Optical System

International Service Department

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

Version: 1.1, Update Date: 2009-3-12

5 DIFF Principle

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

Blood Cells

Gra

Eos

RBC

Mon
PLT

Bas

Normal blood cell volume range:RBC 60-120fl

WBC
120-1000fl
Version: 1.1, Update
Date:
2009-3-12,PLT 2-30fl

Lym

WBC 5-part proportion

Five groups of leukocyte (stained)
Lymphocyte
Monocyte
%

25 ~ 30%
3 8

Neutrophil

50 ~70%

Basophil

0 1%

Eosinophil
3%

0.5

Version: 1.1, Update Date: 2009-3-12

Morphological

Version: 1.1, Update Date: 2009-3-12

Conclusion:

Version: 1.1, Update Date: 2009-3-12

How can we identify

the five groups?

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

Flow Mode of Cell Suspension

Normal flow

Version: 1.1, Update Date: 2009-3-12

Sheath flow

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: 1.1, Update Date: 2009-3-12

Multi-angle Laser Scatter

High Angle Scatter 8~20
reflects granularity and
complexity

1 Sample
2 Laser light
3 Lens

Version: 1.1, Update Date: 2009-3-12

Low Angle Scatter 1~5

reflects the cell volume

Laser Scatter: Mie Scatter

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

Version: 1.1, 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: 1.1, 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: 1.1, 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: 1.1, 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: 1.1, Update Date: 2009-3-12

Flow cytometry + Laser Scatter

Flow cytometry + Laser Scatter

Version: 1.1, Update Date: 2009-3-12

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

Morphological treatment

Version: 1.1, Update Date: 2009-3-12

4-DIFF Channel- 4 diff differential

Blood
LEO(I)

Lym

RBC

Gran

Mon

LEO(II)

Eos

Neu

Dilute ratio 1:135(348)

Version: 1.1, Update Date: 2009-3-12

DIFF channel
Morphological treatment
Basophile

Nucleus

Scatter
intensity

Scatter
intensity

Time

Version: 1.1, Update Date: 2009-3-12

Time

DIFF channel
Morphological treatment
STAIN

SIZING

Eos
Neu

... . .. .
. ..
.

Version: 1.1, Update Date: 2009-3-12

... . .. .
. ..
.

... . .. .
. ..
.

Mon
Lym

4-DIFF Channel-Scatter gram

Neu
Mon
Lymph

Eos

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: 1.1, Update Date: 2009-3-12

BASO/WBC channel-Impedance Method

Pulse graph
40fL
35fL
30fL
25fL
20fL

Dilunt
LH

Aperture

Dilute ratio 1:500(753)

Histogram
Version: 1.1, Update Date: 2009-3-12

Oscillograph

BASO/WBC channel-Baso differential

Blood
LH

RBC

Baso

Lym, Mon, Neu, Eos

Dilute ratio 1:500(753)

Version: 1.1, Update Date: 2009-3-12

BASO channel
Morphological treatment
SIZING
Eos
Neu
Bas
... . .. .
. ..
.

Version: 1.1, Update Date: 2009-3-12

.... ..
..

Mon
Lym

Baso/WBC Histogram

Version: 1.1, Update Date: 2009-3-12

Optical System Structure

Version: 1.1, Update Date: 2009-3-12

Optical System Structure

Note: MAS=SS
Version: 1.1, Update Date: 2009-3-12

LAS=FS

Optical System Components

SS Beam Stop
Flow Cell
Assembly

Back Lens Assembly

Forward
Version:
1.1, Optical
Update Assembly
Date: 2009-3-12

Splitter

Backward Optical System

FS PD Assembly

FS Beam Stop

Laser control board

SS PD Assembly

Forward Optical Assembly

Function: generate facula to irradiate the blood
cell.

Version: 1.1, Update Date: 2009-3-12

Forward Optical Assembly

Version: 1.1, Update Date: 2009-3-12

Laser Source
Semiconductor laser technology
small
Semiconductor more stable, low
quantity of heat
long life and low cost for maintenance

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

Sheath fluid bath

Version: 1.1, Update Date: 2009-3-12

Sheath fluid observation

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

Backward optical system optical path

Version: 1.1, Update Date: 2009-3-12

Back lens assembly

Block the direct light, and then collimate.

Version: 1.1, Update Date: 2009-3-12

Splitter assembly
Divide into two directions.

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

PD Assembly

Remove the stray light and convenient to focalize.

Version: 1.1, Update Date: 2009-3-12

Optical System Adjustment

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment
1. Adjust light source collimation and focusing

Pitch Pin

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment
2. Adjust flow cell bath (1)

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment
3. Adjust separate light prism and beam stop
Symmetric

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment

4. Adjust back light collimation

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment

5. Adjust flow cell bath (2)

Flow cell edge

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment

6. Adjust incidence light beam stop

Version: 1.1, Update Date: 2009-3-12

Optical system adjustment primary

adjustment

7. Adjust photoelectric diode

Low angle scatter

High angle scatter

Version: 1.1, Update Date: 2009-3-12

Adjust Focus Position

1. Adjust forward light focus position
Tools:

FLUKE124

Version: 1.1, Update Date: 2009-3-12

Adjust Focus Position

1. Adjust forward light focus position
Test points:
FS Pre-amplify board

Version: 1.1, 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: 1.1, Update Date: 2009-3-12

Adjust Focus Position

2. Adjust back light axis position

7um standard particle

Version: 1.1, Update Date: 2009-3-12

Sample

Adjust Focus Position

2. Adjust back light focus position
Test points:
SS Pre-amplify board

Signal Processing board

SS TP44---MASIN

Version: 1.1, Update Date: 2009-3-12

Adjust Focus Position

Let pulse of SS be biggest.

Oscilloscope settings AC Coupler , Voltage 100mv/div Time 500ns/div

Version: 1.1, Update Date: 2009-3-12

Scatter gram and histogram

Version: 1.1, Update Date: 2009-3-12

Normal Scatter grams and histogram

Version: 1.1, Update Date: 2009-3-12

Normal scatter gram of standard particle

Standard particle target value

7um particle

Version: 1.1, Update Date: 2009-3-12

Param

Before version
1.4

After version
1.4

FS

26 3

182

SS

117 5

1046

SS 0.1Max
Width

18

15

FS 0.1Max
Width

13

Abnormal scatter gram

Version: 1.1, Update Date: 2009-3-12

Abnormal samples: Flag

Left Shift
LIC

Mon

Neu
Eos

ALY

Lym ALY
NRbc
Ghost

Version: 1.1, Update Date: 2009-3-12

Abnormal scatter gram

Left shift

LIC

Version: 1.1, Update Date: 2009-3-12

Abnormal scatter gram

ALY

Version: 1.1, Update Date: 2009-3-12

Gain Improper

Gain low

Version: 1.1, Update Date: 2009-3-12

Gain high

Flow cell clog and dirty

Version: 1.1, Update Date: 2009-3-12

Troubleshooting

Version: 1.1, Update Date: 2009-3-12

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

Version: 1.1, Update Date: 2009-3-12

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

7um particle
Version: 1.1, Update Date: 2009-3-12

Param

Before
version 1.4

After
version 1.4

FS

26 3

182

SS

117 5

1046

SS 0.1Max
Width

18

15

FS 0.1Max
Width

13

6.5

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

Version: 1.1, Update Date: 2009-3-12

Adjust Forward Focus

Forward optical assembly
First loosen these two
screws

Flow cell

Then adjust this screw

Version: 1.1, Update Date: 2009-3-12

Clean Flow Cell

After clean
and
adjustment

Version: 1.1, Update Date: 2009-3-12

Clean Flow Cell

After clean
and
adjustment

Version: 1.1, Update Date: 2009-3-12

Version: 1.1, Update Date: 2009-3-12