Optical System

International Service Department

 Contents

1. 5DIFF Principle
2. Optical System Structure
3. Adjustment
4. Scatter gram
5. Troubleshooting

Version: 1.2, Update Date: 2009-3-12

 5 DIFF Principle

Version: 1.2, Update Date: 2009-3-12

 Blood Cells

 Blood consists of 40-50% blood cell and 50-60% plasma.

 Three main parts of blood cell:
red blood cell, white blood cell and platelet.

Version: 1.2, Update Date: 2009-3-12

Blood Cells Gra

Eos
RBC

Mon

PLT

Bas

Normal blood cell volume range:RBC ： 60-120fl Lym

WBC ： 120-1000fl ,PLT ： 2-30fl
Version: 1.2, Update Date: 2009-3-12
WBC 5-part proportion
Five groups of leukocyte (stained)

Lymphocyte 25 ~ 30%
Monocyte 3 ～8
%

Neutrophil 50 ~70%
Basophil 0 ～1%
Eosinophil 0.5 ～
3%
Version: 1.2, Update Date: 2009-3-12
 Morphological

Version: 1.2, Update Date: 2009-3-12

 Conclusion:

Version: 1.2, Update Date: 2009-3-12

 How can we identify
the five groups?

Version: 1.2, Update Date: 2009-3-12

 5Diff Principle
4-DIFF channel: Lymph, Mono, Neut, Eos:
Flow cytometry
Laser scatter
Morphological treatment: DIFF channel
4 Diff scatter gram
BASO/WBC channel:
Impedance method
Morphological treatment: WBC channel
WBC histogram

Version: 1.2, Update Date: 2009-3-12

 Flow Mode of Cell Suspension

Normal flow Sheath flow

Version: 1.2, Update Date: 2009-3-12

 Hydrodynamic Foucsing (Sheath Flow)
 Sheath fluid surrounds and forces blood cells suspended in dilution to pass
through the center of flow cell one by one.

• Blood cells pass through the facula of flow cell with high speed.

• Sample flow width 20-30um.

20~30 um
Version: 1.2, Update Date: 2009-3-12
 Multi-angle Laser Scatter
High Angle Scatter 8~20
reflects granularity and
5
complexity

3 4
Low Angle Scatter 1~5
reflects the cell volume
1 Sample
2 Laser light
3 Lens

1
Version: 1.2, Update Date: 2009-3-12
 Laser Scatter: Mie Scatter

Mie scatter:
 Cells are bigger than the laser wave length.

Version: 1.2, Update Date: 2009-3-12

 Mie Theory
 For different cells of different size
 Low angle can better reflect the information of size

Laser
LAS

Version: 1.2, Update Date: 2009-3-12

 Low Angle Scatter (LAS)

The low angle scatter signal (1~5) along the direction of incidence light is
called forward scatter.
It reflects the volume of cell.

Version: 1.2, Update Date: 2009-3-12

 Mie Theory

 For different cells of same size

 High angle can better reflect the information of structure

Laser

MAS

Version: 1.2, Update Date: 2009-3-12

 4-DIFF Channel-Side Scatter

Laser
FS

SS

The side angle (8~20) scatter signal along the direction of incidence
light is called side scatter.

It is related to cellular contents (granularity, nuclear content, and so

forth ). It reflects the internal complexity of cell.
Version: 1.2, Update Date: 2009-3-12
 Flow cytometry + Laser Scatter

 Flow cytometry + Laser Scatter

Version: 1.2, Update Date: 2009-3-12

 FAQ
 Is Flow cytometry + Laser Scatter enough
to have 5-part?

Morphological treatment

Version: 1.2, Update Date: 2009-3-12

 4-DIFF Channel- 4 diff differential
Blood
LEO(I) RBC

Lym Gran Mon

LEO(II)

Eos Neu

Dilute ratio 1:135(348)

Version: 1.2, Update Date: 2009-3-12
 DIFF channel

Morphological treatment
Nucleus Basophile

Scatter Scatter
intensity intensity

Time Time

Version: 1.2, Update Date: 2009-3-12

 DIFF channel

Morphological treatment
SIZING STAIN
Eos

Neu

... . .. . ... . .. . ... . .. . Mon

. .. . .. . ..
. . Lym
.

Version: 1.2, Update Date: 2009-3-12

 4-DIFF Channel-Scatter gram

Neu
Mon
Eos
Lymph

Ghost

Horizontal axis: High angle scatter signal reflects the internal complexity of
cell.
Vertical axis: Low angle scatter signal reflects the volume of cell.

Version: 1.2, Update Date: 2009-3-12

 BASO/WBC channel-Impedance Method
Pulse graph

40fL
35fL
30fL
25fL
Dilunt 20fL

LH
Aperture Oscillograph

Dilute ratio 1:500(753)

Histogram

Version: 1.2, Update Date: 2009-3-12

BASO/WBC channel-Baso differential

Blood

LH RBC

Baso Lym, Mon, Neu, Eos

Dilute ratio 1:500(753)

Version: 1.2, Update Date: 2009-3-12

 BASO channel

Morphological treatment
SIZING
Eos

Neu

Bas

... . .. . .... .. Mon

. .. ..
. Lym

Version: 1.2, Update Date: 2009-3-12

 Baso/WBC Histogram

Version: 1.2, Update Date: 2009-3-12

 Optical System Structure

Version: 1.2, Update Date: 2009-3-12

 Optical System Structure

Note: MAS=SS LAS=FS

Version: 1.2, Update Date: 2009-3-12

 Optical System Components
Laser control board

SS PD Assembly

FS PD Assembly
FS Beam Stop
SS Beam Stop

Flow Cell
Assembly

Back Lens Assembly Splitter

Forward
Version: Optical
1.2, Update Assembly
Date: 2009-3-12 Backward Optical System
 Forward Optical Assembly

■ Function: generate facula to irradiate the blood

cell.

Version: 1.2, Update Date: 2009-3-12

 Forward Optical Assembly

Version: 1.2, Update Date: 2009-3-12

 Laser Source

Semiconductor laser technology

small
Semiconductor – more stable, low qua
ntity of heat
long life and low cost for maintenance

Version: 1.2, Update Date: 2009-3-12

 Flow Cell Assembly
■ Function: generate stable sample flow; make
blood cell in the sample flow pass trough laser
irradiation area one by one.

Version: 1.2, Update Date: 2009-3-12

 Sheath fluid bath

Version: 1.2, Update Date: 2009-3-12

Sheath fluid observation

Version: 1.2, Update Date: 2009-3-12

 Backward Optical System
■ Function: first collimate the scatter light, then use prisms
to separate the light into two directions, and use beam stop
in each direction to collect the light at certain angle.

Version: 1.2, Update Date: 2009-3-12

 Backward optical system optical path

Version: 1.2, Update Date: 2009-3-12

 Back lens assembly
• Block the direct light, and then collimate.

Version: 1.2, Update Date: 2009-3-12

 Splitter assembly
• Divide into two directions.

Version: 1.2, Update Date: 2009-3-12

 Beam Stop Assembly
• Block the scatter light at certain angle, and then focus onto
the PD.
SS Stop Assembly

FS Stop Assembly

Version: 1.2, Update Date: 2009-3-12

 PD Assembly

• Remove the stray light and convenient to focalize.

Version: 1.2, Update Date: 2009-3-12

 Optical System Adjustment

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
1. Adjust light source collimation and focusing

Pitch Pin

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
2. Adjust flow cell bath (1)

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
3. Adjust separate light prism and beam stop

Symmetric

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
4. Adjust back light collimation

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
5. Adjust flow cell bath (2)

Flow cell edge

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
6. Adjust incidence light beam stop

Version: 1.2, Update Date: 2009-3-12

 Optical system adjustment － primary
adjustment
7. Adjust photoelectric diode

Low angle scatter High angle scatter

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
1. Adjust forward light focus position

Tools:

FLUKE124

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
1. Adjust forward light focus position

Test points:
FS Pre-amplify board

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
1. Adjust forward light axis position
Let FS pulse be biggest, width smallest(1.3us), and stable.

Oscilloscope settings ： AC Coupler ， Voltage 50mv/div ， Time 500ns/div

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
2. Adjust back light axis position

7um standard particle Sample

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
2. Adjust back light focus position

Test points:
SS Pre-amplify board

Signal Processing board

SS TP44---MASIN

Version: 1.2, Update Date: 2009-3-12

 Adjust Focus Position
Let pulse of SS be biggest.
Oscilloscope settings ： AC Coupler , Voltage 100mv/div ， Time 500ns/div

2
2

Version: 1.2, Update Date: 2009-3-12

 Scatter gram and histogram

Version: 1.2, Update Date: 2009-3-12

 Normal Scatter grams and histogram

Version: 1.2, Update Date: 2009-3-12

Normal scatter gram of standard particle
Standard particle target value
Param Before version After version
1.4 1.4
FS 26 ± 3 18±2

SS 117± 5 104±6

SS 0.1Max ≤18 ≤15

Width
FS 0.1Max ≤13 ≤8
Width
7um particle

Version: 1.2, Update Date: 2009-3-12

 Abnormal scatter gram

Version: 1.2, Update Date: 2009-3-12

 Abnormal samples: Flag

Left Shift
LIC

Neu
Mon
Eos
ALY

Lym ALY

NRbc
Ghost

Version: 1.2, Update Date: 2009-3-12

 Abnormal scatter gram

Left shift

LIC

Version: 1.2, Update Date: 2009-3-12

 Abnormal scatter gram

ALY

Version: 1.2, Update Date: 2009-3-12

 Gain Improper

Gain low Gain high

Version: 1.2, Update Date: 2009-3-12

 Flow cell clog and dirty

Version: 1.2, Update Date: 2009-3-12

 Troubleshooting

Version: 1.2, Update Date: 2009-3-12

 Troubleshooting

Adjust Gain
Adjust forward focus
Clean flow cell
Adjust Direct beam stop

Version: 1.2, Update Date: 2009-3-12

 Adjust Gain
1. First try to adjust Gain by software (Run particle)

Standard particle target value

Param Before After
version 1.4 version 1.4
FS 26 ± 3 18±2
SS 117± 5 104±6
SS 0.1Max ≤18 ≤15
Width
FS 0.1Max ≤13 ≤6.5
Width
7um particle

Version: 1.2, Update Date: 2009-3-12

 Adjust Gain

1. First try to adjust Gain by software (Run normal fresh

blood)

Version: 1.2, Update Date: 2009-3-12

 Adjust Forward Focus

Forward optical assembly

First loosen these two
screws

Flow cell

Then adjust this screw

Version: 1.2, Update Date: 2009-3-12

 Clean Flow Cell

After clean
and
adjustment

Version: 1.2, Update Date: 2009-3-12

 Clean Flow Cell

After clean
and
adjustment

Version: 1.2, Update Date: 2009-3-12

Version: 1.2, Update Date: 2009-3-12