Comprehensive Notes on Fluorescence and Confocal Microscopy

Fluorescence and Confocal Microscopy

Lecture Overview

• What is fluorescence?
• Wide-field fluorescence microscopy
• Confocal and Multi-photon microscopy
• Pros, cons & applications
• Advanced Imaging Techniques

Fluorescence

• Light can be a particle (photons).
• The Photonic Spectrum Reference Chart displays wavelengths from VUV to FIR with laser lines from 1 nm to 100 μm.
• Includes major commercial laser lines.

What is Fluorescence?

• Absorption of light results in the emission of light of a different wavelength (usually longer).

Single Photon Excitation

• Single photon excitation involves:
  • Absorption of a photon ($hv$) leading to an excited state ($S$).
  • Thermal relaxation.
  • Fluorescence emission ($hv$).

Excitation and Emission

• Short wavelengths in, long wavelengths out.

Basic Fluorophores

• Can get wide range of emissions through the range
• Usually R, G, B (FR)
• Alexa’s are stated by their excitation $\lambda$

Fluorescence Excitation & Emission Spectra

• Key features:
  • Excitation spectrum: Shows the wavelengths that effectively excite the fluorophore (EX1, EX2, EX3).
  • Emission spectrum: Shows the wavelengths of light emitted by the fluorophore upon excitation (EM1, EM2, EM3).
  • Stokes shift: The difference between the excitation maximum and the emission maximum.
  • Quantum yield

Wide-field Epi-Fluorescence Microscopy

• Bright source of illumination (white light)
• Objective acts as the condenser and objective
• Dichroic mirror (within a filter block):
  • Reflects the illuminating light (Ex) to the sample and lets the returning light (Em) back.
  • This leads to near-perfect separation.

Dichroic Mirrors and Filter Blocks

• Components include:
  • Microscope Objective
  • Excitation Filter
  • Dichroic Mirror
  • Emission Filter

Widefield

• Detector: usually CCD allowing fast acquisition of the whole field simultaneously.
• Light source: usually mercury or xenon lamp, high flexibility with many excitation and emission wavelengths possible when used in combination with appropriate filter sets. Excitation switching is fast using filter wheels or a monochromator.
• Widefield systems provide a highly flexible system for live cell imaging with fast acquisition and flexible excitation at low cost. Photobleaching experiments are not practical.
• Advantage: You only see specifically labeled parts of your sample.
• Widefield microscopes collect light emitted from the entire depth of the specimen. Acquisition is fast.

How Do We Label Structures?

• Direct Live or fixed
  • Cells and Tissues
• Immunolabelling Fixed
• Genetic Tags Live and fixed
  • Whole cells
  • Cell organelles
  • Proteins
  • Lipids
  • Ions/ pH/ voltages

Direct Labelling

• Histological stains (fixed tissue or cells)
• Ion indicators
  • Live cell imaging
• Organelle labels
  • Live cell imaging and fixed preparations

Histological Staining

• Example: Buccal cells stained with Dane's stain, imaged using brightfield microscopy.

Live Cell Labelling

• How do the probes get through the cell membrane?
  • AM esters (acetoxymethyl esters)
  • Microinjection
  • Electroporation

Cell Structure

• Lists different types of dyes for different organelle staining

Fluorescent Ion Indicators

• Ions: Ca, Mg, Zn
• pH indicators
• Membrane potential
• How do they work?

Cell Labelled with Indo-1-AM

• Pseudocolour change in $Ca^{2+}$ over time.
• Imaged using confocal microscopy every 3 seconds

Indirect: Immunolabelling

• Fixed cell preparations (usually)
• Uses primary antibodies raised against protein of interest
• Detect with secondary antibodies (chromagen/fluorescent)
• Note: labelling can sometimes be 3 steps

Indirect Immunolabelling

• Dual immunolabelling
• Primary antibody (mouse monoclonal) binds to protein of interest in cell or tissue.
• Secondary antibody (rabbit polyclonal) with fluorescent label (FITC - GREEN) binds to primary antibody.
  *Primary antibody (rabbit polyclonal) binds to different protein of interest in cell or tissue.
• Secondary antibody (mouse monoclonal) with fluorescent label (TRITC - RED) binds to primary antibody.

Types of Antibody Detection

• Horseradish peroxidase + DAB chromagen (brown - light microscopy)
• Fluorescent detection
  • Alexa Fluors or DyLights dyes
  • Traditional fluorescent probes - FITC, TRITC
  • Quantum dots
• Electron Microscopy
  • Nanogold labelling
  • DAB
  • Qdots

3 Examples of Immunolabelling

• tubulin - DAB chromagen - LM
• actin & tubulin - Fluorophores - Confocal
• tubulin - nanogold particles - TEM

Secondary Antibodies: Fluorophores

• Usually R, G, B (FR)
• Alexa’s are stated by their excitation λ
• DyLights/ Cy dyes
• FITC/ TRITC/ Texas red
• Quantum dots

Fluorescence Emission: The Selection for Detection

• Consider microscope and spectral properties.

Genetic Labelling: Fluorescent Proteins

• Fluorescent proteins are autofluorescent.
• Derived from:
  • Jelly fish Aequorea victoria (GFP)
  • Corals (DsRed, ‘fruities’)
• DNA sequence for GFP is attached (fusion) to gene of interest.
• When protein is made ⇒ its fluorescent

Jelly Fish and Coral

• Jelly fish Aequorea victoria (GFP)
• Coral Discosoma sp (DsRed)

GFP Applications

• Protein localisation/ tracing
• Cell lineage tracer
• pH & $Ca^{2+}$ indicators
• FRAP
• Whole animal studies
• FRET applications

GFP-Mouse

• Example of GFP use in animal study

Colors of Mutated GFP

• BFP (Y66H…)
• CFP (Y66W…)
• Brighter GFP (S65T)
• YFP (T203Y…)

Agar Plate with Fluorescent Bacteria

• E. coli

Examples of Fruity FP's from Roger Tsein

• mHonydew
• mTomato
• mCherry
• mBanana
• mRFP1 mutants cover the visible spectrum

Considerations When Using Fluorescence

• Photobleaching
• Bleed-through of fluorophores
• Specimen thickness and light scattering

Photobleaching

• Fading of the fluorophore with prolonged exposure to light or high intensity light.
• Dependent on the amount of fluorophore, time of exposure.
• Can be reduced with antifade.

Bleed-Through of Fluorophores

• Spectral Overlap in Paired Alexa Fluor Probes
• Choose pairs carefully OR narrow the bandwidth of emission collection (compromise in signal)

Specimen Thickness and Light Scattering

• If specimen is > approx. 3um - you get out of focus light which can make the image fuzzy
  • Reduced contrast; reduced S/N
• Full illumination of specimen - leads to photobleaching

Confocal Microscopy

• Removal of ‘out-of-focus’ light from above and below the focal point ‘removal of the blur’
• Minsky (1960’s)

Widefield vs Confocal

• Confocal microscopy improves resolution compared to widefield by removing out-of-focus light.

Confocal and Widefield Fluorescence Microscopy

• Comparison of images

Types of Confocal Systems

• Laser Scanning
  • Scans laser spot across specimen
  • Uses a pinhole
• Nipkow spinning disk
  • Several ‘pinholes’ remove out of focus light
• Multi-photon
  • Far-red pulsed laser. NO out of focus light as only enough light is used to excite at the focal plane - no pinhole

Basic List of Components

• Light source – LASER
• Objectives – same as fluorescence/ high NA
• Filters for fluorescence
• Detectors – PMT (LSM) or CCD (Nipkow)
• Scanning mirrors (LSM)
• Prisms for spectrophotometric detection of Em
• THE PINHOLE

Scanning Confocal

• Detector: usually a photomultiplier which has reduced sensitivity compared to CCD-based systems. Some recent systems eliminate the need for a dichroic by using an acousto-optical beam-splitter instead. This increases light throughput and flexibility of detection.
• Scanning of illumination beam across sample limits acquisition speed. Provides flexibility of illumination area needed for photobleaching experiments.
• Laser illumination: excitation wavelengths limited to laser lines available. Excitation beam switching is slow, restricting speed of acquisition. (AOTF - helps control intensity)
• Scanning confocal systems are now a general tool for live cell imaging. Multiple probes can be imaged simultaneously, and the ability to restrict illumination to small regions enables photobleaching experiments such as FRAP. Increasing complexity of hardware increases cost.
• Scanning confocal microscopes include a pinhole to eliminate out-of-focus light from the detector.

Widefield versus Confocal Point Scanning of Specimens

• Widefield illumination (large volume).
• Point scanning (small volume).

Image Formation in Confocal

1. Excitation light is scanned across in a raster fashion using 2 scanning mirrors
2. Emitted light ⇒ objective & is separated by a dichroic mirror
3. ‘In-focus’ light passes through the pinhole
4. Detected and amplified by a Photo-Multiplier Tubes (PMT)
5. Image presented on the screen

Confocal Image Formation

• Laser light source provides excitation light.
• Scanning mirrors control the position of the raster scan.
• Objective focuses light onto the specimen.
• Beam splitter separates excitation and emission light.
• Confocal pinhole (iris) blocks out-of-focus light.
• Light detector (photomultiplier tube, PMT) measures the light intensity.
• Image displayed on a computer screen.

Airy Disk & Pinhole Size

• Airy disk: diffraction pattern of an object (property of the microscope)
• Resolution: Two points may be resolved - see two Airy disks.
• Pinhole: Set to the width of one Airy disk. Dependent on NA of lens

Point Spread Function (PSF)

• PSF is what the microscope does to the object to produce an image
• Microscope PSF is 3D

Beads in Glycerol (PSF)

• Example of PSF

PSF of a 4um Bead on SP2

• Raw, Decon, XY, XZ, Decon + GB images

Spinning Disk Confocal

• Broad laser illumination (limited excitation wavelengths): single color acquisition is very fast, slow switching between laser lines can also limit acquisition speed.
• CCD detector captures light from all pinholes rapidly and simultaneously.
• Nipkow (spinning) disk systems enable rapid live cell imaging with significantly reduced photodamage at an intermediate cost. Photobleaching experiments are not possible.
• Spinning disk confocal microscopes incorporate a rotating array of microlenses to focus illumination. A second array of simultaneously rotating pinholes generates confocality

Nipkow Disk Optical Configuration

• Key components include:
  • Microlens Array
  • Nipkow Pinhole Disks Array
  • Objective
  • Specimen
  • Dichromatic Mirror
  • CCD
  • Lens

Modes of Confocal Imaging

• Fluorescence (confocal + non confocal)
• Reflective (confocal)
• Transmission (NOT CONFOCAL)

What Can Confocal Do?

• Optical sectioning
• 3D reconstruction
• Excellent resolution
• Use of specific wavelengths (mutli-labelling)
• Very high sensitivity
• Digital Images
• Computer controlled systems
• Advanced imaging techniques

Optical Sectioning – 3D Information

• ‘z-series’ – axial resolution is less
• Extended Focus
  • Images are ‘stacked’ on top of each other
• Series of projections from different angles
  • Can be rotated and spun around
• 3D stereo images
  • Need 3D glasses
• ‘X-Y’ = 0.2\mum
• ‘z’ resolution 0.5-1\mum

Pollen Grain Serial Optical Sections by Confocal Microscopy

• Shows a series of optical sections

Three-Dimensional Volume Renders from Confocal Optical Sections

• Shows different volume renderings

Z Series 3D Reconstruction

• eGFP-Vimentin in chondrocyte from Z series

Multi-Labelling - Spectral Separation of Wavelengths

• Selection the bandwidth collection to the PMT (SP2)
• Sequential scanning
• See spectrophotmetric detection in lab handouts for diagram of SP2
• Spectral unmixing

Red, Green and Blue Fluorescence

• Confocal microscope separates emissions and removes blur

Calcium Waves

• Cell labelled with fluo-4 dye. Pseudocolour change in $Ca^{2+}$ over time. Imaged using confocal microscopy every 100 ms apart

Confocal - Reflectance Mode

• Collagen fibres of a rat tail tendon.
• Surface morphology

Applications of Confocal (Just a Few)

• Immunolabelling
  • Single and multiple labels
• Protein trafficking
  • Fluoresent proteins
• Organelle identification
• Live cell Imaging
• Subcellular function
• Ion concentrations
• Molecular mobility - FRAP

Some Limitations

• Resolution limited by wavelength
  • Theoretical limit is 0.1\mum, practical ~0.25 \mum
• Expensive machinery/ software
• You need a fluorescently labelled tag
• Laser light penetration is limited
• Laser light can be damaging

Multi-Photon Microscopy

• Two (multi) photon excitation

Multi-Photon Excitation

• From Cannell & Soeller (1997). Micros Res Tech
• Absorption of 2 photons in a single event using a high energy pulsed laser (ti-sapphire)
• Energy of photon inversely proportional to the wavelength. Eg. fluorophore that normally absorbs UV light (350 nm) can also be excited by 2PE by near-infrared light (700 nm) if both photons reach the fluorophore at the same time
• NO PINHOLE - only light in focus is excited
• Improved point spread function (PSF)/ improved axial resolution

Multi-Photon System

• Multiphoton excitation: Simultaneous absorption of two photons of longer wavelengths stimulate fluorescence.
• Emission remains the same as SPE

Point Spread Function (PSF)

• PSF is what the microscope does to the object to produce an image
• Microscope PSF is 3D

Fluorescence Micrograph of Myocyte

• Fluorescence micrograph from a stack of images of a quiescent rat ventricular myocyte.

Three-Dimensional Skeleton of Myocyte

• Three-dimensional skeleton of the t-tubular system in a rat ventricular myocyte.

2PE: Advantages Over Conventional Confocal

• Higher axial resolution (improved PSF)
• No Pinhole
• Greater sample penetration (far red light)
• Reduced photobleaching of marker dyes
• Increased cell viability

Applications

• Thick tissue imaging.
• In vivo live animal imaging
• Light scattering samples
• Live cell imaging

Resources

• MicroscopyU (Nikon)
• Zeiss
• Evident

For Further Reading

• Advanced fluorescence techniques

Further Reading: Advanced Fluorescence

• TIRF - Total Internal Reflectance Fluorescence
• FRAP - Fluorescence Recovery After Photobleaching
• FRET - Fluorescence Resonance Energy Transfer
• FLIM - Fluorescence Lifetime Imaging Microscopy

TIRF Theory

• Visualisation of molecules near the surface of the coverslip
• Incident light greater than the critical angle – causing internal reflection and an evanescent field of illumination
• Does not replace confocal- ‘completes the picture’

TIRF

• When light at high angle (termed the critical angle) travels from a high RI to a lower RI medium its reflected rather than refracted.
• An evanescent wave is emitted adjacent to the interface (in the low RI medium) and excites fluorophores within only 100nm in the z-axis.

TIRF vs Epi-Fluorescence

• Comparison of Epi-fluorescence image, TIRF/epi-fluorescence image overlay (pseudo color), and Laser TIRF image

TIRF - Examples

• DIC of growth cone structure, TIRF time lapse of actin filaments being laid down for growth cone formation
• TIRF of a protein moving along an actin filament

Exocytosis Using TIRF

Example images

Fluorescence Recovery After Photobleaching (FRAP)

• A defined area is photo- bleached by high levels of laser light
• Bleach a fluorescently labelled protein/molecule (good if you have AOTF)
• Monitor fluorescence in defined area
• % recovery = Y/X x 100

FRAP: ‘Measure of Molecular Dynamics’

• Kinetic properties of proteins/molecules in the aqueous and membrane environment of the cell
  • Cytoskeletal turnover/ stability
• Protein dynamics within cell organelles
• Protein activity and interaction
• Non-diffusional movement
  • (e.g. vesicle transport - neurotransmitter transport)

FRET: Fluorescence Resonance Energy Transfer

• Resonance Energy Transfer Jablonski Diagram
• Donor & acceptor fluorophores need to close: 1-10 nm

Fluorescence Lifetime Imaging Microscopy (FLIM)

• Uses fluorescence lifetime of a dye to assess changes in the environment
  • e.g. ions. pH or FRET
• Lifetime: time between excitation of fluorophore & emission (Emission behaviour)
• When electron is excited state, it can interact with other molecules such as $Ca^{2+}$ or $O_2$ or other probes (FRET)

Advantages of FLIM

• 5 to 6 dyes can be imaged simultaneously with no confusion of colour
• Improved spatial resolution
• Monitor live cells changes – ions etc
• Independent of :
  • Dye concentration
  • Photobleaching
  • Light scattering
  • Excitation light
• Dependent on :
  • Dye type
  • pH, ion concentration
  • Oxygen
  • Proximity to energy acceptors (FRET)

Examples of FLIM Images

• Pseudocolour scales for fluorescence times

FLIM-FRET Detection of CAMP

• Example image.

Light Sheet Microscopy

• Ultra thin light sheet and wide field of view.
• Light Sheet coming to the BIRU 2024!

Next Lecture

• Beyond the diffraction limit
• Super-resolution microscopy
• Confocal
• STED