Light Microscopy

Introduction to Microscopy

• What is Imaging?
• Reflection, refraction, and how does a lens work?
• Magnification, microscope tube length, aberrations, and microscope objectives
• Köhler illumination
• Microscope optical train
• Abbe's resolution limit
• Diffraction angle, resolution and numerical aperture

Contrast Enhancement

• Specimen properties effect on light: absorption, phase shift, and other interactions
• Why do we need contrast?
• Phase contrast microscopy: concept, phase rings, phase components, and limitations of phase contrast
• Dark-field microscopy
• Differential interference contrast (DIC) microscopy
• Light polarity, polarizers, and birefringence
• Polarized light microscopy

Considerations in Objective Design

• The purpose of a microscope objective lens: magnification, resolving details, and minimizing aberrations
• How lens functions
• Common optical aberrations: chromatic, axial chromatic, spherical, coma, and astigmatism
• Correcting spherical aberration with a correction collar

• Advantages of fluorescence microscopy
• What is fluorescence? (excitation and emission, Jablonski diagram, fluorescence spectra)
• Types of fluorescent dyes
• Photobleaching (mechanism, minimizing photobleaching)
• Fluorescent labeling (direct immunofluorescence, indirect immunifluorescence, site specific labeling)
• Single molecule imaging
• Fluorescent proteins (nonoligomerizing fluorescent proteins, swtichable fluorescent proteins, fluorescent proteins as sensors)
• Fluorescent filters (excitation and emission filters, interference filters, matching filters and fluorophores)
• Multiple band pass filters

• Photomultiplier tube (PMT)
• Imaging cameras (CMOS, CCD, EMCCD)
• CCD cameras (CCD architecture, binning, color CCDs, magnification and pixel size, Nyquist–Shannon sampling theorem)
• Quantum Efficiency (QE)
• Sources of noise, noise-to-signal ratio (SNR)
• Scientific CMOS
• Distinguishing intensity levels, intensity scaling, and histogram

• Complete microscope
• Koehler illumination
• The light path and microscope
• Performing Koehler illumination

Introduction to Microscopy & Contrast Enhancement and Considerations in Objective Design: Global BioImaging YouTube channel

Fluorophores, Detectors and Imaging Automation: Global BioImaging YouTube channel

Light Microscopy module on MyScope: MyScope online learning environment

• Introduction to confocal microscopy and the power of optical sectioning
• Detecting the signal in laser scanning confocal microscope using PMT and GaAsP detectors
• How to read fluorescence spectra
• Overview of multifluorescence imaging
• Overview of FRAP, Photoconversion, FCS, and RICS
• Understanding your imaging needs - speed, resolution, and sensitivity
• Airyscan imaging - enhanced resoluion and sensitivity

• Properties of fluorescence microscopy
• Basic properties of light
• Spectral separation, excitation & emission crosstalk, and bleed through
• Multicolor labelling and sequential image acquisition
• What is a spectral system?
• Spectral separation
• Acquisition of lambda stack
• Linear unmixing

The following topics are covered within the confocal microscopy module are available on MyScope:

1. Components of the confocal microscope
2. Practical image acquisition
3. Scanning and resolution
4. Detection parameters
5. Sequential and simulataneous imaging
6. Collecting Z-stacks

Introduction to Laser Scanning Confocal Microscopy, Multi-colour Imaging: Global BioImaging YouTube channel

Confocal Microscopy module on MyScope: MyScope online learning environment

• Resolution and circumvention of diffraction limit
• Overview of laser scanning confocal microscopy and optical sectioning
• What is deconvolution?
• Methods that improve resolution such as pinhole, Airyscan, STED, and structured illumination
• Stimulated Emission Depletion (STED)
• Structured Illiminaion Microscopy (SIM)
• Lattice SIM
• Single Molecule Localization Microscopy (SMLM)

The following super-resolution microscopy modules are available on MyScope:

1. Principles of super-resolution microscopy
2. STED/RESOLFT techniques
3. Single molecule localization techniques (PALM, STORM, DNA-PAINT, 3D SMLM)
4. Sample preparation for super-resolution microscopy
5. Super resolution image acquisition

Super-resolution Microscopy: Global BioImaging YouTube channel

Super-resolution Microscopy module on MyScope: MyScope online learning environment

• How to optimally acquire an imaging volume
• Widefield microscopy
• Parallelized excitation leads to fast acquisition
• Confocal microscopy
• Confocal scanning
• Serial excitation leads to slow acquisition
• Alternative solution: light sheet excitation
• History of light sheet microscopy
• Anatomy of light sheet excitation
• Light sheet beam shaping optics
• Detection: pixels, speed, sensitivity, noise
• Selective Plane Illumination Microscopy (SPIM)
• Objective Coupled Planar Illumination (OCPI) microscopy
• Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM)
• Inverted microscope based SPIM (iSPIM)
• Other light sheet microscopy implementations
• Oblique plane microscopy
• What does light sheet microscopy enable?
• Chellenges in light sheet microscopy: striping in images, scattering, lower axial resolution, diffraction of light sheet
• Solutions to challenges in light sheet microscopy: 2 photon excitation, excitation from multiple directions, engineering a thinner light sheet
• Anisotropic resolution in SPIM
• Dual view iSPIM system (diSPIM)
• Image registration post diSPIM
• Joint deconvolution

• Refractive index matching
• What gives a body maximum transparency?
• Domains of average optical density
• Tissue clearning methods (Benzyl alcohol/benzylbenzoat, 3DISCO, 2Eci, TDE, ScaleS, SWITCH, CUBIC)
• The refractive index is wavelength dependent
• Optics for Light Sheet

• Concept: fluctuation
• Concept: correlation
• Autocorrelation function
• Confocal set-up for FCS
• Model for 1 particle in confocal volume
• Model for 2 particles in confocal volume
• Imaging Fluorescence Correlation Spectroscopy
• Fluorescence Cross-Correlation Spectroscopy (FCCS)

• The difference between FLIM and confocal imaging
• How does a detector convert photons into an image?
• Photon counting in FLIM: time-correlated single photon counting
• Fluorescene lifetime decay curve
• Different fluorophores give rise to different decay curves
• Mathematical modelling of the fluorescence decay curve
• What is mean fluorescence lifetime?
• Advantages of fluorescence lifetime analysis
• What are parametric maps?
• Environmental factors affect fluorescence lifetime
• NAD(P)H & FAD metabolism and metabolic ratio
• Phasor analysis fluorescence lifetime
• Controls are the key to interpretation of phasor analysis
• Phasor analysis of FRET

• Jablonski diagram
• Spectroscopic properties of fluorescence
• Fluorescence and de-excitation processes
• Fluorescence and FRET processes
• Basic properties of FRET
• Förster-Radius
• Förster-Radius - Examples
• FRET probes
• Measuring FRET
• Acceptor photobleaching
• Applications of FRET in structural dynamics

Fluorescence Correlation Spectroscopy (FCS), Fluorescence Resonance Energy Transfer (FRET), Optical Tweezers: Global BioImaging YouTube channel

Fluorescence Lifetime Imaging Microscopy (FLIM): Microscopy Facility of the Institute for Molecular Bioscience YouTube channel