Episcopic-Fluorescence AttachmentEF-UPR-III for BA410E Instruction Manual

If the equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.Note

1

This instruction Manual has been prepared for users of the EF-UPR-llI Epi-Fluorescence

Attachment used in conjunction with the Motic BA410E Upright Microscope.

For your own safety, read this manual carefully and throughly before using the product. Do

not discard this manual. Always keep it near the product for easy reference.

We are constantly endeavouring to improve our instruments and to adapt them to the requirements of

modern research techniques and testing methods. This involves modification to the mechanical

structure and optical design of our instruments.

Therefore, all descriptions and illustrations in this instruction manual, including all specifications are

subject to change without notice.

Fluorescence

The optical phenomenon that occurs when light absorbed by a material, creates a molecular excitation

that causes the material to re-emit light at a different wavelength.

Fluorescence Microscope for Epi-Fluorescence

The technique of fluorescence microscopy with Epi-illuminators is based on the adaptation of the

vertical illuminator used in reflected light microscopy. The radiation emitted by the light source first

passes through the exciting filter and is incident to a short-pass filter* (dichroic beam splitter). The

radiation is then reflected into the objective, which also serves as a condenser. The objective

concentrates the exciting radiation in the object field. Fluorescent light emitted from there is collected by

the objective and returned in the opposite direction to the short-pass filter, which directs it into the

eyepiece through the barrier filter.

* A filter designed to allow the passage of radiation of wavelengths shorter than a given limit.

** This manual may not be reproduced or transmitted in whole or in part without Motic’s express

permission.

2

NOTES FOR SAFETY

To avoid being electrocuted, do not touch the lamp socket while the light is on as a high

voltage current is being supplied to the bulb.

Because the bulb produces a large amount of heat, do not touch the housing, socket or bulb

when on.

This instruction manual needs to be consulted in all cases where this symbol is used, in order

to find out the nature of the potential HAZARD and any actions which have to be taken.

This product has been tested to the requirements of CAN/CSA-C22.2 No. 61010-1, second

edition, including Amendment 1, or a later version of the same standard incorporating the

same level of testing requirements.

Protective grounding is essential for the use of this product; Ensure the user insert it with

good protective earthing socket.

When you place the device, ensure the plug or appliance coupler easy to dial out when the

state of emergency.

The equipment should be used on the desk; it cannot be used on the ground.

The MXH-100 starter should be connected with lamp house first, then plug in the cord to an

AC outlet with ground conductor.

To ensure safety, only use lamp house supplied by Motic.

3

WARNING

• If the mercury lamp burst, the broken glass may be scattered and hurt human beings and/or the

instruments. The gas inside the lamp will be released into the air bringing about the danger of

inhaling the mercury vapor. So all the personnel must immediately leave the room so as not to

inhale the mercury vapor. Further, thoroughly ventilate the room for at least 30 minutes.

• When the lamp is lighting, it emit ultraviolet rays harmful to human eyes and skin. Do not looking

directly at the ultraviolet rays. Please keep the lamphouse attached to the microscope.

To avoid being electrocuted, do not touch the lamp socket while the light is on as a high

voltage current is being supplied to the bulb.

Because the bulb produces a large amount of heat, do not touch the housing, socket or bulb

when on.

Make sure that the switch of voltage range is set to matches your available line voltage.

Disconnect the power supply before replacement of fuse.

To avoid the risk, make sure the MXH-100 starter is used with Motic lamp house.

4

TABLE OF CONTENTS

1. NAMES OF COMPONENT PARTS 7

1.1 BA410E 7

1.2 Epi-fluorescence Attachment Main Body 8

1.3 MXH-100 Power supply unit 9

2. SETTING UP THE INSTRUMENT 10

3. ASSEMBLY 10

3.1 Microscope assembly 10

3.2 Installing the Epi-fluorescence attachment 10

3.3 Epi-fluorescence attachment 11

3.4 Installing the shutter slider with the filter holder 12

3.5 Light cut-off slider 12

3.6 Installing the Filter cubes 12

3.7 Filter cube ID tags 14

3.8 Arrangement of the excitation method ID tags 15

3.9 Attaching UV light protective shield 15

3.10 Mounting UV light blocking tube 16

3.11 Installing the light source 16

3.11.1 Attaching the collector lens to the lamphouse 16

3.11.2 Installing the collector lens in the lamp house 17

3.11.3 Attaching the lamp house to the microscope 18

3.11.4 Installing the lamp 18

3.12 Connecting the lamp house to the power supply 20

3.13 Check the input voltage 20

3.14 Turning On and Off the Lamp 21

3.15 The LCD Display 21

3.15.1 Working mode 22

3.15.2 PIN CODE Protection 22

3.15.3 Light types 23

3.15.4 LCD 23

3.15.5 Brightness adjustment 24

3.15.6 Setup routine Menu (RST operation) 25

5

3.16 Aligning the lamp 27

3.16.1 Names of Components - Lamp housing 28

3.16.2 Procedure of Alignment the Mercury Arc Lamp and Mirror Image 29

3.16.3 Centering the Mirror Arc Image 30

3.17 Changing the size of the field diaphragm 34

3.18 Epi-fluorescence Attachment Main Body 34

3.19 Centering the field diaphragm 35

3.20 Adjusting the shutter slider 35

3.21 Neutral Density Filters 35

3.22 Heat absorbing filter 36

3.23 Filter Blocks 36

4. MICROSCOPY 37

4.1 Selecting fluorescence filters: 37

4.2 Selecting excitation filters (EX) 37

4.3 Selecting barrier filters (BA) 38

4.3.1 Longpass 38

4.3.2 Band pass 38

4.4 Using an oil immersion objective 40

4.5 Fluorescence Photomicrography 41

4.6 Video Fluorescence Microscopy 42

5. TROUBLESHOOTING TABLE 43

5.1 Electrical 43

5.2 Optical 43

6. CARE AND MAINTENANCE 45

6.1 Lenses and filters 45

6.2 Cleaning or painted or plastic components 45

6.3 When not in use 45

6.4 For MXH-100 Power supply unit 46

6

STANDARD FLUORESCENCE FILTER SETS 47

NOMENCLATURE 49

DEFINITIONS OF FILTER TERMINOLOGY 49

SPECIFICATIONS (MXH-100) 54

7

1. NAMES OF COMPONENT PARTS

1.1 BA410E

Fig.1

8

1.2 Epi-fluorescence Attachment Main Body

Fig.2

9

1.3 MXH-100 Power supply unit

Fig.3

10

2. SETTING UP THE INSTRUMENT

• Avoid placing the instrument in areas exposed to dust, vibration, high temperature and high

humidity.

• Avoid locations exposed to direct sunlight, under room lights.

• Select location which allows easy access to power cord from products AC inlet in the event of an

emergency.

• Do not place power supply on a table mat.

3. ASSEMBLY

3.1 Microscope assembly

Follow the instructions provided in the microscope manual.

For the purpose of performing simultaneous observation with phase contrast, attach the appropriate

phase contrast components referring to the specific instructions provided.

3.2 Installing the Epi-fluorescence attachment

Please consult the illustrations provided while assembling the attachment

Required tools: Allen hex keys: 2.5mm and 5mm (supplied with attachment).

Before Starting: Turn off the microscope power switch and unplug the power cord

11

3.3 Epi-fluorescence attachment

1. Position Epi-fluorescence attachment parallel to the microscope arm (Fig.4a) and secure with

5.0mm hexagonal clamp screws. (Fig.4b)

2. Mount eyepiece tube on the Epi-fluorescence attachment and secure with 2.5mm hexagonal clamp

screws. (Fig.4c)

3. Use the spot stickers to cover the two countersink holes.

Fig.4a Fig.4b

Fig.4c

12

3.4 Installing the shutter slider with the filter holder

Loosen one of the slider knobs.

With the filter slider slot to the left and facing the operator, insert the slider into the opening of the

main body of the fluorescence attachment.

Tighten the loosened knob.

Fig.5

3.5 Light cut-off slider

To prevent the specimen from fading during temporary pauses in observation, slide the shutter to

the left.

When using transmitted light illumination, slide the shutter to the left.

3.6 Installing the Filter cubes

Caution

Always keep four blocks on the slider. Do not leave any of the filter slots empty as strong light

may enter and damage the user’s eyes when shifting between excitation methods.

If fewer than four filter cubes are required for microscopy. Use a “DIA-ILL” dummy cube to ensure

that the open space in the slider is filled.

If the filter slider is not filled with four filter cubes, filter cubes may shift sideways by the motion of

excitation method changeover movement, UV rays will reach the eyes through an empty space,

causing serious harm.

Never perform lamp centering with the UV filter cube in the optical path as harmful UV radiation

from the lamp may enter the eyes, possibly resulting in loss of vision.

13

Remove the filter cube cover. (See Fig.6a)

Unscrew the right filter cube cover knurled retaining screw. (Fig.6a)

Slide the excitation method changeover knob to position “4”. (Fig.6b)

Fig.6a Fig.6b

Slacken the filter cube clamp plate hexagonal clamp screw at the end of the filter slider.

(Fig.7a)

While holding the excitation method changeover knob (Fig.7c), slide the filter cube into the

dovetail mount of the filter slider. (Fig.7b)

Position the remaining filter cubes using the same procedure so that there are four filter cubes on

the slider.

• If fewer than four filter cubes are required for microscopy, use a “DIA-ILL” dummy cube to

ensure that the open space in the slider is filled.

Remember the position of the filter cubes for the installation position of the excitation ID tags.

If the filter cubes are to be switched frequently, locate those filter cubes at both ends of the filter slider.

Return the filter cube clamp plate to the horizontal position and tighten the hexagonal clamp plate

clamp screw. (Fig.7d)

Replace the filter cube cover and secure with the knurled retaining screw.

14

Fig.7a Fig.7b

Fig.7c Fig.7d

3.7 Filter cube ID tags (Fig.8)

Insert the Filter cube ID tag into the pocket on the front face of the epi-fluorescence attachment.

Select the ID tags showing the excitation method cubes on the slider and drop them into

corresponding pockets.

Fig.8

15

3.8 Arrangement of the excitation method ID tags (Fig.9)

Fig.9

Insert ID tag in reverse order into ID tag pocket

Positions “1”, “2”, “3”, “4” show the location of the Filter cube on the filter slider

3.9 Attaching UV light protective shield

Loosen UV light protective shield Clamp screw under Epi-fluorescence attachment body.

(Fig.10a)

Insert the UV light protective Shield – slots in from the front. (Fig.10b)

Secure in place with the clamp screw. (Fig.10c)

Fig.10a Fig.10b

16

Fig.10c

3.10 Mounting UV light blocking tube

Mount UV light blocking tube on the microscope condenser carrier and secure in place with

condenser clamp screw. (Fig.11)

Fig.11

3.11 Installing the light source

3.11.1 Attaching the collector lens to the lamphouse

Remove the rubber cap that covers the collector lens focus knob.

Loosen the set screw at the base of the focusing knob with a 2.5mm Allen hex key.

While pulling out the collector lens focus knob, insert the collector lens into the lamp house in the

direction indicated by insertion arrow.

Always attach a collector lens to the lamphouse

This type of collector lens does not have the ability to cut ultraviolet rays; the filter cube

used during fluorescence microscopy does include a method for cutting ultraviolet rays.

17

3.11.2 Installing the collector lens in the lamp house (Fig.12)

Aligning the pin on the collector lens, with the collector lens positioning groove of the lamp house.

Fig.12

Return the focusing knob to the original position with focus knob pin in focusing groove of the

collector lens.

Tighten the set screw at the base of the focusing knob with a 2.5mm Allen hex key.

Tighten the cheese-head slotted screw, following lamp alignment and focusing.

Affix the rubber cap covering collector lens focus knob.

18

3.11.3 Attaching the lamp house to the microscope

Attach the lamp house to the microscope using the dovetail on the Epi-fluorescence attachment.

Align the positioning pin on the lamp house flange with the positioning groove on the dovetail mount of

the fluorescence attachment (See Fig.17a)

Secure the dovetail with the provided Allen Key as shown in Fig.17b.

Fig.13a Fig.13b

3.11.4 Installing the lamp

In order to prevent electric shock always turn the power switch off, and unplug the power cord

before installing or replacing the lamp.

Loosen the lamp house cover clamp screw (Fig.14a) and remove the cover and the lamp socket

assembly. (Fig.14b)

Loosen the lamp clamp screws at both ends of the lamp socket and remove the installed “dummy

lamp”. (Fig.14c)

Before handling the lamp, read the handbook supplied with the lamp.

When installing the lamp, avoid applying force that might cause the lamp to break.

Match the large and small diameter metal ends on lamp and lamp socket. (Fig.14d)

Insert the lamp into the lower flexible lamp clamp base and tighten the clamp screw. (Fig.14e)

Insert the lamp into the upper lamp socket hole supported by the metal plate with the cooling fins

and fasten the clamp screw. (Fig.14e)

Insert the bottom tab of lamp house cover over the inner metal surround of the lamp house and

pivot the lamp socket cover to close (Fig.14f). Secure with the clamp screw. (Fig.14g)

The safety cut-off micro switch will “click” as you secure the lamp house cover clamp screw.

19

Fig.14a Fig.14b

Fig.14c Fig.14d

Fig.14e Fig.14f

20

Fig.14g

3.12 Connecting the lamp house to the power supply

Turn OFF the power switch on the power supply.

Mercury Lamp Power Supply

Fig.15

3.13 Check the input voltage

Confirm that the input voltage marked on the rear panel of the power supply matches your available

line voltage.

Use a power cord that is rated for the voltage used in your area and that has been approved to

meet local safety standards. Using the wrong power cord could cause fire or equipment

damage.

21

When using the extension cord, only use a power supply cord with a protective earth (PE) wire.

Plug the lamp input connector into the output connector on the rear panel of the power supply and

secure by tightening the locking ring.

Plug in the power cord supplied with the power supply into the AC receptacle on the rear panel of

the power supply.

3.14 Turning On and Off the Lamp

A. Before turning on the power switch, the voltage range is checked if it is suitable for the local power

supply voltage. The Input voltage range of MXH-100 is 100-240VAC, 50-60Hz.

B. When the power switch on the power supply switches to “ I ”, then the power is turn on, the

mercury lamp will be lighted up within a few seconds.

When the power switch switches to “O”, the power is turn off.

C. Tuning Intensity of Lamp

On the panel there are 2 buttons called DEC and INC, DEC button can be reduced the intensity of

lamp, the INC button can be added the Intensity.

Note:

1. If the light does not light up, the POWER Switch can be pressed more times until it lights up. If the

POWER Switch is pressed more than 5 times and the light does still not work, you can check the

lifetime of the lamp (200 hours) whether it is within the period of validity or check the housing and

socket are properly connected.

2. When the light will be turned on again, it must be cool down before lighting up again.

3.15 The LCD Display

The LCD display can be indicated the Lifetime of lamp and intensity (10-100) of the lamp. The counter

is able to display from 000: 00 to 999:59 (in hours and minutes on each side of the colon

respectively). When a new lamp has been installed, click the RST button within 3 seconds to reset

the counter, when the LCD will be showed “NEW LAMP OR NOT? Please press RST key to confirm”.

User can have 10 seconds to confirm. When the RST button is pressed, the LCD will show the lamp

type can be chosen as follows: USH-102D—RST, HBO 103W/2—DEC, user can press the buttons of

RST, INC and DEC on the panel to choose the lamp type installed in 10 seconds, if user doesn’t press

any buttons, the default choice will be USH-102D.

22

3.15.1 Working mode

MXH-100 has three working modes: Auto Intensity/ LifeTimeExt/ Manual mode. MXH-100’s working

mode settings will be saved and valid until system reset.

**System settings can be protected by using the “pin code” function to avoid unauthorized resetting of

the system**

A. Auto Intensity mode

• Every Mercury lamp bulb has a decay over its lifetime. Brightness can decay about 20% over the

specified lifetime hours.

MXH-100 can be used in Auto Intensity mode to compensate this decay by Software and keep the

brightness at a constant level over the whole lifetime cycle.

• If auto Intensity mode is active, user still can adjust brightness manually through the INC and DEC

buttons to override the value.

The brightness will be automatically reset back to the Auto Intensity mode, one hour after the last

manual override occurred.

The timeframe until automatic reset will be indicated on the LCD Display as a time count down.

• Auto Intensity mode is MXH-100 default working mode, the unit can be setup to different lamps and

working modes through the Setup function.

B. LifeTimeExtmode

• Under LifeTimeExt mode, MXH-100 is set to work at 80% brightness level, under this mode the

lamp’s life can be extended significantly.

• If LifeTimeExt mode is active, user still can adjust brightness manually using the INC and DEC

buttons to override the automatic setting. The brightness will be automatically reset back to the Auto

Intensity mode, one hour after the last manual override occurred.

The timeframe until automatic reset will be indicated on the LCD Display as a time count down.

C. Manual mode

• Under Manual mode，MXH-100 is working according to user intensity setting.

Intensity settings can be chosen between 80 and 100% brightness.

• User can adjust brightness through INC and DEC any time and the setting will be stored in the

system and persist until next change.

3.15.2 PIN CODE Protection

A. Some user may wish to protect the MXH-100 settings from being altered by unauthorized users.

Therefore MXH-100 System is equipped with PIN CODE protection functionality. If the PIN CODE is

set, all of operations must input the correct PIN CODE for next step. Of course PIN CODE

23

protection is a optional feature and can be ignored or deactivated at any time (see PIN CODE

setting operation).

B. PIN CODE is a 6 digital data，user can input PIN CODE through three buttons on the panel, RST is

regarded as ‘R’, INC is regarded as ‘I’, DEC is regarded ‘D’.

C. The input interface of PIN CODE on the LCD is as below. It is required user must finish the input of

6 digits PIN CODE in 15sec at count down. After 15sec the display will be came back to working

interface.

D. If the PIN CODE is forgotten, please contact Motic after sales department to get help.

3.15.3 Light types

MXH-100support three types of light：

● USH-102D, normally work life is 200 hours.

● HBO 50W/3, normally work life is 200hours.

● HBO 103W/2, normally work life is 300hours.

3.15.4 LCD

LCD shows MXH-100’s work status and related information and guides the user with interactive

behavior to provide easy access to its features.

A. Start display

When the power supply turns on, the display is as below. user can see initialization rate of progress,

MXH-100implementsinitial operation about 5sec, after initialization is finished unit will enter into

normal work display.

24

B. Working display

Working display is as below (contents)：

1st line: shows current light type；

2nd line: shows “light theoretic work life” and “real cumulate work time”, both of switch to show

about 3sec per once;

3rd line: shows brightness percent value, to adjust brightness under Auto Intensity mode or

LifeTimeExt mode, unit will switch to show 1 hours count down time about 3sec per once.

4th line: show current working mode.

3.15.5 Brightness adjustment

Brightness illumination is divided to 100 levels，0% is the darkest, 100% is the brightest. The

increment is +/- 1%, when the INC/DEC button is clicked. User can enter into brightness adjustment

display through INC and DEC (see below brightness adjustment display), If unit is under PIN CODE,

user need to input correct PIN CODE. INC key is to increase brightness, DEC key is to decrease

brightness.

Brightness adjustment changes 1% per once, when user press INC or DEC key continually, user

can see brightness display rate of progress and brightness value changing.

There is 9sec count down on brightness adjustment display, if INC or DEC key is not pressed in

time, unit will come back to work display, contrarily count down time is refresh in 5sec, after then

count down time will come back to work display.

25

3.15.6 Setup routine Menu (RST operation)

To enter the MXH-100 Setup functionality from actual working mode,

please press the “RST” button. If the PIN CODE protection is set, user need to input correct PIN CODE

within 15seconds.

MXH-100 Setup functionality can display three operations:

1. Timer and light setting operation

2. PIN CODE protection setting

3. Working mode selection

To navigate through the guided setup routine please use the INC or DEC keys to go up and down, and

RST as a confirmation button.

A. Timer clear and light setting operation (Reset)

After entering the reset option in MXH-100 Setup routine, display shows as below: there is two

options，when RST key is pressed to option “Yes”, then the timer will be reset, in the same time

unit enters into light setting confirmation display. When RST key is pressed to option “No”, then the

timer will be not reset, in the same time unit enters into light setting confirmation display.

The time for Timer clear display is 10sec, if the key is not pressed in time, unit will enter into light

setting confirmation display.

26

Light setting confirmation display is as below. When RST key is pressed, and choose “Yes”, unit

will enter into light option display; When INC key is clicked and choose “No” then the unit will come

back to work display. The operation time for light setting confirmation display is 10sec, if the key is

not pressed in time, unit will come back to work display.

Light option display is as below, there are three light type options：

RST key to select light type is USH-102D；

INC key to select light type is HBO 50W/3；

DEC key to select light type is HBO 103W/2；

After finishing light option operation, the timer will be reset. When work mode sets as Auto Intensity

mode, initial brightness is 80%. The operation time for light option operation is 10sec, if the key is

not pressed in time, unit will come back to work display.

B. PIN CODE operation

On setting display to click INC key unit will enter into PIN CODE operation display, display shows as

below. There are two option on PIN CODE operation display: when RST key is pressed, then unit

enter into PIN CODE setting display. To click INC key, PIN CODE protect function will be canceled

(not need to input PIN CODE when operate). Operation time is 10sec, if the key is not pressed in

time, unit will come back to work display.

27

PIN CODE setting display is as below, user can click RST key, INC key, DEC key to setup 6 digits

PIN CODE. It needs to input twice for confirmation, if the setting is succeed, PIN CODE protect

function take effect, the operation time for PIN CODE is 15sec. if the key is not pressed in time, unit

will come back to work display.

Note：if the PIN CODE is forgotten, please contact Motic after service department.

C. Work mode option (Working Mode)

Work mode option display is as below, there are three work mode option：

RST key to select Auto Intensity mode；

INC key to select LifeTimeExt mode；

DEC key to select Manual mode.

The operation time for the Mode option display is 10sec, if the key is not pressed in time, unit will

come back to work display.

3.16 Aligning the lamp

DO NOT perform the lamp centering procedure with the UV filter cube in the optical path as harmful UV

radiation from the lamp may enter the eyes, possibly resulting in loss of vision.

Please note: A UV excitation filter cube cannot be used in this instance since an arc image will not

appear in the window of the centering tool.

28

The mercury lamp consists of two electrodes sealed in a glass bulb under high pressure, which

contains mercury. When the power supply is turned on, a high voltage pulse is sent to the electrodes,

which in turn ionize the gas in the bulb, igniting the lamp. These ions, under low voltage, carry the

current that generates the light between the electrodes. The lamp gets very hot during the vaporization

of the mercury; creating high pressure inside the glass bulb. Avoid applying mechanical force that might

cause the lamp to explode.

The average lifetime of a mercury arc lamp varies between 200 and 400 hours, depending upon design

specifications, burns and the switch cycle.

Avoid touching the lamp with bare fingers as the oils from the fingers may etch the glass surface

resulting in light loss.

When the lamp has been allowed to stabilize it gives off high intensity light concentrated at certain

discrete wavelengths (e.g. 365, 400, 440, 546 and 580nm) making it an ideal source of illumination for

fluorescence microscopy.

3.16.1 Names of Components - Lamp housing

Fig.16a

Lamp Housing

29

Fig.16b

Centering tool

3.16.2 Procedure of Alignment the Mercury Arc Lamp and Mirror Image

Slide the shutter to the left to block the optical path of the fluorescence excitation.

Attach the centering tool to the nosepiece.

Turn the nosepiece to bring the centering tool in the optical path.

Insert any fluorescence filter cube (except UV/DAPI cube) into the optical path.

Set the fluorescence (incident light) field aperture diaphragm to open position.

Slide the shutter to the right to open the optical path.

If the projected image of the arc is too bright, insert ND filter into the light path.

Loosen the hexagonal collector lens focus knob resistance control screw at base of the

collector lens focus knob.

Turn the collector lens focusing knob on the lamp housing to bring the lamp arc image into sharp

focus in the widow of the lamp centering tool. (Fig.17)

Fig.17

30

Turn the lamp vertical (Fig.18a) and horizontal (Fig.18b) centering knobs until the direct arc image

is at the centre of the centering tool window. (Fig.18c)

Fig.18a Fig.18b

Fig.18c

3.16.3 Centering the Mirror Arc Image

The Motic lamp housing mirror system is designed to direct more intense illumination into the objective

rear aperture. Microscopes equipped with this type of lamp housing will produce two arc images.

Use the mirror centering and lamp centering knobs to position the direct arc (lamp) image and its mirror

image (which is usually less intense) side-by-side. Use the mirror focus knob (No. 5 in Fig. 21) to adjust

the intensity of both until they are approximately equal.

Turn the lamp horizontal centering knob (Fig.19a) to move the real arc image slightly off-centre; the

mirror arc image is not yet focused. (Fig.19c)

31

Turn the back mirror focusing knob (Fig.19b) on the lamp housing to focus the reflected arc image

on the centering tool window. (Fig.19c)

Fig.19a Fig.19b

Fig.19c

32

Turn the back mirror centering knobs (vertical or horizontal) (See Fig.20a) so that the direct arc

image and the reflected image are symmetrically positioned. (See Fig.20b)

(Back mirror centering knob – vertical) (Back mirror centering knob – horizontal)

Fig.20a

Fig.20b

33

Turn the horizontal lamp centering knob (Fig.21a) to bring the arc image to the centre of the

centering window so that the real arc image touch the reflected arc image. (Fig.21b)

Fig.21a Fig.21b

Gradually defocus the collector lens with the focus knob. Enlarge the arc image until it fills the

entire window of the lamp centering tool with a uniform field of illumination. (Fig.21c)

Fig.21c

34

3.17 Changing the size of the field diaphragm

The field aperture diaphragm determines the illuminated area on the specimen.

Operating the field aperture knurled ring changes the size of the field aperture diaphragm. For

normal observation, the diaphragm is set just slightly outside the edge of the field of view.

If a larger than required area is illuminated, extraneous light will enter the field of view. This will

create a flare in the image and lower the contrast.

Cutting out the excessive light is useful in preventing contrast from being diminished.

Decreasing excessive illumination is useful in protecting the specimen from fading.

3.18 Epi-fluorescence Attachment Main Body (Fig.22)

Fig.22

35

3.19 Centering the field diaphragm (Fig.23)

Move the field aperture diaphragm knurled ring to stop down the diaphragm.

If the field aperture diaphragm is off the centre of the field of view, centre the field aperture

diaphragm with centering screws.

Open the field aperture diaphragm just slightly outside the edge of the field of view.

Fig.23

3.20 Adjusting the shutter slider

Whenever observation is interrupted, slide the shutter into the light path by moving the slider to the

extreme left in order to prevent fading of the specimen.

The shutter is also used to cut off the reflected light illumination when observing the specimen by

transmitted light illumination (e.g. as in Phase contrast microscopy).

3.21 Neutral Density Filters

Neutral density filters equally reduce the intensity of all wavelengths of light. These filters are used for

reducing the intensity of the exiting light to prevent photo-bleaching of the specimen.

Relationship between the ND filters and the brightness

Filter

ND2 (T=50%)

ND4 (T=25%)

ND16 (T=6%)

36

3.22 Heat absorbing filter

The heat-absorbing filter is placed in front of the light source to reduce heat transfer to the excitation

filter, and damage to the interference filter coatings.

When utilizing the infrared excitation method remove the heat-absorbing filter, as it will not transmit the

near infrared and infrared light.

3.23 Filter Blocks

A cube shaped modules that hold a matched set of fluorescence filters including excitation (EX),

barrier-emission (BA) and a dichroic mirror (DM).

The excitation filter allows transmission of light at a wavelength within the absorption spectrum of the

dye and rejects light at wavelengths within the emission spectrum of the dye, which could be reflected

by the specimen and incorrectly detected as emission energy.

A barrier filter allows the transmission of light at wavelengths within the emission spectrum of the dye

and rejects light at wavelengths within the absorption spectrum of the dye, which could be detected as

emission energy.

A dichronic mirror is placed at a 45º angle relative to the incoming excitation light; the dichroic mirror

performs two functions: it reflects the shorter wavelength exciting radiation light to the specimen and

transmits the longer wavelength fluorescence.

37

4. MICROSCOPY

Epi-fluorescence Micoscopy

4.1 Selecting fluorescence filters:

For best results, select excitation and emission filters with centre wavelengths as close to the

absorption and emission peaks as possible.

Note: The centre wavelength is situated at the midpoint of the bandwidth. It is not necessarily the peak

transmission wavelength although with a symmetrical band the centre wavelength and the peak

wavelength are equal.

4.2 Selecting excitation filters (EX)

Excitation filters selectively pass the light within the range of wavelengths needed to cause the

specimen to fluoresce and filter out other light.

To maximize the brightness of the desired fluorescence (the signal) relative to brightness of the

background (the noise), one can choose excitation and emission filters with wide bandwidths.

However this may result in unacceptable overlap of the emission signal with the excitation signal,

resulting in poor resolution and wide bandwidth which also leads to a high level of selffluorescence and

severe fading.

To minimize spectral overlap, one can instead choose excitation and emission filters that are narrow in

bandwidth and are spectrally well separated to increase signal isolation. This will conversely yield a

dark image. Since little excitation light reaches the specimen, selffluorescence and fading are minimal.

38

4.3 Selecting barrier filters (BA)

The barrier filter prevents the excitation light from reaching the observers eye. Its transmission should

be as low as possible in the range of light used for excitation and as high as possible within the spectral

range of the emission from the specimen.

Barrier filters may be Longpass (LP) or Bandpass (BP).

4.3.1 Longpass

Longpass filters only allow wavelengths above a certain

Wavelength to pass through and will prevent light of lower

wavelength from passing through.

Longpass filters should be used when the application requires

maximum emission and spectral differentiation is not necessary.

This is generally the case for fluorophores that generate a single

emitting species in specimens with reasonably low levels of

background auto fluorescence.

Fluororophore or fluorescent probe:

Fluorochromes when conjugated to other originally active substances, such as a protein, antibody, or

nucleic acid, in order to selectively stain targeted substances within the specimen

Longpass filters are also useful for observing all the stains on multi-stained specimen such as FITC and

TRITC .

4.3.2 Band pass

Filters that pass light of a certain restricted range of wavelength.

Bandpass filters are useful for observing a certain stain on multi-stained specimen such as when two

stains FITC and TRITC are used.

Bandpass filters are designed to maximize the brightness of the desired fluorescence (signal) to

brightness of the background (the noise) ratio for applications where discrimination of signal component

is more important than overall image brightness.

39

Selecting fluorescence filters requires a thorough understanding of filter technology. This will enable the

user to utilize stain and illumination selection to improve the image quality of the desired fluorescence

signal.

Selection of filter combinations also requires knowledge of the excitation and emission spectra of the

stain.

Epi-fluorescence and Phase Contrast Microscopy

In addition to epi-fluorescence microscopy, phase contrast microscopy can be used to search an

organelle or the location of a particular molecule in a cell instead of epi-fluorescence microscopy which

bleaches the specimen.

Combing epi-fluorescence microscopy with phase contrast microscopy makes it possible to

compensate for the limitations of each method.

40

4.4 Using an oil immersion objective

Oil immersion objectives are labelled with the additional engraving “Oil” and are to be immersed in oil

between the specimen and the front of the objective.

The immersion oil supplied by Motic is synthetic, non-fluorescing and non-resining oil, with a refractive

index of 1.515

Normally, a cover glass must be used with oil immersion objectives with a few exceptions.

Deviations from thickness are not important as a layer of immersion oil acts as compensation above the

cover glass.

The small bottle of oil supplied with every immersion objective facilitates application of the oil to the

cover slip.

Remove any air bubbles in the nozzle of the oil container before use.

Immersion oil must be used sparingly.

Freedom from air bubbles must be ensured. To check for air bubbles, remove one eyepiece, open the

field diaphragm as far as possible and look at the exit pupil of the objective within the eyepiece tube

(the exit pupil will appear as a bright circle).

If it is difficult to see if there are any bubbles, use a phase centering telescope and rotate the eyepiece

part of the centering telescope to focus on the exit pupil of the objective. Air bubbles in the oil will

deteriorate the specimen image. To purge bubbles, swing the immersed objective forward and

backwards by rotating the revolving nosepiece, add more oil, or wipe off the oil and apply new oil.

Place a single drop of immersion oil on the cover glass.

Make contact with the cover glass, and focus.

View and wipe clean the objective with a lens cleaning tissue.

Any residual film of oil remaining on immersion type objective, or where it has spread to the surface of

dry type objective will have a discernible, negative effect on the image.

41

To remove a film of oil, moisten a lens tissue or clean cloth with petroleum benzine and lightly wipe the

lens surface, wipe the lens surface with absolute alcohol (ethyl alcohol or methyl alcohol).

Petroleum benzine and absolute are highly inflammable. Use great care when handling them.

4.5 Fluorescence Photomicrography

For the basic procedure and key points of photomicrography, see the manuals provided with the

photomicrographic equipment

Since the specimen colour may fade effort must be made on minimizing the exposure of the specimen

to irradiation both before, and during exposure.

Select the area of interest without using fluorescence, use phase contrast or interference contrast.

Select an appropriate filter combination for the specimen (whether single, dual or triplepass filter sets

are used, exposure times for acquiring video or photographic images will increase for dual and

triplepass filter sets when compared to singlepass filter sets).

The magnification of the image at the film plane affects the intensity of the image; the latter is inversely

proportional to the magnification. To minimise the magnification required to fill the picture with the

desired object, the format chosen should be as small as considered practical and hence have shorter

exposure times.

Exposure time varies for each objective and projection lens combination even if the total magnifications

are the same. The objective with higher numerical aperture (N.A.) should be chosen over increasing the

magnification by projection lens. (The numerical aperture of the objective increases with increase in

magnification and higher the numerical aperture, the brighter the image).

Excessively bright excitation light will cause the specimen to fade, adjust the brightness by inserting a

neutral density filter in the light path as these filters give a constant transmission over a wide range of

wavelengths.

42

Focus collector lens give brighter or more even illumination.

Avoid bleaching of the specimen by blocking the excitation light when not viewing or photographing the

specimen.

4.6 Video Fluorescence Microscopy

Electronic photomicrography utilizes electronic detection devices to acquire images, inserting an IR

(infrared) barrier filter in the light path before the detection device may produce better results by

avoiding erroneous readings caused by infrared light.

43

5. TROUBLESHOOTING TABLE

As you use your microscope and the epi-fluorescence attachment, you may occasionally experience a

problem.

The troubleshooting table below contains the majority of frequently encountered problems and the

possible causes.

5.1 Electrical

Lamp does not light

Power supply not plugged in

Lamp not installed

Lamp burnt out

Fuse is blown

Inadequate brightness Specified lamp not being used

Lamp blows out immediately Specified lamp not being used

Lamp flickers

Connectors are not securely connected

Lamp near end of service life

Lamp not securely plugged into socket.

5.2 Optical

Image not visible

Shutter slider in light path.

Filter cube unsuitable for specimen.

Filter cube not completely in light path.

Lamp on but image is unclear or has no contrast

Light source is not centred

ND filter in optical pat

Filter cube unsuitable for specimen

One component of filter cube is missing

Objective or filters are dirty

Specimens slide or cover glass dirty

Slide glass is fluorescing

Cover glass is missing

Greasy residue on eyelens

Room light is too bright

44

Field of view is partially obscured

Revolving nosepiece not clicked into position.

Shutter slider in intermediate position

Filter cube not installed correctly

Filter cube in intermediate position

Field diaphragm out of centre

Field diaphragm is stopped down

The excitation filters deteriorate with time due to exposure to heat and intense light.

Replace when necessary.

Handle Multi-Band filters with great care as the very complex coatings wear easily. Special

attention should be given to moisture. When filter is not in use, store in a desiccators.

45

6. CARE AND MAINTENANCE

6.1 Lenses and filters

To clean lens surfaces or filters, first remove dust using an air blower. If dust still persists, use a

soft/clean brush or gauze.

A soft gauze or lens tissue lightly moistened with pure alcohol should only be used to remove

grease or fingerprints.

Use petroleum benzine to clean immersion oil.

Use petroleum benzine only to remove immersion oil from objective lenses.

Because petroleum benzine and absolute alcohol are both highly flammable, be careful handling

around open flame.

Do not use the same are of gauze or tissue to wipe lens more than once.

6.2 Cleaning or painted or plastic components

Do not use organic solvents (thinners, alcohol, ether etc.) doing so could result in discoloration or in

the peeling of paint.

For stubborn dirt, moisten a piece of gauze with diluted detergent and wipe clean.

6.3 When not in use

When not in use, cover the instrument with vinyl dust cover and store in a place low in humidity

where mold is unlikely to form.

Store the objectives, eyepieces and filters in a container or desiccator with drying agent.

Proper handling of the microscope will ensure years of trouble free service. If repair becomes

necessary, please contact your Motic agency or our Technical Service directly.

The fluorescence vertical illuminator can accommodate three filter blocks and a dia-filter dummy block

(devoid of filters) that enables normal brightfield observation. Filter blocks are mounted on a sliding rack

and can be inserted into the optical path by means of a lever that is utilized to control the rack position.

Each block has an accompanying identification plate that can be inserted into a slot on the illuminator

exterior housing in sequential order to enable the operator to easily select the proper block for

fluorescence observation.

46

6.4 For MXH-100 Power supply unit

1. Ensure the fan on the back panel of the power supply is kept free of obstructions. Do not cover with

cloth or other items that may block the flow of air.

2. Ensure air is able to freely flow through the lamp housing. Do not cover with cloth or other items

that may block the flow of air as this will affect heat dispersal.

3. If there is dust on the outer casing, wipe it off using a dry cloth.

4. Do not touch the bulb or collector lens directly with your fingers. Use a dry cloth to wipe the bulb

and clean the collector lens.

5. When both in use and in storage, always protect the microscope from dust and moisture in order to

keep the electrical components running smoothly.

6. When cleaning, ensure the power has been turned off and the plug removed from the power supply.

7. Turn the power on and off as little as possible to preserve the life of the lamp. If the instrument is

damaged, do not take the instrument apart or attempt to repair. Contact your local distributor or a

professional to help repair the instrument.

8. No liquid is allowed for cleaning any part of this product.

9. Disconnect the power supply before replacement of fuse.

47

STANDARD FLUORESCENCE FILTER SETS

MF31000 DAPI and Hoechst Set: Exciter D350/50x Dichroic 400DCLP Emitter D460/50m MF31001 FITC FITC/RSGFP/Fluo 3/DiO Acradine Orange(+RNA) set: Exciter D480/30x Dichroic 505DCLP Emitter D535/40m MF31002 TRITC (Rhodamine)/DiI/Cy3 Set: Exciter D540/25x Dichroic 565DCLP Emitter D605/55m MF31004 Texas Red® / Cy3.5 Set: Exciter D560/40x Dichroic 595DCLP Emitter D630/60m MF41008 Cy5, Alexa Fluor 633, Alexa Fluor 647 Set: Exciter HQ620/60x Dichroic Q660LP Emitter HQ700/75m The emission at these wavelengths will be better detected by camera than by the unaided eye. Only a small percentage of humans can detect these wavelengths. MF31044 Cyan GFP Set: Exciter D436/20x Dichroic 455DCLP Emitter D480/40m This set will typically exclude the signal from YFP but not from GFP. MF41017 Endow GFP Bandpass Emission Set: Exciter HQ470/40x Dichroic Q495LP Emitter HQ525/50m This is the recommended filter set for the newer S65T GFP mutants. It is also recommended for wtGFP for which it was originally designed.

48

MF41028 Yellow GFP BP (10C/Topaz) Set: Exciter HQ500/20x Dichroic Q515LP Emitter HQ535/30m

This set is designed to image YFP. It will not, in most cases, detect CFP when co-

expressed in cells. By eye, fluorescence in the 535 nm pass-band will be seen as green.

Arrangement of Filters in a Fluorescence Cube

Upright Microscope

It is often the case that specific combinations of excitation filter, emission filter and dichroic mirror are

needed to visualize and/or quantitate the fluorescence emission from a particular fluorescent species.

In newer models of fluorescence microscopes, manufacturers have provided a means to change these

optical elements in a convenient manner by arranging a set of four or more filter cubes in a circular (or

linear) turret under the objective. With a turret arrangement, a specific filter cube can be selected in a

manner similar to that of selecting a specific objective.

49

NOMENCLATURE

Excitation Filter - EX A filter used in fluorescence microscopy designed to pass only those wavelengths, which excite

fluorescence.

Excitation Filter D350/50x

The center wavelength is at 350nm; full bandwidth is 50nm [= ±25nm].

In some cases when the bandwidth is not specified, the letter ”x” is used to define the filter as an exciter

filter and is generally used for narrow band UV excitation filters, e.g. D350x.

Dichroic Mirror - DM

The dichroic mirror is the optical component that separates the excitation light from the fluorescence.

Dichroic mirror is designed to reflect selectively the shorter wavelength exciting radiation and transmit

the longer wavelength fluorescence.

Dichroic mirror are placed in a 45° incidence angle to light, allowing the mirrors to act as a precise

discriminator of excitation and fluorescence wavelengths.

Light passing through the excitation filter is reflected 90° toward the objective and the specimen.

Finally, light emanating from the specimen is passed through and directed toward the emission filter

and observation method.

Dichroic multi-layer thin-film coatings makeup typically consists of one of the following design types:

short wave pass, long wave pass, or bandpass filter. These design types comprise the basis of color

determination and color separation and this is determined by the transmittance and/or reflectance of a

band of wavelengths.

Dichroic Beamsplitter 505DCLP

The cut-on wavelength is approximately 505nm for this dichroic longpass application.

Emission Filter - EM (Barrier filter)

Emission filter is the last component in a fluorescence cube, which transmits fluorescence emission

wavelengths while blocking excitation wavelengths. Emissions filters are coloured glass or interference

filters that have transmission properties of a bandpass or longpass filters.

50

Most interference are mounted at a slight angle to allow for better imaging by suppressing ghost

images.

Emission filter D460/50m*

The center wavelength is at 460nm; full band is 50nm [= ±25nm].

*m –emission

51

DEFINITIONS OF FILTER TERMINOLOGY

Bandpass Filters

Bandpass filters transmit a band of wavelengths and block all light above and below the specified

transmission range. These filters are characterized with respect to optical performance by their centre

wavelength (CWL) and bandwidth, also referred to as the full width at half of maximum transmission

(FWHM). (See Figure Below)

CWL

Center wavelength

For optical bandpass filters, the arithmetic mean of the cut-on and cut-off wavelength at 50% of peak

transmission.

DCLP

Dichroic Longpass.

DCXR

Dichroic Longpass, extended reflection including the UV.

FWHM

Bandpass filters are usually named by their filters center wavelength (the arithmetic means of the

wavelengths at 50% of center peak transmission) and by range of wavelengths (bandwidth) of light they

transmit at 50% peak transmittance (full width half maximum, FWHM). Example D350/50x is an

interference filter with maximal transmission at 350nm and transmits light from 325 and 375nm. (See

Figure Below)

GFP

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is used as a fluorescent

indicator for monitoring gene expression in a variety of cellular systems, including living organisms and

fixed tissues.

The wild type GFP has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm.

Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.

Due to this widespread usage different mutants of GFP have been engineered over the last few years:

52

Color mutants have been obtained from the GFP gene as well: in particular the cyan fluorescent

protein (CFP) and the yellow fluorescent protein (YFP) are two colour variants employed for

fluorescence resonance energy transfer (FRET) experiments.

GG

Green Glass – Longpass absorption glass from Schott Glassworks with cut-on wavelengths in the violet

and blue-green regions

HQ

A designation for high-performance filters with narrow cuton and cutoff wavelengths

LP

Longpass Filters

Longpass filters transmit a wide spectral band of long wavelength radiation while blocking short

wavelength radiation. (See Figure Below)

Narrowband Filters

Narrowband filters with a very narrow band, typically 1 to 3nm wide. They can be used successfully

only with high intensity light sources because of the narrow bandwidth.

ND

Neutral density filters are designed to reduce transmission evenly across a portion of the spectrum.

OG

Orange Glass - Longpass absorption glass from Schott Glassworks with cut-on wavelengths in the

green, yellow and orange regions

PC

Polychromatic mirrors or beamsplitters.

These beamsplitters reflect and transmit more than two bands of light. Dual-band or Triple-band filter

sets includes three carefully balanced combinations that contain dual or triple bandpass excitation and

emission filters, these dedicated filter sets also incorporate polychromatic mirrors or beamsplitters with

multiple bandpass characteristics, having transmission and reflection regions that are matching to the

specific excitation and emission filters employed.

53

RG

Red Glass - Longpass absorption glass from Schott Glassworks with cut-on wavelengths in the red and

far red regions

SP

Shortpass Filters

Shortpass filters transmit a wide spectral band of short wavelength radiation and block long wave

radiation. (See Figure Below)

S/N

Signal to Noise Ratio

Ratio of intensity of signal to that of the background.

Stokes shift

The distance between the peak absorption and emission of a dye, usually in nm.

Filters for isolating the wavelength of illumination:

Shortpass and longpass filters, block or transmit wavelengths at specific cut-off wavelengths.

Bandpass filters exhibit broadband or shortband transmission centered on a particular band of

wavelengths. Filters performance is defined by the central wavelength (CWL) and by the full width at

half maximum (FWHM).

54

SPECIFICATIONS (MXH-100)

Input Rating 100-240V~±10%, 150W (max), 50~60Hz.

Internal Fuse T2.5AL 250V

Output Rating 100W

Light Source Lamp rating: 100W super high pressure mercury lamp. Lamp type: super high pressure mercury lamp. USHIO USH-102D. OSRAM HBO 103 W/2.

Protection Class Class I

Operating Environmental Conditions

Temperature:5 to +40°C Humidity: 80% relative humidity for temperatures up to 31°C decreasing linearly to 50% relative humidity at 40°C. Altitude: 2000m max. Pollution Degree: 2. Indoor use only

Storaging Environmental Condition

Temperature: 2°C to 60°C. Humidity: 80% relative humidity for temperatures up to 31°C decreasing linearly to 50% relative humidity at 40°C.

Conforming Standards UL listed product.

Outlook Dimensions: 256(D) x 111(W) x 190(H) Weight: Approximately 2.75Kg

Power Cord Use only the following power supply cord. Using the wrong power cord could result in danger or fire. The protection Class 1 equipment should be connected to PE (protective earth) terminal.

Update: 26.06.2015

(BA410E EF-UPR / MXH-100)

Motic Incorporation Ltd. (Hong Kong)Rm 2907-8, Windsor House, 311 Gloucester Road, Causeway Bay, Hong Kong Tel: 852-2837 0888 Fax: 852-2882 2792

Motic Instruments Inc. (Canada)130-4611 Viking Way, Richmond, B.C., V6V 2K9 Canada Tel: 1-877-977 4717 Fax: 1-604-303 9043

Motic Deutschland GmbH (Germany)Christian-Kremp-Strasse 11 D-35578 Wetzlar, Germany Tel: 49-6441-210 010 Fax: 49-6441-210 0122

Motic Spain, S.L. (Spain)Polígon Industrial Les Corts, Camí del Mig, 112 08349 Cabrera de Mar, Barcelona Spain Tel: 34-93-756 6286Fax: 34-93-756 6287

Website: http://www.motic.comE-mail: info@motic.com.hk

Motic China Group., Ltd. (China)Motic Building, Torch Hi-Tech Industrial, Development Zone, Xiamen P.R.C. Tel: 86-0592-562 7866 Fax: 86-0592-562 7855

© 2007-2012 Motic China Group Co., Ltd. All rights reserved. Motic is a registered trademark and service mark of Motic China Group Co., Ltd. Microsoft Windows logo is a registered trademark of Microsoft Corporation. All other trademarks are the property of their respective owners.

Design Change: The manufacturer reserves the right to make changes in instrument design in accordance with scientific and mechanical progress, without notice and without obligation.