MICROSCOPY PRIMER„Learn to live with Uncertainty“

Josef GotzmannHead of Bio-Optics FacilityMax F. Perutz Laboratories

Vienna BiocenterJosef.gotzmann@meduniwien.ac.at

March 2020

Download Lecture/ check Training Workflow and Technical details at the homepage:https://www.maxperutzlabs.ac.at/research/facilities/biooptics-light-microscopy

© Josef Gotzmann

Technical details

1

2

34

Download, Read AND Understand

Administrative Rules@ the homepage

Read them thoroughly BEFORE you

come for any training session!!© Josef Gotzmann

Trainees

MUST

• provide an organized experimental strategy to discuss with the facility staff

• already have own samples for a specialized training session

“INDIVIDUALIZED” TRAINING STRATEGY

LEGIBILITY FOR TRAINEES(valid for central facility!)

© Josef Gotzmann

“INDIVIDUALIZED”

TRAINING STRATEGY

Administrative Rules

1. Attend the Introductory Lecture

2. 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 [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

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).

Cancelation policy:

HIVE Acquisition Server

MFPL BioOptics – Light Microscopy

HIVE: Purpose

• Saving acquisitions directly on the server (except very fast acquistions)

• Not for long-term storage.

• Images are deleted automatically after 6 months

• One account per MFPL group (Default password: "biooptics1„)

HIVE: At the microscope• Connection window pops up on Windows login

• Group‘s password + UniVie ID + password for authentication

• Maps as <myGroup>\<UniVieID> as drive Z:\

• Save images on Z:\

• To change group/user or disconnect: click "HIVE" icon on the desktop

• Mandatory: Log-off from „HIVE“, when your session is finished !!!

HIVE:What if no connection software pops up?

If the server is down or something else is wrong:

• Save images locally on hard drive

• Connect to the VBC share ("Login" icon)

• Transfer data via 1Gbit connection

Lecture Contents

• General (physical) principles – nearlyformula- free

• Microscope hardware considerations(Illumination, objectives, detection)

• Fluorescence and Tools

• Confocal Microscopy

• Laser Safety Instructions

© Josef Gotzmann

Light

Light is an electromagnetic wave: radiation (direction and speed) and wave properties (intensity and wavelength)

Geometric OpticsAnd Wave Optics

Refraction (Brechung) and Reflection

Interference, Diffraction (Beugung), Polarisation,

Energy

© Josef Gotzmann

REFRACTION

Refractive Index(= measure for optical density):

Air: 1,0003Water: 1,3333Silica glas: 1,459Immersion-oil: appr. 1,52Diamond: 2,417

Refraction varies by energy = frequency = wavelength

less dense

more dense

https://www.flickr.com/photos/121935927@N06/13580411493; https://ingeniumcanada.org/education/try-this-out/broken-pencil-illusion

Lenses and Aberrations

Can be longitudinal (as shown) and lateral (perpendicular to focal point)

Reason: lens failures-glass inconsistencies, reflection, RI mismatches, sample thicknessOptical solution: aspheric lenses (cheaper: apertures)

Reason: prism-effect at lens edgesOptical solution: achromatic or apochromatic lenses (2 types of glass) – Fluorescence !!

Often aspherics are combined with „plan“-lenses:Correction for „field curvature“

Other aberrations include: Distortion (fish-eye) / astigmatismhttps://kvond.wordpress.com/2008/06/24/spherical-aberration-descartes-solution/

https://www.globalspec.com/learnmore/optical_components_optics/optical_components/

optical_lenses ; https://i.ytimg.com/vi/GCISX9id86I/maxresdefault.jpg

http://www.funsci.com/fun3_en/ucomp1/ ; https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/fieldcurvature/https://www.fujifilm.com/products/digital_cameras/xf_lens/about/aspherical_lens/

Perfect lens !!CHROMATIC ABERRATION

SPHERICAL ABERRATION

Essential Wave Properties• Wavelength=ENERGY (nm):

• Amplitude – Intensity:

• Phases/Interference

• DIFFRACTION

Constructive Interference Destructive

l / 2-shift

Diffraction Patternhttp://www.a-levelphysicstutor.com/wav-light-diffr.php; http://www.sgha.net/articles/diffraction.jpg ; https://dsp.stackexchange.com/questions/23773/fourier-transform-of-an-image

https://www.researchgate.net/figure/Transmission-of-light-through-a-circular-aperture-or-radius-r-in-an-infinitely-thin_fig1_264417726

Light Point

The Microscope

http://micro.magnet.fsu.edu/primer/anatomy/bh2cutaway.html

1Illumination

3Detection

© Josef Gotzmann

Light Sources / 1

Mercury Arc Metal HalideLampsHalogen Lamps Xenon Lamps

www.olympus.de

-High power

-200-500h

-Peak intensities

- < power than mercury

-1500-3000 h

-Uniform spectrum

-Weaker emission intensities- ultralong lifetime (>10kh)-Narrow spectra (lack betw. 530 and 580nm) Individual bulbs – user specs.

LEDs

http://www.olympusmicro.com/; http://zeiss-campus.magnet.fsu.edu/articles/lightsources/metalhalide.html; https://theislandpond.com/2016/06/12/how-bright-is-your-light-part-iii-led-conversion/

Power:

Lifetime:

Spectral Characteristics:

Brightfield Fluorescence

Temporal Resolution and Fluorescence Illumination Sources

• Power Loss during Lifetime

• Output-Stability („Flickering“)

http://www.photonics.com/Article.aspx?AID=58093; http://zeiss-campus.magnet.fsu.edu/articles/lightsources/metalhalide.html

I

time

Light Sources / 1a

Mind Your Time

Resolution

Long Term

High Speed

Laser

• Light amplification by stimulated emission of radiation• Medium for amplification can be gas (HeNe, Ar, Kryptone…), liquid

(chemical lasers) or solid (Al2O3-rubene, corunde, titan-sapphire,semiconductor lasers, diodes…)

• Can be continuous wave (cw) or pulsed (photonic packages down to fs)

PROPERTIES

• COHERENT: means waves maintain the same phase relationship while traveling

• COLLIMATED: coaxial paths of propagation through space –>

indep. of l, phase or polarization

• Laser light is also monochromatic (one wavelength) and LINEARLYpolarized (E-vectorial propagation in parallel planes)

Light Sources / 2

© Josef Gotzmann

https://www.univie.ac.at/mikroskopie/1_grundlagen/mikroskop/licht/4b_opt_resonator.htm

Objectives

D.B.Murphy, „Fundamentals of Light Microscopy and Electronic Imaging“; Wiley-Liss, 2001; http://zeiss-campus.magnet.fsu.edu/tutorials/basics/objectivecolorcoding/index.html; https://www.olympus-ims.com/en/microscope/terms/feature15/

© Josef Gotzmann

Infinity-Correction

Coverslips• # 0 : 0.08 – 0.13mm

• # 1: 0.13 - 0.16 mm

• # 1.5: 0.16 - 0.19 mm• # 2: 0.19 – 0.25 mm

- Conventional TC plastics not useful for fluorescence applications (absorption, scattering!)

- Permanox plastics has glass-like properties and can be used as cheap alternative.

- For live imaging use glass-bottom dishes, chambered coverglass or chamber slides (cave:

working distance)

http://www.ibidi.de/products/p_disposables.html // http://www.glass-bottom-dishes.com/

© Josef Gotzmann

Resolution

Resolution thus depends on:1. The wavelength of light that reaches the objective2. Numerical Aperture (NA) ---> Property of the objective3. Immersion medium (part of NA calculation)

• Definition: the smallest distance between two points that can be displayed

0.61 l

NAd =

Numerical Aperture

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 1.6585

Methylene iodide 1.740

Numerical Aperture (NA) = n(sin µ)

http://micro.magnet.fsu.edu/index.html

u

NA = n · sin u

objective

1,0 ­ n ­ 1,5

un = 1,0

objective

n = 1,5

dry immersion

µ

Resolution

High Aperture Low Aperture

!!!!!Magnification identical !!!!!!

© Josef Gotzmann

Counts for transmitted and reflected light microscopy© Josef Gotzmann

http://www.alevelphysicsnotes.com/astrophysics/telescopes.html

; http://micro.magnet.fsu.edu/index.html ;

http://www.olympusmicro.com/primer/

Depth of Field and Numerical Aperture

http://www.olympusmicro.com/primer/anatomy/objectives.html, https://www.quora.com/How-do-you-increase-the-depth-of-field-on-a-microscope; https://slideplayer.com/slide/5063693/

Magnification

NumericalAperture

Depth ofField(mm)

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

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 !

© Josef Gotzmann

Often mixed up with “Depth of Focus” = the thickness of the image plane itself.

Largely dependent on Magnification !

Info:

• Axial Resolution is worse than lateral: minimum distance two diffraction images of “points” can approach each other along the z-axis

• Z shrinks inversely proportional to

the 2nd power of the NA

Axial Resolution

2 l n

(NA)2zdistance =

http://zeiss-campus.magnet.fsu.edu/ © Josef Gotzmann

DetectionSimple Geometry of a Microscope

Magnifying glas)

© Josef Gotzmann

DETECTIONDigital 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 grey levels)

2 bit 00, 01, 10, 11 (4 grey levels)

N bit: 2N grey levels

© Josef Gotzmann

Cameras: „ARRAY DETECTORS“

PMTs: „POINT DETECTORS“

http://micro.magnet.fsu.edu/primer/digitalimaging/concepts/

Nyquist-Sampling Theorem – or how many pixeldo I need for a resolution representative image ?NYQUIST CRITERION:

the sampling rate 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 example: 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 !

E.g.: ½ inch chip -> 6,4mm x 4,8mm: minimum # of pixels

horizontally = 6400 (4800) / 11,5 calculates to 557 x 417

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 Gotzmannhttp://micro.magnet.fsu.edu/primer/digitalimaging/concepts/

Nyquist

Conversely you can use the formula todetermine the need for additionalmagnification for proper sampling

R * M = 2 * pixel size M = 2 * pixel size / R

Let‘s say you have an NA 1,4 objective at 500nm and a camera with 6,5µm

pixel pitch:

M = 2*6500 / (0,61*500/1,4) = 13000 / 218 = 59,6

Magnification must be higher than calculated value!

Means a 60x objective with that NA would be fine – a 40x objective with that

NA would need an 1,5 times extra magnification

(Confocal) Point DetectorsPhoto Multiplier Tubes (PMTs)

• 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

http://de.academic.ru/pictures/dewiki/80/Photomultiplier_schema_de.png;

© Josef Gotzmann

http://micro.magnet.fsu.edu/primer/java/digitalimaging/photomultiplier/channel/index.html

Fluorescence

1) Molecule absorbs Light = Energy

2) Excitation of electrons

3) Relaxation of energized electrons

4) Emission of fluorescent Light of higher

wavelength than exciting light

Stokes (1852)

Jablonski (1935)

© Josef Gotzmann

Fluorophores - Spectra

lEm > lExc

© Josef Gotzmann

Principle of Fluorescence1) Excitation with light of proper

energy/wavelength lifts electronsfrom basal (S0) to S1 excited levels(green arrows)

2) Emission free conversion to loweststate energy level. Process calledInternal Conversion (yellow arrows)

3) Return to energy state S0 byemission of a photon (red arrows; D

Energy = lem) Fluorescence

© Josef Gotzmann

https://static2.olympus-lifescience.com/data/olympusmicro/primer/java/jablonski/jabintro/jablonskijavafigure1.jpg?rev=26F4

• Molecules capable to fluoresce are termedFLUOROPHORES

• each of the energy levels is sub-divided intoseveral possible vibrational states of themolecule -> spectra are probability statistics!

• Else routes, some non-radiative:

a) Blinking: Intersystem Crossing(ISC): between a „dark“ triplet state(T1) and S1.

b) Conversion to T1 and

b1) return to S0 without anyradiative emission

b2) Long lasting emission oflight as „phosphorescence“

1

2

3

a

b2

b1

Fluorophores Quantum Efficiency• Only emitted light is relevant for fluorescence detection in microscopy –

intersystem conversion processes equals to loss of fluorescence efficiency

• Quantum yield (QY) or Quantum efficiency (QE) in steady state:

Number of emitted photons QE = --------------------------------------

Number of absorbed photons

• QE is essential for a fluorophore to qualify

for optimal use in microscopy

Quantum Yield[Q.Y.] Standards

Q.Y. [%]

Conditions for Q.Y. Measurements

Excitation [nm]

Ref.

Cy3 4 PBS 540 2

Cy5 27 PBS 620 2

Cresyl Violet

53Methanol

580 3

Fluorescein

950.1 M NaOH, 22oC

496 3

POPOP 97Cyclohexane

300 3

Quinine sulfate

580.1 M H2SO4, 22oC

350 3

Rhodamine 101

100 Ethanol 450 4

Rhodamine 6G

95 Water 488 4

Rhodamine B

31 Water 514 4

Tryptophan

13Water, 20oC

280 3

L-Tyrosine

14 Water 275 3

© Josef Gotzmann

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.

• Emission spectra of Donor and Excitation spectra of Acceptor

molecules must overlap significantly

• Works only over a limited narrow spatial neighborhood in the

range of 20 – 70 Angström

– Photobleaching: Interaction with light– ROS – can lead to

photochemical changes in molecule structure and in worst case to

loss of fluorescent properties

– Power saturation-Damage: power limit at specimen over which fluorophores

become destroyed (1mW – confocal / 50 mW for 2-photon)

– Red shifting by solvent relaxation: Interaction with solvent dipoles reduces the

energy of emitted photons

© Josef Gotzmann

https://www.qmul.ac.uk/blizard/media/blizard/tmp/migration-files/blizard/images/flow/FRET-500x517.jpg; http://www.olympusconfocal.com/theory/fluorophoresintro.html ;

FLUORESCENCE MICROSCOPY

© Josef Gotzmann

http://themurderofmeredithkercher.com/Luminol_Traceshttp://www.olympusmicro.com/primer/

Types of Filters

• Shortpass-filter

• Bandpass-filter

• Longpass-filter

http://www.semrock.com/http://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html

Longpassfilter

cut-on point

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

© Josef Gotzmann

Shortpassfiltere.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

Bandwith

median wavelength

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)

© Josef Gotzmann

Full Cube assembly

Emissionfilter

Dichroic mirror

Excitationfilter

31001 (Chroma)

400 700600500http://www.chroma.com/;

© Josef Gotzmann

http://www.olympusmicro.com/primer/

Multi-Colour 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

Multi-Colour Problem 2: Cross emission (emission bleed-through)

When emission spectra of two fluorophores overlaps, emission from one channel will extend to another channel.

FITC TRITC emission peaks

DAPI-FITC emission peaks

http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/IMAGES/

82000v2 Filtersatz

von Chroma

Für DAPI, FITC, TRITC

Emissionfilter

Dichroic mirror

Excitationfilter

400 700600500http://www.chroma.com/

© Josef Gotzmann

Filter set for simultaneous detection of triple fluorescence

MULTICOLOUR-FILTER-

BASED IMAGING IS ALWAYS

A TRADE-OFF!!

ORGANELLES

Endosomes

Mitochondrien(rot)

+Lysosomen (grün)

(MitoTracker – Lysotracker)

ER (membrane stains like

DiOC6, ConA)

Golgi

Cytoskeleton (Phalloidin, Taxol)

Organelle Lights ™

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-

Tissue-Analysis/Cell-Structure/CS-Misc/Organelle-Lights-reagents.html http://celldynamics.org/celldynamics/

Nuclei (Syto, Yopro,

Topo, histone-FP)

© Josef Gotzmann

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)

FUNCTIONAL ANALYSES

Fluorosensors

(cAMP)

Enzyme Activities

(alk-Phosphatase)

Ionic and pH Indicators

(FURA, Indo, pHrhodo)

© Josef Gotzmannhttps://www.sciencedirect.com/science/article/pii/S0012160604008553

https://lsbio-7d62.kxcdn.com/image2/plap-alkaline-phosphatase-antibody-aa56-83-clone-421ct8.1.1-ls-c156297/214053_1616953.jpg

Optical Highlighters

• Fluorescent proteins

• Spectral properties can be manipulated by illumination with specific wavelengths

PhotoactivationirreversiblePA-GFP

dark + 405 nm → green emission

PhotoconversionirreversibleDendra, Eos, Kaede

green + 405 nm → red

Photoswitchingreversible cis-trans isomerizationsDronpa

dark + 488 nm → greengreen + 405 nm → dark

Source: Zeiss

Special (Dynamic) Fluorophores

The Point-Spread Function

The point spread function (PSF) is the image ofa point source of light from the specimen

projected by the microscope objective onto theintermediate image plane.

PSF of a system is the three dimensionaldiffraction pattern generated by an ideal pointsource of light.

PSF depends on: numerical aperture

illumination wavelength

contrast mode.

http://www.biomedical-engineering-online.com/content/5/1/36

© Josef Gotzmann

DeconvolutionLimitations 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, filters, glass, or from optical defects in objectives.Highly sophisticated software calculations can be applied to „reverse“ these convolution effectsand 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

https://www.leica-microsystems.com/fileadmin/academy/user_upload/Fig7_Introduction_to_Widefield_Microscopy_Deconvolution.png

Confocal Microscopy

http://www.olympusmicro.com/primer/techniques/confocal/index.html

Marvin Minsky, Harvard, 1957

© Josef Gotzmann

Confocal: Overview•Laser Illumination

•Illumination pinhole

•Scanning mechanism

•Detection 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-of-focal-plane light, supression of stray light in the final image© Josef Gotzmann

http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/IMAGES/cf_tut1.gif

Confocal - Pinhole

© Josef Gotzmann

"optical sectioning“

1

AIRY

unit

50% FWHMFull width

Half maximum

E-cadherin ß-catenin Desmoplakin

Tight junctions

Adherens junctions

Desmosomes

© Andreas Eger

Z – sectioning

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-scannersArea and ROI scanning

© Josef Gotzmannhttps://static2.olympus-lifescience.com/data/olympusmicro/primer/techniques/confocal/images/scanningsystemsfigure2.jpg?rev=42FB

point-wise ROI-selection capabilities

Filters / Sliders guide the light

LSM 700 LSM 980

© Josef Gotzmannhttp://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/lsm700/indexflash.html

http://confocal-microscopy-list.588098.n2.nabble.com/Spectral-Detector-in-Zeiss-700-td4869090.html

Alternatives to select light before detectionGrating / Prism Spectrometer

Advantages: • Large splitting

• Linear splitting• Much higher transparency

• For all wavelengths and polarizations

Grating spectrometer: Prism spectrometer

© Josef Gotzmannhttps://www.researchgate.net/profile/Rolf_Borlinghaus/publication/257868431/figure/fig4/AS:297415925092355@1447920890193/SP-Detector-Colored-emission-is-dispersed-by-a-prism-and-guided-to-sensors-Band.png

Spectral Detection and Emission Fingerprinting

l-scanDefine fluorophores and spectra

unmixwww.zeiss.de© Josef Gotzmann

Zeiss AIRY-SCAN 2

• 32 GaAsP detectors

• Multiplexing option (faster -2Y, 4Y, 8Y modes)

• Superresolution (x1,7)

• Virtual Pinhole

• Confocal -GaAsP

TECHNICAL FEATURES APPLICABILITY

Superresolution GaAsP

Virtual Pinhole

LSM980-”Airy2”

www.zeiss.de

Virtual PinholeMode

3.0 AU 1.0 AU

3.13 AU 1.3 AU

PMT vs. GaAsPConfocal Mode; 1.0 A.U.

SENSITIVITY OF GaAsP Detector

Superresolution

AIRY SCAN 2 / MULTIPLEXING

Multiplex Mode of the ZEISS LSM 9 Family

© Science vol 300 David j. Stephens and Victoria J. Allen

Spinning Disc Microscopy

The spinning disk confocal microscope collects multiple points

simultaneously rather than scanning a single point at a time

http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html

single Airy pattern unit in diameter

with reference to the focal plane

© Josef Gotzmann

1

2 3

Live Imaging in a quasi-

confocal regimen at HIGHLY

reduced photodamage (appr. 4

orders of magn. less power comp. to confocal)

Total internal reflection fluorescence microscopy

(TIRFM)

http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html

WF - TIRF

http://www.zeiss.de/c12567be0045acf1/Contents-Frame/649722ef8c671420c1256dd6004b7751

Single molecule

„Background free“!!

Only at the surface

sensitive detection

Basis for superres.-techniques

Critical alignment Laser artefacts/ringing

Refractive index change

OIL

WA

TE

R

FACILITY ROOMS

• 2 Confocal Microscope Sytems

– Zeiss LSM700 (inverse, 2014)

– Zeiss LSM980 (inverse, June 2020)

Applications:

Routine high resolution imaging, z-sectioning, FRAP, optical highlighters; 4D and 5D-imaging,

LSM980: AIRY SCAN technology / z-piezo stage

© Josef Gotzmann

Facility Portfolio/1Room 1.223

• Live Imaging Unit Olympus Cell-Sens plus TIRF (2015):

– Long-term Live imaging (T- and CO2-control)

– Hardware autofocus / Focus maps (multiwellscreening)

– sCMOS + color camera; multiple positions, multicolor, z-Stacks, time-lapse; count & measure,

– (2017) TIRF module (3 lasers, 405,488,561; 405nm also for manipulation) - ((PALM/STORM))

© Josef Gotzmann

Facility Portfolio/2Room 1.223

• Spinning Disc Microscope (2012)

- dedicated for short-term live confocal imaging;

- only 63x, 1.4 oil objective;

- FRAP/PA/PC (405nm);

- 405/488/561 nm lasers

- No red laser

- two camera solution:

sCMOS / EM-CCD

- Cherry Temp: cooling device© Josef Gotzmann

Facility Portfolio/3Room 1.223

• Spinning Disc Microscope II (2014)

- dedicated for long-term live

confocal/WF imaging (Hardware-autofocus)

- 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 -> WF live imaging

- WF: split imaging for ratiometric data© Josef Gotzmann

Facility Portfolio/4Room 1.318

Deconvolution Microscope “Deltavision” (2010)

• Widefield fluorescence, multi-positioning, 5D imaging

• no environmental control

• critical illumination

• “online”-deconvolution

Facility Portfolio/5Room 1.320

Deltavision „Ultra“ (2018)

Additional features:

• Environmental control (T/CO2)

• sCMOS camera

• Hardware Autofocus

• Multiwell option

Zeiss Cell Discoverer 7 (2018)

– Fully automated high content microscope

– Sample measurements

– Autocorrection objectives

– Water immersion 50x

– Full Env. Control/Long-term imaging

– Diverse focus strategies (hardware/software combined)

No individual training – workflow setup by facility personnel

Facility Portfolio/6Room 1.320

http://www.abberior-instruments.com/products/compact-line/stedycon/ ; http://zeiss-campus.magnet.fsu.edu/tutorials/superresolution/stedconcept/indexflash.html

Abberior STEDYCON– 2D Stimulated Emission

Depletion superresolution microscopy

– XY 30-75nm

– Fixed samples / 2 channels

– 405/488/561/640nm

– 775nm pulsed STED laser

– 100x oil, 1.46 objective

– Standard Confocal possible

– (orange/red/deep red) STED dyes needed

Room 1.723 Facility Portfolio/7

• Fully automatized inverse stand

• Long distance (dry) objectives• Phase Contrast• CCD detection (Orca-ER)• Pinkel setup• µManager-driven (more or

less facility controlled)

• SCREENING (multi-well)• STANDARD IMAGING(ON DEMAND-FLEXIBILITY)

TECHNICAL FEATURES APPLICABILITY

Facility Portfolio/8Room 1.723

Home-built “High Content Imaging” (HCM; 2017)

Let us know what you need !

NOTE: In-house microscopes have their own administrative rules!!

Training sessions must

be organized with

Irmgard Fischer !!

7

2020

2020

Literature

• http://www.probes.com/

• http://micro.magnet.fsu.edu/primer/techniques/confocal/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/

• 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

© 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 RulesDownload 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

• Power / Energy

• Mode of Operation

• Beam Quality

© Josef Gotzmann

Power / Energy

• Energy: in Joule (J)

• Power : Energy/Time = J/sec = Watt (W)

• Irradiant dose: Energy per Area (J/m2)

• Irradiance: Power per Area (W/m2)

most important parameters to calculate laser hazard

© Josef Gotzmann

Mode of Operation

• Continuous Wave: a continuous beam of laser light is emitted

• Pulsed: the laser light is emitted in pulses at defined/variable frequencies (down to fs); in addition energy, power and pulse duration can be variable

Time Time

Ener

gy

Ene

rgycw pulsed

© 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 absorption2. Energy 3. TIME !!!!

© Josef Gotzmann

Laser Wavelength Region

IR-C = 1 mm to 1400 nm

IR-B = 3000 nm to 1400 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

Absorption of Light by the EyeLens

Cornea Retina

Mid and Far

IR(1400 nm-1

mm)

Mid UV

(180 nm-315 nm)

Near UV(315 nm-400 nm)

Visible and

Near IR

(400 nm-1400

nm)

700-1400nm

most

dangerous

since no

natural

defense

mechanisms!!!

© Josef Gotzmann

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†

© Josef Gotzmann

* (reaction time to avert from irradiation source)

† Maximum time to focus at a point

Nominal Ocular Hazard Distance (NOHD)

• Calculated distance where it is safe for individuals: depends on

– Laser power

– MPE-values

– Divergence (beam width)

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 !

Rad = 360o/2P

© Josef Gotzmann

Laser Classification / 1• Class 1-Exempt Lasers

– Class 1 laser cannot, under normal operating conditions, produce damaging radiation levels (40-400µWvisible). 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 harmful diffuse reflections. Visible lasers: 5-500 mW radiant power.

• Class 4-High Power Lasers and Laser SystemsA high power laser (>0.5W) or laser system that can

produce a hazard not only from direct or specularreflections, 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

RISK ASSESSMENTClassification

P maximal(mWatts)

Eye Risk (long term)

Eye Risk (shortterm)

Skin Risk 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 facilityCW:

• 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

Pulsed:

• 2 x 355nm pulsed TEM00; 50µJ

80Hz, pulselength 1ns

• 488nm, pulsed 40MHz, 5mW

• 561nm, pulsed 40MHz, 5mW

• 640nm, pulsed 40Mhz, 5mW

• 775nm, pulsed 40 MHz, 1,25W average

Class 3B

© Josef Gotzmann

Class 4

Laser Classification / 4• Note 1: 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 2: “embedded” lasers must be equipped with interlocks (shut off lasers, when active) and make embedded lasers Class 1 equipment

© Josef Gotzmann

LB.. Protection Level= OD

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)

• Laser safety eyewear is required when ACCESSIBLE class 3B and 4 lasers are in use – within the NOHD.

• Laser Safety Glasses will be provided in every room – the protection for wavelength bands will be provided! (see Admin Rules)

Filtered

unfiltered

Fassung OD SKYLINE

Artikelnummer 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 1-20,01W 2x10E-6J RB1

D..”Dauerstrich”-cwI..”Impulse Laser”R..”Riesenimpulslaser”M..”mode-coupled”

© 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 !!

• Labmates, Friends, visitors or any other untrained individuals are NOT allowed to enter the microscope rooms without the permission of the LSO!

• Wear laser safety glasses, whenever you are not sure, if yourself or your kind of manipulation is safe.

• 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/evident failures immediately to the laser safety officers or any other

administrative unit. You can be held responsible for not reporting accidents/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)

© Josef Gotzmann

Chief LSO:

• Josef Gotzmann

Head of Biooptics Facility

josef.gotzmann@meduniwien.ac.at

+43-1-4277-61672 or

+43-664-8001635200

Deputy LSOs:

• Thomas PeterbauerBiooptics Facility

Thomas. peterbauer@univie.ac.at

+43-1-4277-61676 or +43-664-8175977

• Irmgard Fischer5th floor; Rooms 5.528/5.530

Irmgard.fischer@univie.ac.at

+43-1-4277-52866