2
0,01W 2x10E-6J
RB1
LB.. Protection Level
= OD
•
Laser safety eyewear is required when ACCESSIBLE class 3b and 4 lasers are
in use – within the NOHD.
•
It is important to wear safety eyewear all the time when using ACCESSIBLE
class 3b and 4 lasers - within the NOHD.
•
Laser Safety Glasses will be provided in every room – the protection for
wavelength bands will be provided! (see Admin Rules)
© Josef Gotzmann
SAFETY RULES / 1
•
Switch on the laser warning light whenever you are about to use a
laser-based microscope
•
Always check for your safety if the laser warning light is “on”,
BEFORE you enter the room!
•
Be aware of the beam’s location. Avoid looking directly into any laser beam or at its
directed or diffuse reflection.
•
Only trained and qualified people should work with lasers – in case of microscopes
means for acquisition purpose only !!
•
Wear laser safety glasses, whenever you are not sure, if yourself or your kind of
manipulation is safe.
•
Labmates, Friends, visitors or any other untrained individuals are NOT allowed to
enter the microscope rooms without the permission of the LSO!
•
Remove all watches, jewelry, mirrors, mobiles and unnecessary specular (shiny)
reflecting surfaces from the work area and store them at a safe place.
© Josef Gotzmann
SAFETY RULES / 2
•
ANY kind of MANIPULATION at the LASERS themselves or any ACCESSORIES
(e.g. light guidance fibers) – e.g .for calibration or adjustment procedures – IS
STRICTLY PROHIBITED and MUST only be done by the LSO or a company
technician!!
•
Report accidents immediately to the laser safety officer or any other administrative
unit. You can be held responsible for not reporting failures!
•
In the case of eye exposure consult an ophthalmologist and report the circumstances of
your accident, as soon as possible (BUT AFTER being treated!)
•
In any case you MUST obey the orders and instructions
of the Laser Safety Officers (LSOs) !!
© Josef Gotzmann
MFPL-LASER SAFETY OFFICERS
(LSOs)
Chief LSO:
• Josef Gotzmann
Head of Biooptics Facility
josef.gotzmann@meduniwien.ac.at
+43-1-4277-61672 or
+43-664-8001635200
Deputy LSOs:
• Thomas Peterbauer
• Irmgard Fischer
Biooptics Facility
Thomas. peterbauer@univie.ac.at
+43-1-4277-61676 or +43-664-8175977
5th floor; Rooms 5.528/5.530
Irmgard.fischer@univie.ac.at
+43-1-4277-52866
© Josef Gotzmann