The Basic Concept of Light Microscope
Taiwan Instrument Co, Ltd
Eva Lin
1
Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
2
Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
3
Different types of light microscopes
Upright Microscope
Inverted Microscope
Stereomicroscope
4
Upright Microscope
• Designed for slide sample
• High magnification
• High resolution in transmitted
light sample
5
Inverted Microscope
• Lower resolution in transmitted
light sample
• Long working distance
• Suitable for petri dish sample
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Inverted Microscope
Micromanipulation
37℃
℃, 5% CO2, Living cell incubation
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Stereomicroscope
• Long working distance
• Low magnification
• Observation with stereo perception
For large object observation:
• Plant, Zebrafish, Drosophila,
Mouse dissection..etc
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Different types of light microscopes
Upright Microscope
Slide sample only
High resolution
Inverted Microscope
Slide, Dish, Multiwell plate
Living cell incubation
Micromanipulation
Stereomicroscope
For large sample
Observation in 3 dimention
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Parts in Upright Microscope
Camera
Eyepiece
Filter Wheel
Lamp house
for Fluorescent sample
Objective
Sample
Condenser
Condenser wheel
Lamp house for
Tramsmitted light
sample
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Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
11
The incident angle determines the size that we see
Incident angle
θ
Two factors are important
for incident angle:
Distance and size of object
θ
θ
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Basic principle of light microscope
- The incident angle is magnified by lens
Condition without microscope
θ
Very Small
object
Microscope helps us to enlarge the viewing angle !!
Very Small
object
θ
Objective
Tube lens
Intermediate
image
Eyepiece
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Basic principle of light microscope
- Magnification of the Microscope
MMicroscope
= MObjective × MEyepiece × MIntermediate Factor
• M = magnification
Example:
Objective = 40 x
Eyepiece = 10 x
Without magnifying glass
Overall M = 40 x 10 x 1 = 400
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Magnification alone is not enough:
the "Resolution" determines what we see.
Numerical aperture and Resolving power
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Numerical aperture
Numerical Aperture = N.A. = n · sin α
α: half the opening angle of the objective
n: the refractive index of the immersion medium
used between the objective and the object
(n = 1 for air; n = 1.51 for oil or glass)
α
■ The N.A. value:
The ability of an objective to gather
the diffracted light at a fixed working
distance.
The higher N.A. value,
the more details you can get from your sample
Numerical aperture
(α)
α
α
α
α
α
α
Magnification:
Working distance:
N.A. value:
Low
Long
Low
High
Short
High
N.A. Value for Condenser
N.A. = 0.9
Max. N.A. = 0.55
What does "Resolution" actually mean?
The shortest distance that you still
can distinguish the two objects.
1 μm
0.2 μm
0.1 μm
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What does "Resolution" actually mean?
Microscope:
Point Spread Function
Airy disks
1 μm
0.2 μm
0.1 μm
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Resolving Power
Example:
Wavelength = 550 nm
N.A. obj = 1.4
N.A. Cond = 0.9
d0 = 1.22 × 550 nm / (1.4 + 0.9) = 291 nm
■ The lower d0 value, the higher
resolution you have
The shortest distance between
the two objects.
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Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
22
Transmitted Light (TL) Path - Color-stained sample and Cell morphology
Reflected Light (RL) Path - Fluorescence sample
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Transmitted-light and Reflected-light in upright microscope
Camera
Eyepiece
RL
Filter Wheel
Objective
Sample
Condenser
Condenser wheel
TL
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Transmitted-light and Reflected-light in inverted microscope
TL
Camera
Condenser Wheel
Eyepiece
Condenser
Stage
Objective
Filter Wheel
RL
25
Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
26
Techniques for Transmitted-light Observation
•
Bright field (H) - Color-stained, high-contrast sample
•
Dark field (D) - Find structure, tiny sample
•
Phase contrast (Ph) - Low-contrast, transparent sample
•
Differential Interference Contrast (DIC)
H
- Low-contrast sample, for surface structure observation
Bright field
Phase contrast
Bright field
DIC
Bright field (H) - Color-stained, high-contrast sample
The most universal technique used in light microscope.
Diagnosis of pathological section stained by HE stainning.
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Dark field (D) - Find structure, tiny sample
Fine structures can often
not be seen in front of a
bright background.
In a dark background, you
can see the fine structure
of your sample.
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Dark field (D)
Objective
Sample
Condenser optics
Annular stop
• Annular stop is needed.
• Aperture: Objective < Condenser
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Dark field (D)
Objective
Sample
Condenser optics
Annular stop
Condenser wheel
Switch to "D"
Phase contrast (Ph)
- Low-contrast, transparent sample
•
•
Used for thin unstained objects.
For example culture cells with
approx. 5 bis 10 um “thick” above
the cell nucleus, but less than
1um “thick” at the periphery.
H
Ph
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Phase contrast (Ph)
Phase ring
(Objective)
Arabic Numeral : Ph1, Ph2, Ph3
Phase stop
(Condenser wheel)
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Phase contrast (Ph)
Phase ring
(Objective)
Arabic Numeral : Ph1, Ph2, Ph3
Phase stop
(Condenser)
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Differential Interference Contrast (DIC)
- Low-contrast sample, for surface structure observation
DIC is used fo unstained, thick samples.
Components :
2 DIC prism, polarizer and analyzer
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Differential Interference Contrast (DIC)
Analyzer:
Filter wheel at DIC position
DIC prism:
Above objective
Polarizer + DIC prism:
Condenser wheel
Roman Numeral : DIC I, II, II
Get Information from Your Objective
The classification of obj.
LD -> Long working distance,
for petri dish sample
Correction ring for
adjusting the thickness
of cover glass.
Standard thickness :
0.17 mm
Magnification: 40
Numerical Aperture (N.A.): 0.6
Phase contrast
Get Information from Your Objective
Use with immersion oil
Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
39
The Priciple of Fluorescence
EGFP (Ex. 488 nm, Em 507 nm)
Excited state
Ground state
Fluorophore
Texas Red (Ex. 589 nm, Em 615 nm)
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The Light Source of Fluorescence
White
light
Specific
wavelength
Filter
Filter
Mercury lamp:
Provide intensive,
full spectrum light
(UV portion)
DAPI
Filter
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The Types of Filters
Transmission
1. Shortpass filter
2. Longpass filter
3. Bamdpass filter
Wavelength
Filter Set
李郁蕙 Grace Li
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Filter Set
2. Emission filter
Transmission
1. Excitation filter
Filter set for EGFP:
Excitation filter: 450-490 nm
Exmission filter: 500-550 nm
Beam splitter filter: > 495 nm
3. Beam
splitter filter
Wavelength
Sample
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Digital CCD Camera
Basic Concept of Digital CCD Camera
Pixel and Resolution
Quantum Efficiency
Image Depth: the bit number
For detecting weak signals:
- Exposure time
- Binning
- Gain
CCD - The Charge Coupled Device
Collected Lens
Color Filter
(absent in monochrome CCD )
Sensor:
Transfer photon to electric signal
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CCD - The Charge Coupled Device
1388 pixels
CCD
Light detecting unit "Pixel"
1040
pixels
CCD is composed of the light detecting unit "Pixel".
- Pixel number
Resolution
- Pixel size
Sensitivity
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CCD - The Charge Coupled Device
1388 pixels
CCD
1040
pixels
AxioCam MRm
AxioCam ICm 1
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Quantum Efficiency
CoolSNAP HQ2 CCD
The ability of CCD to transfer Quantum to Electric signal
Quantum Efficiency : Monochrome v.s. Color CCD
Mono
Color
The QE value of Monochrome CCD is usually higher than color CCD.
The Image Depth - For Quantification
Scaling the Gray level from dark to brightness
1 bit：
：21 = 2 Gray Scale
2 bit：
：22 = 4 Gray Scale
8 bit：
：28 = 256 Gray Scale
12 bit：
：212 = 4096 Gray Scale
1 bit Image
8 bit Image
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The Image Depth for Color Image
R: 8 bit, 0-256 intensity gray
G: 8 bit, 0-256 intensity gray
B: 8 bit, 0-256 intensity gray
RGB：
：8 × 3 = 24 bit
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Basic Concept of Digital CCD Camera
Pixel and Resolution
Quantum Efficiency
Image Depth: the bit number
For detecting weak signals:
- Exposure time
- Binning
- Gain
For detecting weak signals..
Increase exposure time:
Gain:
Binning:
• Photobleach
• More noise
• High speed
• More noise
• Background
• Reduce resolution
• Cannot keep up with
reaction time
• Loss contrast
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Binning
Binning: Decreasing resolution to Increase signal intensity.
Resolution
Binning mode
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Monochrome v.s. Color CCD
Type
Monochrome
Color
Color
Pseudo-color
True color
Sensitivity
Higher
Lower
Signal
Sharp
Blunt
Application
Fluorescence sample
李郁蕙 Grace Li
Color-stained sample
58
Today's Outline
•
Different types of light microscopes
– Upright microscope
– Inverted microscope
– Stereomicroscope
•
Basic concepts of light microscope
– Magnification
– Resolution power and Numerical aperture
•
Two kinds of light path : transmitted light and reflected light
•
Transmitted Techniques in Light Microscope
– Bright field, Dark field, Phase, DIC
•
•
Fluorescence and Digital CCD Camera
Optical section: Spinning Disk and TIRF
59
What is an Optical Section?
Qualitative…
Optical Section
Conventional image
• Removed “out-of-focus” light
• Only the light from a thin
region near the focal plane
of the objective remains in the
final image
Optical section
• Improved image quality:
better signal-to-background
more information
李郁蕙 Grace Li
60
General Optical Sectioning Methods
General Overview
Optical Sectioning Methods
Avoiding out-of focus light
(excitation strategy)
Multi-Photon
Total Internal
Reflection
Blocking out-of focus light
(detection strategy)
Confocal
Methods
Removing out-of focus light
(downstream strategy)
Structured
Illumination
Deconvolution
Confocal Point Scanner
Confocal Line Scanner
Spinning Disc Systems
李郁蕙 Grace Li
61
General Optical Sectioning Methods
General Overview
Optical Sectioning Methods
Avoiding out-of focus light
(excitation strategy)
Multi-Photon
Total Internal
Reflection
Blocking out-of focus light
(detection strategy)
Confocal
Methods
Removing out-of focus light
(downstream strategy)
Structured
Illumination
Deconvolution
Confocal Point Scanner
Confocal Line Scanner
Spinning Disc Systems
李郁蕙 Grace Li
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Spinning Disk
Spinning Disk Confocal Microscopy
Principle
• parallel scanning of ~ 1000 points
Microlens Array
arranged on a disk
Excitation Light
Beamsplitter
• rotation of the disk scans the pinholes
over the sample
Lens
• microlenses focus the incident laser
light through the pinholes to increase
light input
• Light from the focal plane passes
Camera
Pinhole Array
Objective
through the pinholes; out of focus light
is rejected
• The in-focus light is then reflected by
the dichroic beam splitter onto an area
detector (CCD Camera)
李郁蕙 Grace Li
Specimen
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Spinning Disk
Spinning Disk Confocal Microscopy
Advantages
• High frame rates due to parallel
scanning of >1000 points
• Low excitation power densities and
long dwell times, therefore low
bleaching and low phototoxicity
• Use of area detectors with highest
quantum yield possible (CCD or
EMCCD)
李郁蕙 Grace Li
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Spinning Disk
Spinning Disk Confocal Microscopy
Drawbacks
• Fixed pinhole diameter: Use with low
magnification objectives results in
suboptimally thick sections
• Low signal to background in thick /
scattering samples due to pinhole
crosstalk
• Artefacts: Stripes and Moire-patterns
李郁蕙 Grace Li
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Spinning Disc
Microlens-enhanced Spinning Disc
Typical Applications
Image subcellular trafficking in 3D
with maximum acquisition speed
Visualize cytoskeletal dynamics
with highest sensitivity
Functional imaging of cellular signal
transduction with high time
resolution
Long-term live cell imaging with
lowest phototoxicity
李郁蕙 Grace Li
66
General Optical Sectioning Methods
General Overview
Optical Sectioning Methods
Avoiding out-of focus light
(excitation strategy)
Multi-Photon
Total Internal
Reflection
Blocking out-of focus light
(detection strategy)
Confocal
Methods
Removing out-of focus light
(downstream strategy)
Structured
Illumination
Deconvolution
Confocal Point Scanner
Confocal Line Scanner
Spinning Disc Systems
李郁蕙 Grace Li
67
TIRF
TIRF
Principle
Evanescent Field
n2=1.33
cell
membrane
coverslip
n1=1.52
objective
Incident
light
Fluorescence
light
Reflected light
The Evanescent Field can be used for fluorescence excitation
李郁蕙 Grace Li
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TIRF
TIRF
Advantages
• Excitation limited to evanescent field
black background
• Highest z-resolution
• Acquisition rates only limited by camera
Conventional HBO-excitation
TIRF
李郁蕙 Grace Li
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TIRF
TIRF
Drawbacks
• Limited to samples at the glass-water interface
• No Z Stacks, so limited experiment potential
• Limited to objectives with NA > 1.4 so limited FOV
Conventional HBO-excitation
TIRF
李郁蕙 Grace Li
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Thank you for your attention~
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