Light microscopy: Basic principles and concepts

Carlo Bevilacqua
Prevedel group

slides prepared by Robert Prevedel

EMBL summer school

15-26 July 2019
 Overview – Table of Contents
— Introduction to ‘Light’ Microscopy

— Optics and Image Formation

— Microscope anatomy & basic principles

— Resolution

— Fluorescence microscopy

— Confocal & other imaging techniques

— Light scattering and multi-photon microscopy

 Light: Wave-particle duality

• Light can be described as an electromagnetic wave

• Light can be described as a particle (photon)

Main properties of light are:

• Intensity
• Frequency or wavelength
• Polarization
• Phase

Study of light is known as optics

 History & Developments
— greek ‘mikros’=small; ‘skopein=to observe’

— most widely used research tools

— requires: 1. Magnification
2. Resolution
3. Contrast

Robert Hooke, ~1665 commercial light commercial fluorescence superresolution fluorescence

~10 µm microsocope, ~1880 microscope, ~1998 microscopes, ~2011
~1 µm ~200 nm <100 nm
 Optics and Image Formation

— Remember Snell’s law

— Focusing works by law of refraction (Snell’s law)

 Optics and Image Formation
— Ray tracing rules of thumb for α~sin(α)~tan(α)

Optical axis

— Rays through center are unaffected — neglect beam shift (thin lens)
 Basic principle of a microscope II
— Image formation in a microscope (finite conjugate)
! ! ! A microscope is an
+ = arrangement of optical
" $ &'
𝑖 elements that provides a
𝑀=−
𝑜 magnified view of an
object with much higher
𝑜 resolution
𝑖
— Image formation in a modern microscope (infinite conjugate)

— allows inserting of additional optics (filters, beamsplitters, etc.)

 Basic principle of a microscope I
— Image formation in a microscope

Here, magnification means

the ratio of viewing angles
with and without microscope.
Major elements are the
objective lens, the eye piece
and (especially in modern
microscopy) a tube lens.
 Optical aberrations (most common)
— Chromatic — Spherical

— Correction using concave flint glass

‘Achromatic doublet’
 Optical aberrations (most common)
— Field curvature

center in focus edges in focus

— Geometric distortions
Computer chip

Object Pincushion Barrel

Pincushion Barrel
 Microscope objectives
— characterized by effective focal length fobj = ftube/M
Microscope anatomy & basic principles

Detection

Illumination
 Illumination – to see an object you need light
— Köhler illumination - even illumination of the sample

Homogeneous illumination of the object is provided by a light source and an optical path
such that the bundle of rays emanating from each spot of the light source illuminates the entire
object to contribute to the image formation.
In other words, the light source generates a field of equally bright spots in the object.
Elements of a Koehler light path are the collector lens, the field stop, the aperture stop
ApSt, and the condensor lens C.
 Optical resolution

The diffraction pattern of an objective lens is described by the Airy function. The central lobe is
called Airy disk with radius d.
d depends on the NA of the lens and on the wavelength of the light. It determines the resolution
of the objective lens – only object points with a distance of d or more can be identified as separate
points. Then the intensity drop between the points is more than 20%, the so-called Rayleigh
criterion.
The optical path can be inverted: luminous points in the object are seen as diffraction patterns,
also called point-spread function PSF.
 Point-spread function (PSF) and image quality
— PSF is 3D focal volume of a microscope
X-Y X-Z
— elongated in axial dimension (z)
— determines image quality

— Convolution
— Deconvolution (postprocessing)

Image x, y =
0 𝑃𝑆𝐹 𝑥 − 𝑥’, 𝑦 − 𝑦 7 𝑜𝑏𝑗𝑒𝑐𝑡 𝑥, 𝑦 𝑑𝑥 7 𝑑𝑦’

microscopysolutions.ca

Wide-field Deconvolved
 Obtaining higher resolution

— increase NA
— immersion objectives (higher n)
— 4 Pi microscopy

Lang NJP 2008

— decrease λ
— electron microscopy

TEM C. elegans embryo 5000x

 Obtaining higher resolution

— super-resolution techniques (localization)

— STED
— PALM, STORM
 Fluorescence
— known since ~1850s (Herschel & Stokes)

— allows specific labeling

— basic principle

400nm 508 nm

Molecular Probes FluoCells

Blue: DAPI (nucleus)

Red: Mitotracker Red (mitochondria)
Green: Alexa488 (actin)
 Fluorescence – basic principle
In 1953, Alexander Jablonski described fluorescence for the first time using
a diagram of the different energy levels involved in the fluorescence process

pictorially:

Jablonski diagram

vibrational
levels { S1,2

absorption/
excitation

relaxation
~10-15 s

emission
~10-12 s

~10-9 s
Olympus Microscopy Resource Center

energy
http://www.olympusmicro.com
electronic
states

• Excitation takes place from the S0 ground level

to an S1 higher level with high efficiency
• Thermal equilibration with the environment: S0
relaxation to ground level
• Emission from S1 ground level to an S0 singlet states
higher level with high efficiciency
 Fluorescence – absorption and emission spectra

• The distribution of
vibrational levels
generally determines S1
the absorption and
emission spectra

• Mirror rule:
similar vibrational
levels of different S0
electronic states result
emission
in very similar though spectrum
mirrored absorption
and emission spectra

• The spectra are specific properties of the fluorophores that allow to identify them
• They can be measured with fluorescence spectrometers
Fluorescence microscopy
 Fluorescence microscopy – choose your filters wisely!
— match filters to exc/em spectrum — dual/multi-color imaging

— consider spectral overlap of fluorophores

Fluorescence microscopy – choose your fluorophore!

Fluorophores emit across the whole spectrum

Shaner et al., Nat. Meth. (2008)

Also: http://www.fpvis.org/FP.html
 Common problems
— Photobleaching
— photochemical alteration
— limited no. cycles
— GFP - 104-105 photons
— organic dye: 105-106 photons
— Quantum dot: 108 photons
— lower intensity – longer imaging time

— Photo-toxicity
— plasma membrane blebbing
— large vacuoles, enlarged mitochondria
— fluorescent protein aggregation (bright spots)
— lower for longer excitation wavelengths

— Light-sheet microscopy
— less out-of-focus light (better contrast)
— reduces light dosage to imaging area
— More details in next lectures
 Further advanced techniques
— Fluorescence-lifetime imaging (FLIM)

— to unmix fluorphores with similar exc/em spectra

— based on different fluorescence lifetimes – decay curves

— requires fast detection + electronics

— Förster resonance energy transfer (FRET)

— between two light-sensitive molecules

— donor-acceptor, matched energy levels

— scales with d6

— to determine co-localization of fluorophores (1-10nm)

 Specialized microscopy techniques
— Confocal microscopy – adding optical sectioning
— reduction of out-of-focus blur
— sharper images – pinhole size defines PSF
Wide-field microscopy

Confocal microscopy

— Spinning disk confocal microscopy – adding speed

— several pinholes – imaged onto camera
Contrast enhancing methods
 Contrast enhancing methods

Bright-/dark-field Phase contrast DIC

 Imaging more complex samples
— from cells to organisms The problem: Light scattering

Problem: Scattered photons lead to blur

surface 100µm in tissue

Two-photon microscopy – point-wise excitation
— improved optical sectioning – focal volume
— improved penetration in tissue (longer wavelength)

one-photon two-photon

Ø Point-wise excitation/detection
Ø no pinhole (all fluorescence recorded)
 Two-photon microscopy – requirements
— coherent light sources (lasers)

— pulsed in time (two-photon advantage)

— photons arrive within virtual state lifetime
Δτ~fs
— ~100fs pulse width, 100MHz rate

Two-photon laser (~100kEUR)

— increased imaging depth to ~0.5-1mm in tissue

— confocal ~100µm
 Two-photon microscopy – in practice
— schematic principle — example image: neurons in mouse brain
— 3D scanning of laser spot — superior resolution at depth

nips.ac.jp
The end… Any questions?