Adjuster for Repeatable Targeting of Local Part of Cell at Stageof Microscope for Biochemical Analysis
Shigehiro HASHIMOTO, Yoshinori YAGUCHI, Yusuke TAKAHASHI, Haruka HINO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/andKana MIYASHITA, Naomi HACHIYA
Department of Pathophysiology,
Tokyo Medical University, Tokyo, 160-8402, Japan

ABSTRACT
Biochemical analysis with molecular biological technique atlocal area of the biological cell is much more informative thanthat at the entire area of the tissue. A devise of arms foradjusting a position of biological specimen has been designed
for repeatable sampling of a local target part of a biological cellat a stage of a microscope. The space for the device is limited
to the small area surrounded by a laser irradiator and by aneedle for collecting sample.
The main part of themanufactured device is made of aluminum, although the screw
is made of stainless steel. The device has been applied to theglass plate or to the culture dish on the stage of the microscope.
After the local part of the cell was cut and taken off for thesample segment, the specimen was re-adjusted for the next
sampling. The local part of the biological tissue on the glass
plate has repeatedly been targeted with the designed device
within 0.05 mm. The device is available for both glass plates
and culture dishes to be re-adjusted on the stage of themicroscope to be focused at the same targeted micro area in theadvanced laser micro dissection system.
Keywords: Biomedical Engineering,
Microscope, Laser and Local Target.

BiologicalCell,

1. INTRODUCTION
Protein relates to the biological function. Analysis of protein
is useful to trace the behavior of biological cells.
Biochemical analysis is useful to detect protein in the biologicaltissue. The conventional technique, however, is not enoughsensitive to detect local distribution of protein within a cell. In
the conventional technique, protein in the biological tissue isdetected after homogenization.
An aggregate of protein might induce neurological diseases.
The aggregation exists at a local part in a tissue. The
concentration of the protein decreases after homogenization of

the entire tissue, which asymmetrically includes the protein.
The low concentration makes difficult to detect protein in thebiochemical analysis. The concentration of protein can beaccelerated, if the sample is collected from the targeted micro
space, which densely includes the protein.
If the position of the micro dissection is traceable, thedistribution of protein can be identified. The identification oflocalization of protein may useful to investigate mechanism ofdiseases.
The advanced laser micro dissection system [1-3] has newly
been designed and applied to biochemical analysis forneurological disease. In the present study, an adjusting device
has been designed for repeatable sampling of a local target partof a biological cell at the stage of the microscope of theadvanced laser micro dissection system.

2. METHODS
Laser Micro Dissection System
The laser micro dissection system consists of a laser oscillator
for dissection, a microscope for observation, and an aspirator
for sample collection. In the system, the sample is cut at theperimeter of targeted micro segment with the laser beam under
microscopic observation, and specified micro dissection iscollected by the aspirator with the micro needle capillary. The
dimension of the laser beam focused on the specimen is 0.005
mm.
The space for the adjusting device is limited to the small areasurrounded by the laser irradiator and by the needle for the
biological micro sample to be collected (Fig. 1).

BA

Adjusting Position of Glass Slide
To adjust the position of a glass slide, one of the following
methods is alternatively applied: the method with two arms, orthe method with three arms.
The method with two arms is as follows. When one of therims of the glass slide is facing along the rim of the thin forearmof one of the arms, the neighbor rim of the glass slide istouching to the tip of the thin forearm of the other arm.

CThe method with three arms is as follows. When one of therims of the glass slide is simultaneously touching to the tips ofthe thin forearm of two arms, the neighbor rim of the glass slide
is touching the tip of the thin forearm of the third arm (Fig. 3).
These thin forearms are useful to save spaces for approaching to
the thin glass slide.
Fig. 1: Adjusting device (A) surrounded by the laser irradiator(B) and by the collector needle (C) on the stage of themicroscope.

Adjusting Position of Culture DishTwo arms are applied to the culture dishes, which has a notchon the outer cylindrical wall. When the notch is touching to
the tip of the short forearm of one of the arm, the other point of
the outer cylindrical wall is touching to the tip of the shortforearm of the other arm. These two points of contact should
not be on the line of the diameter of the dish (Fig. 4). The
short arms have enough height to fit to the notch.

BA

Fig. 2: Device for adjustment. Numbers are millimeter.

Device for Adjustment
The adjusting device consists of a stage and several arms (Fig.
2). The stage is made of aluminum and fixed to the original
stage of the microscope with screw of M4. The stage has arectangular center hole, where a glass plate or a culture dish isplaced.
The arms are made of aluminum. They are placed around thehole. These arms are fixed on the stage with screws of M3.
Both the internal and the external screws are made of stainlesssteel to get enough strength for clamping. The internal screw
is inserted in the stage of aluminum. Each arm has rectangularhole, through which the screw is inserted. The screw can slide
along the hole. The position of fixation of each arm is
adjusted with variation of position between the arm and thescrew. The position of these arms traces that of the glass slideor the culture dish.
Each arm consists of two rods: a forearm and an upper arm.
These two rods are connected with a screw of M2, with which
the angle between the two rods varies.

Fig. 3: Glass slide (A) adjusted with three forearms (B).

BAFig. 4: Dish (A) adjusted with two short forearms (B).

Trial for Adjusting Position
The glass slide with a freeze-dried biological specimen was set
with the designed device. Once the position of the arms is set
at the first trial, the position of the arms is maintained withtightening the screws of the device. A target area wasdissected with the laser beam. A droplet was supplied with themicro pipette to drift the dissected micro sample. The
dissected micro specimen was collected with the micro needle
capillary. The position of the glass slide was memorized withthe position of the arms. With the same position of the arms as
the first sampling, the position of the slide was successively
readjusted on the stage of the microscope.
By the device, the glass slide was tried to be adjusted to thesame position as that of dissection. The displacement between
the targeted point at the second trial and the dissected point at
the first trial was measured (Fig. 5).
The displacement was measured in several tests. Each test
consists of two successive trials of adjustment of the position.
The displacements were quantified as coordinates of twodimensions in x-y plane, when the position of the first trial is set
as the origin. The x-y plane is defined in parallel to the stageof the microscope. The direction of x axis is defined inparallel to one of the rim of the stage. The direction of y axis
is defined in perpendicular to that of x axis, and in parallel tothe stage.

3. RESULTSIn Figs. 5-7, the painted dye of red is irradiated by the laser fordemonstration.
Figs. 5 & 6 exemplify the microscopic viewing fields ofsuccessive adjusting of dish with the designed arms. The sametarget in the biological tissue is able to be easily traced in thefigures.

Fig. 6: Distance between the dissected point at the first trial(circle) and the targeted point at the third trial (square) of
adjusting of dish. The square shows the target point for laser
beam. The arrows show x and y coordinates, respectively.

Fig. 7 exemplifies the microscopic viewing fields of successive
adjusting of dish with the designed arms. The same dissectedarea (square) is able to be easily traced in these figures.
Data of displacement are summarized in Figs. 8 & 9. Data arescattered and smaller than 0.05 mm. Each datum shows thedistance between the cut area and the successively focused area.
The local part of the biological tissue on the glass plate has
repeatedly been targeted with the designed device within 0.01
mm (Fig. 8). The local part of the tissue on the dish, on theother hand, has been targeted within 0.05 mm (Fig. 9). The
dimension is smaller than that of cells.

4. DISCUSSION
Laser has widely been used in a lot of applications: to measure
micro movement [4], and to cut the biological tissue [5].
Scanning system is important to trace target in microscopic
studies [6]. In the trials of the glass slide, displacement of
0.01 mm is smaller than width of 0.06 mm in the observation
area of the microscope. In the trials of the culture dish,displacement of 0.05 mm is also smaller than width of 0.06 mm
in the observation area of the microscope. The displacements
are smaller than 0.1 mm of the viewing field for microscopic
observation.

Fig. 5: Distance between the dissected point at the first trial(circle) and the targeted point at the second trial (square) of
successive adjusting of dish. The square shows the target
point for laser beam. The arrows show x and y coordinates,
respectively.

Fig. 9: Distance between the dissected point at the first trial andthe targeted point at the successive second trial on the dish.
Displacements are shown with the combination of x and ycoordinates. Numbers show micrometer. The origin shows
the dissected point at first trial.

Fig. 7: Microscopic viewing fields of successive adjusting of
dish: the first trial (upper) and the second trial (lower).

Micro debris adhered on the rim of the glass slide might cause
the micro displacement of the readjusted position. The
position of the forearm is not stable, when the friction between
screws is low. Stainless steel is selected for the material of thescrew to strengthen the morphology of the surface of the screw
against strong tightening of the screw. The larger diameter ofthe screw is selected to get higher stability of the arm. Thestability of the arm is improved, when the diameter of the screwincreases.
Micro debris adhered on the outer wall of the culture dish mightalso cause the micro displacement of the readjusted position.
The fitting between the notch of the dish and the tip of theforearm affects the preciseness of the adjustment of the positionof the dish on the stage of the microscope.
While the micro dissection is collected, the operation produces
several forces. When the droplet of liquid is supplied on theglass slide to drift the dissected micro sample, vibration can beoccurred on the glass slide. The vibration generates horizontal
forces against the arms. When the position of the glass slide isreadjusted on the stage, the sliding action produces horizontal
forces against the arms. The position of the arms has
successfully been fixed with enough strength on the stage of the
microscope with the screws (M2, M3, M4) of stainless steel ofagainst these forces.
The micro technology for biochemical analysis contributes toevaluation of behavior of biological cells on engineered surface[7]. The dimension of 0.05 mm for readjustment is the sameorder of cells, and available for analysis of local part of tissue.

Fig. 8: Distance between the dissected point at the first trial andthe targeted point at the successive second trial on the glass
slide. Displacements are shown with the combination of x andy coordinates. Numbers show micrometer. The origin shows
the dissected point at first trial.

5. CONCLUSION
A devise of arms for adjusting a position of biological specimen
has been designed for repeatable sampling of a local target partof a biological cell at a stage of a microscope. With themanufactured devise, the position of the target point ofbiological sample has been adjusted in the area within 0.05 mm.
The devise is applicable to adjust the position on the stage ofthe microscope.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities from the Japanese
Ministry of Education, Culture, Sports and Technology. This
study was also supported by a Grant-in-Aid for Exploratory
Research in Japan (25461275 to NH, 26660176 to NH) and The
Ministry of Health Labour and Welfare (NH).

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