Lecture 18

Principles of microscopic optical

imaging
Light-tissue interactions
Optical methods are based on different types of light-matter interactions to
provide structural, biochemical, physiological and morphological
information

• scattering
– elastic scattering
• multiple scattering
• single scattering
Epithelium

• absorption

• fluorescence
Connective Tissue
Biomedical optical imaging: Use
photons only in the 270-800 nm
range
 Why optical microscopic imaging?
• Potential for very high resolution (current limit: 80 nm)
• Potential for no or minimal effects on sample
• Can be performed using entirely endogenous sources of
contrast or non-toxic exogenous chromophores
• Can be adapted to sample/problem specifications

Applications:
• Basic molecular and cell biology studies
• Understanding disease processes
• Drug development screening and efficacy
• Human disease diagnostics (cancer, diabetes,
atherosclerosis)
• Human therapeutics (Dosimetry, Response monitoring)
 H&E Confocal in vivo
“En face”
SECTION of
human skin

4-Pi microscopy
Mitochondrial network
of live yeast cell
80 nm res

Tumors and
blood vessels
imaged in vivo

Triple stained endothelial Engineered tissue:

Cell of pulmonary artery Fibroblast (red) in
collagen matrix (green)
Endogenous signal
 Engineering challenges in optical
microscopy
• Improved resolution
• Faster image acquisition
• Multi-color (spectroscopic) information
• Miniutarization
• Portability
• Multi-modality imaging
 Some types of microscopes
• Brightfield/Transillumination (absorption)
• Phase contrast (scattering/phase retardation)
• Differential Interference Contrast
(scattering/refractive index gradient)
• Fluorescence
• Confocal (depth resolution; reflectance/fluorescence)
• Multi-photon fluorescence (depth resolution)
• Second Harmonic Generation (depth resolution;
scattering)
 Main aim for microscopy
• To see the finer details
• Main performance characteristic of a
microscope is its resolving power, which
depends not only on magnification

Same magnification, but better Same optics, but improper settings of

quality optics for lab microscope microscope components resulted in improper
yields better image quality illumination and poorer image quality
Light as a wave: Basic concepts
• Phase of a wave is the offset of
the wave from a reference point fo
• We typically talk about a phase
shift
• When light interacts with matter
(e.g. as it travels through a
biological specimen), its speed of
propagation slows down. The
wave emanating from the
specimen exhibits a phase shift
when compared with the initial
wave
• The refractive index , n, and the
thickness of a specimen
determine by how much the wave
is retarded
c
n , where c  speed of light in vacuum
 Green-incident wave
Blue-wave after passing
through specimen shifted by
l/4
 Optical image formation: Basic concepts
• Refraction: When light enters a medium of different refractive index,
it is bent, i.e. its direction of propagation changes, by an amount that
is given by Snell’s law
n1sinq1=n2sinq2

• Dispersion: Different colors of light are refracted by different

amounts because the magnitude of the refractive index is
wavelength dependent. Blue light gets “bent” more than red light

http://www.microscopyu.com/tutorials/java/lightandcolor/refraction/index.html
 Optical image formation: Basic concepts
Geometrical optics, assuming perfect lens
• If a parallel beam of light is
incident on a lens it will get
focused to a spot at the focal
length of the lens
http://www.microscopyu.com/tutorials/java/components/perfectlens/index.html
• To determine the location and
size of the image of an object,
find point of intersection of two
light rays traced using two rules: Focal length
---light ray traveling parallel to
the optical axis, will go through
the focus after it hits the lens
---light ray traveling through the center
of the lens will continue along the
same path beyond the lens L=lens
O=object Im=image
C=lens center
http://www.microscopyu.com/tutorials/java/components/characteristicrays/index.html
 Optical image formation: Basic
concepts
si
xo

so

1 1 1 F=focal length
  So=distance of object from principal plane of lens
f s o si Si=distance of image from principal plane of lens

si f
M  
so x0 M=magnification
X0=distance of object from focal back focal plane of lens
f y f - sign because of inverted image
 i 
s0  f yo si  f
 Numerical aperture
• Numerical aperture (NA) of a lens is a measure
of its ability to gather light and resolve fine
specimen detail at a fixed object distance

NA=n*sina
n=refractive index of medium
a=half angle of light collection cone

Typically, as magnification increases, the working distance, i.e. the distance

from the edge of the objective to the sample, decreases

http://www.microscopyu.com/tutorials/java/objectives/nuaperture/index.html
 Resolution
• The resolution of an optical microscope is defined as the shortest distance
between two points on a specimen that can still be distinguished by the
observer or the camera as separate entities
• For an ideal optical system, resolution is limited by the process of
diffraction, which results in the formation of an Airy disk pattern when we
focus a beam of light onto a spot

r=1.21l/(NAcond+NAobj)=0.61*l/NAobj

l=wavelength
NA=numerical aperture

• The shorter the wavelength and the

higher the NA the better the resolution

• For standard light microscopy, diffraction

limited resolution is on the order of 200 nm

http://www.olympusfluoview.com/java/resolution3d/index.html
http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
 Point Spread Function
Point source Diffracted rays interfere, either in a constructive or
In a destructive way, and give rise to interference rings.
Objective
The mathematical representation of this phenomenon
lens is called the Point Spread Function (PSF)

Converging rays
Image formation by an objective
lens

Diffracted rays

The diffraction pattern of a point source of light. (a) Intensity profile of a diffraction
spot. The central spot and surrounding rings are evident. The seperation distance between
The center of the spot and the first minimum depends on the angular aperture of the lens.
(b) Diffraction pattern showing central diffraction disk surrounded by diffraction rings.
Airy disk in 2D and 3D: Axial resolution is inversely proportional to the
squared NA

Lateral resolution
rlat=1.21*l/(NAcond+NAobj)
=0.61*l/NAobj
Axial resolution
rax=2*l*n/(NAobj)2
n=refractive index of the object medium
With electron microscopes which operate
Whereas with light microscopes
Under vacuum
we can study living cells
We can only study dead cells
 Contrast
• Contrast refers to the ability of an individual specimen detail to be
distinguished when compared to the background or other adjacent
features.

• In effect, contrast is defined as the difference in light intensity

between the specimen image and the adjacent background relative
to the overall background intensity.

• Specimen properties that produce changes in brightness, or color

differences, arise from light absorption, reflection, spatial variation in
refractive index, scattering, diffraction, birefringence, fluorescence
and similar optical phenomena.

• In general, contrast is measured by the relationship between the

highest and lowest intensity in an image, and can be described by a
simple formula:
Percent Contrast = [(Ib - Is)/Ib] x 100
where I(b) is the background intensity and I(s) is the intensity of
specimen features for which contrast is being investigated.
 Major components of a
Forms specimen image microscope
Eyepiece:

on retina in combination
with eye lens;
Additional 10x Camera:
magnification Image storage

Observer

Objective:
Main imaging/magnifying
lens; collects light from
sample
Stage:
Specimen location

Condenser: Illumination
Lens for uniform Source
sample illumination
 Types of illumination: Trans
vs. Epi
Epi-
illumination
Source

Brightfield/Transillumination
Source

• Trans-illumination: Image is formed by light that goes through specimen;

condenser and objective lenses are two distinct lenses
• Epi-illumination: Illumination and imaging light paths are on the same side of
specimen; one lens serves as both the condenser and objective lenses
 Upright vs. Inverted Microscope
Upright microscope:
Epi-
objective is above
illumination
objective Source specimen
•histopathology (fixed
specimen specimens)
Brightfield •Thick specimens
Source
•Animal imaging

Brightfield Inverted microscope:

Source
Objective is below
specimen specimen
objective Epi- • live cell specimens
illumination
Source
Microscope optical train
Component Attributes

Light Source, Collector Lens, Field

Illuminator Diaphragm, Heat Filters, Light Balancing
Filters, Diffuser, Neutral Density Filters

Condenser Iris, Darkfield Stop, Aperture

Light Mask, Phase Annulus, Polarizer, Off-Center
Conditioner Slit Aperture, Nomarski Prism,
Fluorescence Excitation Filter

Numerical Aperture, Focal Length,

Condenser Aberrations, Light Transmission,
Immersion Media, Working Distance

Slide Thickness, Cover Glass Thickness,

Immersion Media, Absorption,
Specimen
Transmission, Diffraction, Fluorescence,
Retardation, Birefringence

Magnification, Numerical Aperture, Focal

Length, Immersion Media, Aberrations,
Objective
Light Transmission, Optical Transfer
Function, Working Distance

Compensator, Analyzer, Nomarski Prism,

Objective Iris, Phase Plate, SSEE Filter,
Image Filter Modulator Plate, Light Transmission,
Wavelength Selection, Fluorescence
Barrier Filter

Magnification, Aberrations, Field Size, Eye

Eyepiece
Point

Table 1
Human Eye, Photographic Emulsion,
Detector Photomultiplier, Photodiode Array, Video
Camera
 Major light paths
• Illumination
– Major goal: provide uniform sample
illumination
• Imaging
– Major goal: reproduce magnified sample
image with minimal distortion, high light
collection efficiency, and high resolution
 Köhler illumination

• Köhler illumination creates an evenly

illuminated field of view while
illuminating the specimen with a very
wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen
and the other the filament from the light
 Illumination conjugate planes
are shown in red; an image of
the lamp filament is in focus at
these planes

Imaging path conjugate planes

are shown in red; an image of
the specimen is in focus at
these planes

http://www.microscopyu.com/articles/formulas/formulasconjugate.html
 Transillumination

Field diaphragm: will affect size of

field that is illuminated at the focal
plane

Condenser aperture: will affect

the numerical aperture of the
condenser

http://www.microscopyu.com/tutorials/java/conjugateplanes/index.html