Introduction to Microscopy I

A Simple Question

Why do you need a microscope?

Image Formation

Light rays travel in straight lines in all directions

Detector
Sample

Light Source
Image Formation

Light rays travel in straight lines in all directions

Detector
Sample

Light Source
Image Formation

Light rays travel in straight lines in all directions

Detector
Sample

Light Source
Image Formation

Light rays travel in straight lines in all directions The pinhole camera

Detector
Sample

Light Source
Camera

Why don’t we
just use a
pinhole?

Diascopic
lamphouse
Image Formation

Light rays travel in straight lines in all directions

Wasted Light
Detector
Sample

The pinhole stops almost all

the light → very dim image.
Light Source
Image Formation

The pinhole stops almost all

the light → very dim image.
Sadly, our samples are not as bright as the sun…
Image Formation

Light rays travel in straight lines in all directions

Wasted Light
Detector
Sample

Light Source
Image Formation

Light rays travel in straight lines in all directions How do lenses bend light? Refraction!

Detector
Sample

Light Source
 Camera

Why not just

use a lens?

You can!
This is just a
magnifying
glass.

Diascopic
lamphouse
Image Formation

Simple Magnifying Glass:

Quiz: How do we focus on the blue cell?
Answer: Move the lens!

With a magnifying
glass, the magnification
and image distance are
directly coupled!
Image Formation

What makes a microscope a microscope:

Vs.
Image Formation

Compound Microscope:

Only move the front

lens to refocus. The addition of the
second lens retains the
Infinity relative object size.
Space

Objective Lens Tube Lens

A Simple Question

Why do you need a microscope?

Magnification

Compound Microscope:

Focal
Length
What happens if we We get magnification!
shrink the focal length?
Magnification

Magnification:
So why do we not magnify the object by 1,000× to see small details and structures of the sample?
Magnification

Magnification:
So why do we not magnify the object by 1,000× to see small details and structures of the sample?
Magnification

Magnification:
So why do we not magnify the object by 1,000× to see small details and structures of the sample?

Your image is not just magnified, but also blurred.

Why is my image blurry?

To understand this, we need to talk about…

Why is my image blurry?

Important Concept 1: Light behaves light a wave

Wavelength
Amplitude

A Random Wave
Why is my image blurry?

Important Concept 1: Light behaves light a wave

Color
Brightness

A Light Wave
Why is my image blurry?

Important Concept 1: Light behaves light a wave

Important Concept 2: Waves can be added and subtracted together
Constructive Destructive
Interference Interference

+ +

= =
Why is my image blurry?

Important Concept 1: Light behaves light a wave

Important Concept 2: Waves can be added and subtracted together
Important Concept 3: When light encounters an edge, it diffracts.
Wavefront
Plane Wave

Diverging
Spherical
Direction of
Wave
propagation
(rays)
Why is my image blurry?

Important Concept 1: Light behaves light a wave

Important Concept 2: Waves can be added and subtracted together
Important Concept 3: When light encounters an edge, it diffracts.
These 3 concepts work together to ruin your images! But how?
Why is my image blurry?

Important Concept 1: Light behaves light a wave Abbe: “The microscope

Important Concept 2: Waves can be added and subtracted together image is the interference of a
Important Concept 3: When light encounters an edge, it diffracts. diffraction phenomenon”
These 3 concepts work together to ruin your images! But how? Detector
1. Light 2. Light encounters an
behaves like edge and diffracts.
3. Constructive
a wave.
Interference

3. Destructive Interference
 Why is my image blurry?
This is your
Point Spread Function (PSF): Point Spread
Function!
The PSF describes how light emitted from a
single point source is detected by the system.

Point Source Lens

The specific
pattern is called
an “Airy Disk”

Image

Intensity
 Why is my image blurry?
This is your
Point Spread Function (PSF): Point Spread
Function!
The PSF describes how light emitted from a
single point source is detected by the system.

The specific
pattern is called
an “Airy Disk”

Intensity
 Why is my image blurry?

Point Spread Function (PSF): +0.6 µm

The PSF describes how light emitted from a

+0.4 µm
single point source is detected by the system.

+0.2 µm

+0 µm

-0.2 µm

-0.4 µm

z
x -0.6 µm
 Why is my image blurry?
This is your
Point Spread Function (PSF): Point Spread
Function!
The PSF describes how light emitted from a
single point source is detected by the system.

Point Source Lens

The specific
pattern is called
an “Airy Disk”

Image

Intensity
 Why is my image blurry?
This is your
Point Spread Function (PSF): Point Spread
Function!
The PSF describes how light emitted from a
single point source is detected by the system.

Point Source Lens

The specific
pattern is called
an “Airy Disk”

Image

Intensity
If you capture
and focus more
light, your PSF
gets smaller.
Why is my image blurry?

The Numerical Aperture (NA) is an indication of how well your objective collects light.

Low NA High NA 𝑁𝐴 = 𝑛 Sin(𝛼)

𝛼 – Collection Angle
𝑛 – Immersion Media
Refractive Index
Why is my image blurry?

NA and the Point Spread Function (PSF):

744 297 200

6100nm 976 440

z
NA = 0.4 1.0 1.49 x
Imaging with a Microscope

Why do you need a microscope?

Resolution

The Effect of Numerical Aperture:

Low NA High NA
Resolution

Resolution:
The minimum distance between two sources of light with which they can be
clearly differentiated in space.
Resolution

Resolvable Distance:

Resolvable
Resolution

Resolvable Distance:

Resolvable
Resolution

Resolvable Distance:

Not
resolvable
Resolution

Class Quiz: Which objective lens provides the best

resolution?

63× 40× 63× 100× 100×

1.0 NA 0.8 NA 1.4 NA 0.9 NA 1.2 NA
Resolution

Class Quiz: Which objective lens provides the best

resolution?

63× 40× 63× 100× 100×

1.0 NA 0.8 NA 1.4 NA 0.9 NA 1.2 NA
Resolution

Theoretical Resolution: Practical Resolution:

𝜆 Diffraction limited resolution is

𝑅𝑥,𝑦 ≈ 0.61 never guaranteed.
𝑁𝐴
Other critical factors:
𝜆𝑛
𝑅𝑧 ≈ 2 • Imaging media
𝑁𝐴2
• Immersion media
NA more dramatically impacts
• Coverslip
axial resolution.
• The Sample
Resolution theoretically • Brightness
depends only on NA and
• Scattering
wavelength
• Aberrations
Practical Use of an Objective

Magnification:

What will influence your choice of magnification?

Practical Use of an Objective

Magnification:

The field of view is proportional to the camera chip size, and inversely proportional to the magnification.

Increasing the magnification will decrease the field of view and reduce the amount of the sample that can be
imaged at any one time, with no other benefit.

Fixed sensor size Increased

magnification Reduced FOV
Practical Use of an Objective

Quiz: Does increasing magnification increase or decrease brightness?

Answer: Increasing magnification reduces brightness. Proportionally fewer

photons strike each pixel of the detector.

Fixed sensor size Increased

magnification Reduced FOV
Decreased brightness
Practical Use of an Objective

Depth of Field:
How much of your sample is in focus at once?
Practical Use of an Objective

Working Distance:

The distance between the front of the objective and the

nearest surface when a specimen is in focus.

Directly related to Numerical Aperture.

Practical Use of an Objective

Immersion Medium:

Immersion media is a requirement for some lenses.

• Air: n = 1.0003
• Water: n = 1.33
• Glycerin: n = 1.47
• Immersion oil: n = 1.51 *oils are matched to specific temperatures
 Practical Use of an Objective

Immersion Medium:

3 2 1 0 1 2 3 4 5
4

n = 1.0 n = 1.5

θ = 50° θ = 65°
Practical Use of an Objective

Immersion Medium:
The oil you choose matters!

Talley Lamber, HMS

Practical Use of an Objective

Refractive Index Matching:

Why does this matter?

Water Water Water

Air Water Oil

What is missing here? The coverslip! Objectives assume you are using a #1.5 coverslip and account for it.
Practical Use of an Objective

Correction Collar:

Spherical Aberration Coverslip correction

Practical Use of an Objective

Refractive Index Matching: What else is missing? Your sample!

Biological material is dense and heterogeneous, so all bets are off…

Water Water Water

Air Water Oil

Practical Use of an Objective

Aberration Correction:

Achromat:
Chromatic aberration correction at 486 and 656nm light; Spherical
aberration correction at 546nm light.
++Fluorite:
Extended spherical aberration corrections (up to 3 colors of light).
+++Apochromat:
Extended chromatic aberration correction (up to 4 colors of light); Extended
spherical aberration corrections (up to 4 colors of light).

Plan:
Additional flat-field correction.
Summary
Image Formation Magnification, PSFs, and Resolution

Waves, interference, and diffraction

Objective Lenses