Microscopy 2:Fluorescence microscopy

Prof. Dr. Dietmar J. MansteinMedizinische Hochschule Hannover, Fritz-Hartmann-Centre for Medical Research

Director, Institute for Biophysical Chemistry, MHHDirector, Division of Structural Biochemistry, MHH

Director, Core Unit Laser Microscopy, MHHFounding Director, Centre for Structural Systems Biology, Hamburg

Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Web: www.mh-hannover.de/bpc.html; www.cssb-hamburg.de/

Main topics of the lecture

• Fluorescence and fluorophores

• Layout of an epifluorescence microscope

• Evanescent field microscopy

• Confocal microscopy

• Multiphoton microscopy

• Nanoscopy - Super resolution microscopy

• Correlative light-electron microscopy (CLEM)

Fluorescence

Fluorescence allows organelles, cells and tissues to be labelledwith great specificity, sensitivity and with high contrast.

Fluorescence quantum yield is the ratio of the number offluorescence photons emitted to the number of photonsabsorbed.

The molar extinction coefficient describes the absorptionpotential of the fluorophore.

Fluorophore brightness is the product of quantum yield andthe extinction coefficient.

The localised environment - in particular the pH - cansignificantly influence fluorophore absorption and emissionspectral profiles.

Modern fluorescence microscopes are built as epi-illuminationstands. The objective acts as its own condenser.

Glossar

Luminescence is the production of light by cold-body radiation.

Photoluminescence is the absorption of light at one wavelength and emission at a longer wavelength.

In chemiluminescence an excited state is created via a chemical reaction, with the emission of light tracking the kinetic progress of the underlying chemical reaction.

Fluorescence ceases within nanoseconds after interruption of the external photon source or by photobleaching of the fluorophore.

A Jabłoński diagram illustrates the electronic states of a molecule and the transitions between them.

The Jabłoński Diagram describes the various types of recombination occurring in luminescent materials

- Excitation of an electron occurs always from the ground state S0

- Recombination and thus the emission of a photon always occurs from the lowest vibrational state (S1)

- Recombination occurs to any state S0n

- Intersystem crossing to the triplet state (3A) that relaxes to the ground state by phosphorescence.

FluorophoresTypes of fluorescent tags

Fluorescein

Organic dyes

Fluorescent proteins

Semiconductor nanocrystals - Quantum dots

KillerRed protein absorption and emission spectra

Illumination Designs

1: Excitation filter2: Dichroic mirror3: Emission (barrier) filter

Modern epifluorescence microscopescontain exchangeable filter cubes that eachcontain three different types of filters:

Detector

Detector

Excitation filter

Emission filter

Transmission design Epifluorescence design

Light source

Light source

Emission filter

Condenser

Epifluorescence Microscopy

• Modern fluorescence microscopes are built as epi-illumination stands.

• The objective acts as its own condenser.

• Image brightness in the epi-fluorescence microscope is proportional to N.A.4/Mag2.

https://www.laser2000.de/de/filter/20230-simulation-eines-fluoreszenzmikroskopie.html © THORLABS.com

Beampath and Sample Illumination in Different Types of Fluorescence Microscopy

Evanescent Wave Microscopy

Toomre D, Manstein DJ. Lighting up the cell surface with evanescent wave microscopy.

Trends Cell Biol 2001; 11(7): 298-303.

Evanescent Wave Microscopy

Toomre D, Manstein DJ. (2001)

Fluorescent microtubuli in Dictyostelium discoideum mutant cell line with cytokinesis defect Images courtesy by Dr. G. Gerisch, Max-Planck-Institute for Biochemistry, Martinsried

Confocal Microscopy

• In standard fluorescence microscopy the entirevolume of the sample is illuminated. The resultingout-of-focus fluorescent light, reduces resolutionand obscures fine details when captured by thedetector.

• Improved imaging resolution is achieved inconfocal microscopy by eliminating fluorescencefrom outside the plane of interest by adding twopinhole apertures to a standard fluorescencemicroscope.

• The first pinhole is placed in front of the excitationsource to excite a diffraction limited point in thesample. In most modern confocal microscopes theexcitation pinhole has been replaced by a singlemode fibre).

• The second pinhole in front of the detector is usedto block out-of-focus fluorescent light fromreaching the detector.

Confocal Scanning Microscopy

• At each given point in time, the detector(photomultiplier tube) collects only lightfrom the focal point.

• Two-dimensional slices are captured byscanning the excitation light in X and Y usingone or more motorised oscillating mirrors(“galvanometer mirror scanners”).

• Three-dimensional images are produced byrecording and computationally stackingsuccessive slices.

http://www.microscopist.co.uk/essential-techniques/confocal-microscopy/

Illumination Unit Specimen Holder & objektive Detector Unit

Multicolor Fluorescence Microscopy

Objective lenses are corrected to the standard cover glass thickness of 0.17 mm.

Typical efficiency of CCDs

Multi-photon Excitation Microscopy

• Multiphoton microscopy is a technique that enables high-resolution fluorescencemicroscopic images of living tissue up to a thickness of about one millimetre.

• The λ−4 dependence of light scattering means that shorter wavelengths are scatteredmuch more than longer ones.

• Images are created by exploiting one of two different physical phenomena:

Multiphoton fluorescence:

Doubling (Second HarmonicGeneration, SHG) or tripling(Third Harmonic Generation,THG) of the oscillation frequencyof the irradiated light:

Multi-photon Excitation Microscopy

• Two photons, each having only about half the energy required to excite the molecule,can excite a fluorophore in a quantum event if the photon density is sufficiently high.

• Femtosecond lasers with peak powers from mega- to terrawatt per cm2 and repetitionrates > 80 MHz are used to achieve the required high flux of excitation photons in thefocus volume (1 femtoliter).

• The probability for two-photon excitation is inversely proportional to the pulseduration

Multi-photon Excitation Microscopy• As the absorption of two photons and the efficient excitation are limited to the

extremely small focus volume, no background signals are generated.

• This allows the use of so-called non-descanned detectors without pinholes, whichsignificantly increases the efficiency of light detection.

• Photobleaching only occurs in the focus volume.

Conventional Fluorescence

1 Short wavelength excitation photon

1 Longer wavelength emission photon

Excitation energy > Emission energy

Multiphoton Fluorescence

2 - 3 Long-wavelength excitation photons

1 Shorter-wavelength emission photon

Excitation energy > Emission energy

https://www.univie.ac.at/mikroskopie/3_fluoreszenz/fluoreszenz_mikroskop/5d_multiphoton.htm

Exitation and emission by 2 photons is confined to thefocus.

Exitation by 1 photonresults in absorption and emission along the entirebeam path.

Nanoscopy - Super Resolution Microscopy

The terms "nanoscopy" or “super-resolutionmicroscopy" are used to refer to microscopicprocedures that are capable of resolving objects belowthe diffraction limit (d = λ/2 N.A.).

All of these methods use techniques to distinguishfluorophores by different states. These can be, forexample S0 and S1, or cis and trans or the occurrenceof light and dark states is exploited.

Rayleigh criterion

The Rayleigh criterion is the generally accepted criterion for the minimum resolvable detail - Two points are regarded as just resolved when the principal diffraction maximum of one image coincides with the first minimum of the other.

Basic Principles of Stimulated Emission Depletion Microscopy

STED is based on confocal laser scanning microscopy

The combination of co-centered excitation and depletion beams produces the phenomenon of stimulated emission depletion. Fluorescence photons are collected only from the region inside of the doughnut-shaped depletion beam.

Multicolor-STEDFluorophores with a large Stokes Shift allow the use of a single STED beam in multicolorexcitation architectures. A triggerable subnanosecond fiber laser eliminates the needfor pulse conditioning. Time-gating detection improves emission depletion.Subnanosecond lasers provide narrow-band STED beams (<1 nm). By combining all laserbeams into one SMF fibre, the system is aligned by design.

Vicidomini, G., Bianchini, P. & Diaspro, A. STED super-resolved microscopy. Nat Methods 15, 173–182 (2018).

Basic Principles of Stochastic Localization Microscopy

Detectedsignals

Positions of the individual emitters

Diffraction limited image

Detection of individual emitters with determination of their

maxima

Resulting high-resolution image

Decision Tree for Selecting SRM Techniques

Schermelleh L, Ferrand A, Huser T, et al. Super-resolution microscopy demystified. Nat Cell Biol 2019; 21(1): 72-84.

Correlative light-electron microscopy (CLEM) • CLEM refers to computationally enhanced combinations of fluorescence and

electron microscopy.

• CLEM and cryoCLEM are used in order to obtain biomarker-enhanced information at different length scales.

• Fluorescence microscopy provides information about the localization of proteins and organelles and defines regions of interest.

• Electron microscopy provides high-resolution information.

• In integrated CLEM system, the sample is imaged using an electron beam and an optical light path simultaneously.

Schematic indication of realizations for integrated fluorescencemicroscopy inside ((a), (b)) scanning or ((c), (d)) transmissionEMs. Electron beam is indicated in green, light beam in blue.

Correlative light and electron microscopy of humanhepatoma cells producing a GFP-tagged viral protein(green) and an EGFP-tagged cellular marker protein; lipiddroplets (red); nucleus (blue).© SFB 1129 Mirko Cortese and Volker LohmannToshio Ando et al. J. Phys. D: Appl. Phys. 51 (2018) 443001 (42pp)

Volume-CLEM provides a direct link between live cell dynamics

and 3D ultrastructure on the single organelle level

(A)Schematic representation of volume-CLEM workflow.

(B) Fluorescence image of cells producingGFP-tagged lysosome-associatedmembrane glycoprotein 1 after incubationwith Dextran-Alexa568 as endocytic marker.

(C) The cell was imaged live for severalminutes, followed by in situ fixation.

After fixation the cell is stained, embeddedin resin, and imaged in Focused Ion BeamSEM (FIB-SEM).

(D) Shows the slices on all three viewingaxes (XZ/XY/YZ) of the reconstructed FIB-SEM dataset containing the live-cell ROI ((B),(C) red square). Both spots 1 and 2 areclassified as late endosomes based on theirhigh number of intraluminal vesicles.

(E) FIB-SEM segmentation and 3Dreconstructions of spots 1 and 2; theorganelles imaged in live-cell fluorescencemicroscopy (1,2) were segmented andcorrelated with reference LM data.

Thank you for your attention