Microscopy from the very beginning and

Spotlight: Introduction of Light Microscopy

Introduction of Digital Image
¾ Different types of light microscopes
ƒ Upright microscope
ƒ Inverted microscope
ƒ Stereo microscope

¾ Upright and inverted microscope

¾ Basic concepts of light microscopy

ƒ Magnification
ƒ Resolution power and N.A. of an objective

¾ Two kinds of beam path: transmission light and reflected light

¾ Transmitted Techniques in Light Microscope

Taiwan Instrument Co, Ltd ƒ Bright field
ƒ Dark field
台灣儀器行股份有限公司 ƒ Phase
科學儀器部 :葉正造
ƒ DIC

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Spotlight
Different types of light microscopes
實驗室中常用的生物顯微鏡

¾ Principle 正立顯微鏡 倒立顯微鏡 解剖(實體,立體)顯微鏡

ƒ Stoke shift Upright Microscope Inverted Microscope Stereo Microscope
ƒ Excitation and emission spectrum

¾ Hardware accessories
ƒ light source
ƒ Filter

¾ How to get the best fluorescence picture?

ƒ Fluorescence Crosstalk (bleed through)

¾ Acquisition a Digital Image by a Digital CCD Camera

¾ Image Digitalization Principle

Acquisition a Digital Image by a Digital CCD Camera

Image Digitalization Principle 3 4

Upright Microscope Inverted Microscope

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Stereo Microscope Inverted Light Microscope ( Axiovert 200M )

HAL Lamp

Field Diaphragm

Aperture diaphragm
Specimen stage
Eyepiece
Condenser
Binocular tube
Objective

Filter turret
Aperture diaphragm
Field diaphragm
Lamp for fluorescence
HBO100
Stand

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Upright Light Microscope The difference between the upright and inverted microscope
Illumination
equipment for
fluorescence

Lamp for
fluorescence
Eyepiece
Binocular tube
condenser
larger
Filter turret Luminous-field and working
aperture diaphragm space
Specimen stage for fluorescence
Nosepiece
Vibration
Transmitted Objective free of
Condenser with
the stage
aperture diaphragm condenser while
focusing
Luminous-Field Light Control
Diaphragm
Illuminator

Stand

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The difference between the upright and inverted microscope Long working Distance objective

¾ Most microscope objectives are designed to be used with a cover glass

that has a standard thickness of 0.17 millimeters and a refractive index of
1.515,

condenser
larger
working
space

Vibration
free of
the stage
condenser while
focusing

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 Spotlight: Introduction of Light Microscopy The incident angle determines the size that we see

¾ Different types of light microscopes

ƒ Upright microscope
ƒ Inverted microscope
ƒ Stereo microscope

¾ Upright and inverted microscope

¾ Basic concepts of light microscopy

ƒ Magnification
ƒ Resolution power and N.A. of an objective

¾ Two kinds of beam path: transmission light and reflected light

¾ Transmitted Techniques in Light Microscope

ƒ Bright field
ƒ Dark field
ƒ Phase
ƒ DIC

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All the magnifier are designed for extending the incident angle What dose the “magnification” actually mean?

¾The incident angle is magnified by lens. ¾ Image in your eyes:M Microscope = M Objective X M Eyepiece
¾Microscopy means seeing a large image of something
¾ TV image from the microscope
eye
small.
Eyepiece •A, microscope beam path
EXAMPLE:
Intermediate •B, see the object directly from a distance of An erythrocyte (Ø 8 μm) is photographed using the
image Achroplan objective 100x. The TV adapter has the factor
approx. 25 cm. 0.5x:
Moptical = 100 x 0.5 = 50 x
•1, object
The active sensor diagonal of the 1/3”-chip CCD-camera is
•2, objective物鏡 (image projected at infinity) 5.3 mm.
The monitor has a useful image diagonal of 61 cm (= 610
•3, tube lens (produce a magnified intermediate mm).
Tube lens
Melectronic = 610 mm / 5.3 mm = 115 x
image)
The overall magnification then is:
•4, intermediate image Moverall = Moptical x Melectronic = 50 x 115= 5750 x
Objective The erythrocyte appears on the screen with 8 μm x 5750
•5, eyepiece = 46000 μm or 46 mm = Ø 4.6 cm
•6, eye
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What does ‘resolution’ actually mean?

Point Spread Function

Magnification alone is not enough:

the resolution determines what we see. Airy disks

ƒ Numerical Aperture and Resolution

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 What does ‘resolution’ actually mean? The numerical aperture of objectives

The resolving power, the limit up to which two

intensity
small objects are still seen separately.
¾The light incident from the objects is deflected
(偏斜) from the original direction. To obtain sharp
images of small structures, the objective must
Airy disk collect as much of this diffracted light as
possible. The term aperture (opening) describing
this property. Numerical aperture (N.A.) : a
measure of the solid angle covered by an
objective.

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The Numerical Aperture (N.A) of objectives The numerical aperture of objectives

The numerical aperture of a microscope objective is a measure of its ability to ¾The light incident from the objects is deflected
gather light and resolve fine specimen detail at a fixed object distance. (偏斜) from the original direction. To obtain sharp
images of small structures, the objective must
collect as much of this diffracted light as
possible. The term aperture (opening) describing
this property. Numerical aperture (N.A.) : a
measure of the solid angle covered by an
objective.

reflective
index=n

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Numerical aperture (N.A.)

A. Low Magnification (10X/0.25)

B. High Magnification (40X/0.75)

glass n =1.51
oil n =1.51
air n =1

cover glass
n =1.51

α
α’ The aperture of the objective
and the resolving power would
be reduced by the reflection.

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 Spotlight: Introduction of Light Microscopy Transmitted-light and Reflected-light in inverted microscope

¾ Different types of light microscopes

ƒ Upright microscope
ƒ Inverted microscope
ƒ Stereo microscope

¾ Upright and inverted microscope

¾ Basic concepts of light microscopy

ƒ Magnification
ƒ Resolution power and N.A. of an objective

¾ Two kinds of beam path: transmission light and reflected light

¾ Transmitted Techniques in Light Microscope

ƒ Bright field
ƒ Dark field
ƒ Phase
ƒ DIC
Transmitted-light Reflected-light (Fluorescence)
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Transmitted Techniques in Light Microscopy

Transmitted-light and Reflected-light in upright microscope

¾ 1.明視野 Bright field – 有染色、有一定厚度的樣本

¾ 2.暗視野 Dark field – 對比差、極微小樣本(微生物等)

¾ 3.相位差 Phase contrast – 較薄、透明、無染色、對比差樣本

¾ 4.干涉位相差 Differential Interface Contrast – 透明、對比極差、表面形態觀察

HBO
HBO
¾ 5.偏光 Polarization – 結晶體、澱粉、聚合物、有偏光反應之樣本

HAL

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Bright Field Phase contrast

¾Phase contrast is ideal for thin
unstained objects, for example culture
cells on glass, which are approx. 5 bis
10 um “thick” above the cell nucleus,
but less than 1um “thick” at the
periphery, and which barely exhibit
any light absorption in the visible part
of the spectrum.

¾The eye can scarcely see them in

bright field and dark field. However,
very small differences exist between
the refractive indices of the cells and
the surrounding aqueous solutions
and within the cells between the
¾Bright Field is the most universal technique used in light microscope. cytoplasm and the cell nucleus.
¾Usually used in samples with colorimetric staining or good contrast.

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Phase Contrast Phase Contrast

Phase stop
in condenser

Phase ring
New path in objective

Phase ring
¾The higher the refractive index of a medium, in objective
the smaller the speed or velocity of light in the
medium.

¾It translates the tiny differences into Phase stop in

condenser
differences in intensity.

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Differential Interference Contrast (DIC) Differential Interference Contrast (DIC)

1. Polarizer

2. Condenser prism

6. DIC prism

7. Analyzer
7. Analyzer

6. DIC prism (slider

behind the objective)

Decomposition and
laterally shift the partial
light beams

2. Condenser prism
1. Polarizer DIC prism

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The way of increasing the optical resolving power

¾Choose a large angle of the ray cone on the illumination side.
¾To use immersion liquids between the front lens of the objective and the cover slip.

¾Use Condenser. max. resolution if N.A.objective=N.A.condenser

Condenser

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Microscopy from the very beginning
Condenser Koehler illumination:顯微鏡之穿透光校正
¾1.eyepiece歸零 2.放置樣本之載物台，選用低倍數物鏡10x 3.選擇明視標準觀察位置
4.對焦至清晰位置將視野光圈轉至最小範圍，同時上下移動聚光鏡位置使視野邊至清晰位置
5.慢慢打開視野光圈，同時判斷中心位置之偏離方向，利用聚光鏡中心校正螺桿調至中心位
置，最後全開視野光圈至剛好覆蓋全視野大小位置

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Koehler illumination:顯微鏡之穿透光校正 Spotlight: Epifluoresence microscopy introduction

¾ Principle
step1 ƒ Stoke shift
ƒ Excitation and emission spectrum
step2 ƒ Bleach of a fluorechrom
step3

¾ Hardware accessories
ƒ light source
ƒ Filter

¾ Fluorescence Crosstalk (bleed through)

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Wavelength in Color Fluorescence of a fluorephore

Wavelength color
1. 340 - 400 nm near Ultraviolet (UV) - Invisible excited state
heat
2. 400 - 430 nm Violet
3. 430 - 500 nm Blue
4. 500 - 560 nm Green
fluoresce
5. 560 - 620 nm Yellow to Orange
6. 620 - 700 nm Orange to Red
ground state
7. Over 700 nm near Infrared (IR) - Invisible
¾ Some molecules are capable of being excited, via absorption of light
energy, to a higher energy state, also called an excited state. The energy
of the excited state, which cannot be sustained for long, “decays” or
decreases, resulting in the emission of light energy. This process is
called fluorescence. To “fluoresce” means to emit light via this process.
http://probes.invitrogen.com/

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Stoke shift Light source

¾ This difference between the excitation and emission maxima is called the
Stokes shift. The magnitude of the Stokes shift is determined by the
electronic structure of the fluorophore, and is a characteristic of the
fluorophore molecule.

40X

1. Quartz Glass bulb

2. Cathode
3. Anode
Stoke shift
4. Burning Chamber contains some Mercury
5. Light Arc

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Light source The principle of fluorescence microscope

HBO XBO

The reflected beam path on light microscope

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Conventional microscope use wavelength filters to Fluorescence filter sets

segment the excitation light and emission light

Types of the filter

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The filters used in conventional light microscope How to get the best fluorescence image?

A:EX BP 450-490 ¾Excite samples with the appropriate excitation wavelength.

B:BS FT 510
¾Detect the strong and pure signals.
C:EM LP 515
¾Eliminate the signals from the out-of-focus plane.

1. Light from HBO Lamp

2. Monochromatic Light
3. Fluorescence Light returning from the
Specimen
A. Excitation Filter
B. Dichroic Beam Splitter The spectra of FITC
C. Emission Filter
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How to get the best fluorescence image? How to get the best fluorescence image?

¾ Excite samples with the appropriate excitation wavelength.

¾ Detect the strong and pure signals.

¾ Eliminate the signals from the out-of-focus plane.

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¾ Emission intensity depends on excitation efficiency

¾ The more efficient excitation induces the stronger signal of emitted light.
The emission spectra of FITC The emission spectra of FITC and
other fluorophore

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The crosstalk problem The crosstalk problem

LP 515
FITC / Rhod FITC / Rhod

exi emi
FITC 495 519
Rhod 547 572

FITC excitation spectrum overlapes with Rhod excitation spectrum.

Excite FITC only at 450~490nm

exi emi
FITC 495 519
FITC FITC
Rhod 547 572

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The crosstalk problem The crosstalk problem
BP 515~565
FITC / Rhod LP BP
exi emi
FITC 495 519
Rhod 547 572

¾Cross talk ¾Avoid cross talk

¾brighter ¾dimmer
FITC

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How to get the best fluorescence image? Then you will get fluorescence images

¾ Excite samples with the appropriate excitation wavelength.

¾ Detect the strong and pure signals.

¾ Eliminate the signals from the out-of-focus plane.

CONFOCAL

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Digital Camera Acquisition a Digital Image by a Digital CCD Camera

Digital camera

CCD chip
(Charge Coupled Device
電荷耦合元件)

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Acquisition a Digital Image by a Digital CCD Camera Introduction of Digital Image

Senser: CCD chip Spotlight

Collected Lens
¾Acquisition a Digital Image by a Digital CCD Camera
Bayer Filter
(absent in monochrome ¾Image Digitalization Principle
CCD )

Sensor
Photon → Electron

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Image Digitalization Principle Acquisition a Digital Image by a Digital CCD Camera

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Analyze Image to Basic Component: CCD: Charge Couple Device

¾Pixel CCD Chip

¾Intensity (Monochrome or Color) 1040

Zeiss AxioCam HRm Photon → Electron

Chip Size: 8.9 * 6.7 mm2
Resolution: 1388*1040 pixels
Pixel Depth: 14 bits
Cooling
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Image Digitalization Principle Image Digitalization Principle

Pixel: the basic element of image Intensity: the basic “color” data of every pixel
Monochrome:
Monochrome Gray Scale from black (dark) to white (brightness)
Pixels are identified by
1 bit： “0” (black) “1” (white) → 2 Gray Scale
their position in a grid
(two-dimensional array),
reference by its row (x),
and column (y).

2 bit： “0”“0”(black), “0”“1”(dark gray),

“1”“0”(light gray), “1”“1”(white) → 4 Gray Scale
4 bit：→ 16 Gray Scale
The more pixels,
8 bit：→ 256 Gray Scale
the better resolution, 12 bit：→ 4096 Gray Scale
the more image size

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Intensity: the basic “color” data of every pixel
Image Digitalization Principle

Color:
Color Red (R), Green (G), Blue (B)

R: Red
G: Green
B: Blue
Y: Yellow
C: Cyan
M: Magenta
W: White

1 bit image 8 bit image

R: 8 bit, 0-255 intensity gray

Eye: 8 bit G: 8 bit, 0-255 intensity gray
CCD camera:12,14,16 bit B: 8 bit, 0-255 intensity gray
RGB：24 bit 16777216 color

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When the signal from samples is very slight…..

Image Digitalization Principle

Increase exposure time gain binning

•Photobleach •more noise
• High speed
•more noise •background
• Redusce
•cannot keep up with reaction •Loss contrast resolution
time

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binning binning

10 10 10

10 10 10 90
10 10 10 3x3
BINNING

Binning: Decreasing resolution to

Increase signal intensity.

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binning Quantum efficiency

Photon → Electron

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Digital Image Measurement & Analysis Digital Image Measurement & Analysis

Spatial Measurement: ※Spatial Calibration

Principle of Digital Image:
Image
Calibration: Pixel → Micrometer (μm)
¾Pixel

¾Intensity

Fundamental of Measurement & Analysis:

¾Spatial: Length, Area, Volume, Diameter, Radius,

Perimeter, Angle, etc.
Base on Pixel

¾Light Density: Average Intensity, Min Intensity,

Max Intensity, etc.
Base on Intensity

Every magnification, every resolution should be done once!

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