www.vwr.com VWR Inverted Fluorescence Microscope
Operation Manual (Version 1.0.0)
VWR Inverted Fluorescence Microscope 89404-464
89404-464 VWR Inverted Fluorescence Microscope
Man.V100 (28 Sept. 2012) ww.vwr.com
TABLE OF CONTENTS
SECTION
I. Safety Instructions 3
II. Nomenclature 4
III. Specification 6
IV. Setting-up the Instrument 7
V. Assembling the microscope Input voltage 7 1. Installing the lamp 8 2. Mounting the condenser 8 3. Installing the Objectives 9 4. Mechanical Stage 9 5. Mounting the eyepieces 9
VI. Microscopic procedure Interpupillary distance adjustment 10 1. Diopter adjustment 10 2. Centering the condenser 11 3. Centering the lamp 11 4. Brightfield microscopy 12 5. Phase-contrast microscopy 13
VII. Photomicrographic procedure 14
VIII. Troubleshooting Table 15
IX. Care and maintenance 16 1. Lenses and filters 16 2. Cleaning of painted or plastic components 16 3. When not in use 16
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I. Safety Instructions
Please read this manual carefully before using this product for optimal use. The indicated cautions are related to safety and you should observe all safety and warning instructions to avoid potential damage to product and injury to operators. Keep this manual for future reference.
Note: Use this product only in a way described in the product literature and this manual. Before using the product, verify that this product is suitable for its intended use.
Do not modify the system components or use the unauthorized parts as this will void the product warranty.
The following warning labels (or symbols) are found on the microscope, Study the meaning of the warning labels (or symbols) and always use the equipment in the safest possible manner.
Warning Label / Symbol Explanation
Indicates that the surface becomes hot, and should not be touched with bare hands.
Indicates that the main switch is ON.
Indicates that the main switch is OFF. ~ Indicates alternating current.
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II. Nomenclature
1. Lamp Socket Clamp Screw Knob 8. Objectives 2. Lamp House Cover Clamp Screw 9. Revolving Nosepiece 3. Annular Diaphragm 10. Coaxial Coarse/Fine Focus Knob 4. Condenser Diaphragm Lever 11. Torque Adjusting Ring 5. Condenser Focus Knob 12. Filter Retaining Ring 6. Condenser Lens 13. Hexagonal Centering Screwdrivers (x2) 7. Stage Plate Insert
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1. Vertical Photo Port 10. Phase Slider 2. Interpupillary Distance Scale 11. Condenser Clamp Holder Screw 3. Diopter Adjustment Ring 12. Condenser Centering Screws 4. Eyepiece 13. Stage Plate 5. Optical Path Selector Lever 14. Fluorescence Filter cassette Mount 6. Filter Slider 15. Power Switch 7. Lamp House 16. Light Intensity Control Dial 17. Brightness Indicator 8. Field Diaphragm Lever (LED Segmented Display) 9. Condenser Clamp Screw
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III. Specifications
Magnification Ratio: 40X-600X Eyepiece : Objective field Ø22 Objectives :
Magnification N.A. W.D. (mm) Plan Achromat 4x 0.1 23.5 * Plan Achromat 10x 0.25 7.5 * Plan Achromat LWD 20x 0.4 7.0 * Plan Achromat LWD 40x 0.6 2.8 * Plan Achromat LWD 60x 0.8 1.4 Plan Achromat Phase 10x 0.25 7.5 Plan Achromat Phase LWD 20x 0.4 7.0 Plan Achromat Phase LWD 40x 0.6 2.8
* is subjected to the region and package you purchase.
● Condenser : 1. N.A. 0.3 / W.D. 72mm ● Electrical Specifications: Input: 90-240V~, 35W, 50-60Hz Lamp: DC6V 30W Halogen Fuse: 250V T2.5A (If the original fuse is damaged, please replace a new one with same specification)
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IV. Setting-up the Instrument
Working environment ● The location should be free from dust, moisture, chemical vapours and mechanical vibrations. ● Do not situate the instrument in a warm and/or humid environment. ● Locate the instrument where the operator’s line of vision is not directed towards a window, a lamp or a well-lit bright wall. The quality of the viewed image from the microscope will deteriorate where there is significant ambient light.
Operating environment ● Indoor use ● Altitude:Max 2000 meters Ambient temperature: 15˚C to 35˚C Maximum relative humidity: 75% for temperature up to 31ºC decreasing linearly to 50% relative humidity at 40ºC ● Supply voltage fluctuations: Not to exceed ±10% of the normal voltage. ● Pollution degree: 2 (in according with IEC60664) ● Installation/Overvoltage category:2 (in according with IEC60664) ● Air Pressure of 75kPa to 106kPa ● No hoar frost, dew, percolating water, rain
V. Assembling the microscope Input voltage
● Automatic voltage selection works with electrical outlets worldwide. However, always 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. ● In case of using the extension cord, use only the power supply cord with the PE (protective earth) wire. ● In order to prevent electric shock, always turn the power switch on the power supply off before connecting the power cord.
Power Cord
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1. Installing the lamp ● In order to prevent electric shock always turn the power switch off and unplug the power cord before replacing the lamp.
Power Switch
● Release lamp housing cover clamp screw using a coin to remove the cover.
Lamp Housing Cover Clamp Screw
● Firmly insert the lamp into the socket pinholes until it reaches the limit, be careful not to tilt the lamp when mounting. ● When installing the lamp, do not touch the glass surface of the lamp with bare fingers. Doing so will cause fingerprints , grease, etc., to burn onto the lamp surface, reducing the illumination provided by the lamp. If surface is contaminated, wipe it clean using lens tissue. ● Close the cover and fasten with it with lamp housing cover clamp screw. ● Insert the filter slider with mat surface of the diffuser turned towards the user.
2. Mounting the condenser ● and index marks facing the front and secure it with the clamp screw. Mount the ELWD condenser on the circular dovetail mount of the condenser holder with the aperture diaphragm lever
Condenser Clamp Screw
● Insert the Ph annular diaphragm slider with centering hexagonal socket head screws facing the front.
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● The centerable condenser mount is height adjustable with rack and pinion and is dovetail mounted on the illuminating pillar with a clamp screw.
3. Installing the Objectives ● Remove the stage plate insert from the stage. ● Install the objectives into the nosepiece so that the magnification increases with clockwise rotation of the revolving nosepiece. ● Replace the stage plate insert.
4. Mechanical Stage ● Secure the mechanical stage to the plain stage using the two mounting screws located beneath the stage on the right side ● Stage: X:108mm; Y:72mm (travel distance)
5. Mounting the eyepieces ● Remove the dust caps from the eyepiece tubes. ● Insert the eyepieces into the eyepiece tubes. ● If the rubber eye guards are to be used, fit them in the groove around the eyepiece.
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VI. Microscopic procedure Interpupillary distance adjustment
● Before adjusting the interpupillary distance, bring a specimen into focus using the 10x objective. ● Adjust the interpupillary distance so that both the right and left field of view become one. ● This adjustment will enable the user to observe the specimen with both eyes.
1. Diopter adjustment ● Diopter adjustment compensates for differences in vision between the left and right eyes. In addition to making observation through both eyes easier, this adjustment also reduces the extent to which focusing is lost when the objective magnification is changed. In particular, this occurs when a low power objective is used. ● Before adjusting the diopter, bring a specimen into focus using the 10x objective. ● Turn the diopter compensation ring on each eyepiece until the adjustment ring is adjusted to “0” position.
Diopter adjustment “0” position
● Position 40x objective into the optical path and bring the specimen image into focus by turning the coarse and fine focus knobs. ● Position either 4x or 10x objective into optical path. Without adjusting the fine and coarse focus knobs, turn the diopter rings on the eyepieces so that the specimen images in the left and right eyepieces are focused individually. ● Repeat the above step twice.
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2. Centering the condenser ● Set the Phase annular diaphragm slider in centre position (O). ● Fully open the field of view diaphragm. ● Move the aperture diaphragm lever in open “O” position. ● Bring the specimen image into focus, using the 10x objective. ● Close the field of view diaphragm to its minimum setting. ● Turn the condenser focus knob so that the image of the field diaphragm forms on the specimen surface.
● Adjust the condenser centering screws so that the centre of the field diaphragm image matches the centre of the field of view. This adjustment is easier to make if the field diaphragm size is stopped down to slightly smaller than the eyepiece field of view. ● For normal observation, the size of the diaphragm should be just outside the edge of the field of view.
3. Centering the lamp ● Set the Phase annular diaphragm slider in the centre position (O).
● Fully open the field of view diaphragm. ● Move the aperture diaphragm lever to the open “O” position. ● Using the 10x Phase contrast objective, bring the specimen image into focus. ● Remove the diffuser filter slider from the light path. ● Remove an eyepiece and insert the phase centering telescope in its place. ● Holding the knurled part of the centering telescope, rotate its eyepiece to focus on the phase plate image of the objective.
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● Release the lamp socket clamp screw using the knob.
Lamp Socket Clamp Screw Knob
● Move the lamp socket with the knurled knob to bring the lamp filament image to the centre of the phase plate image of the objective. ● After finishing the above lamp centering procedure, insert the filter slider with mat surface of the diffuser turned towards the user.
4. Brightfield microscopy ● Set the Phase annular diaphragm slider in the centre position (O). ● Bring the specimen image into focus. ● Adjust the opening of the field of view diaphragm, for normal observation the size of the diaphragm should be just outside the edge of the field of view. ● The condenser aperture diaphragm is provided for adjusting the numerical aperture (N.A.) of the illuminating system of the microscope. It is important because it determines the resolution of the image, contrast, depth of focus and brightness.
● Stopping down the aperture diaphragm will lower the resolution and brightness but increase the contrast and depth of focus. By stopping down the N.A. of the condenser to 2/3 of the N.A. of the objective, a good image of suitable contrast will be obtained.
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5. Phase-contrast microscopy ● Phase contrast objectives are labelled “Ph”: Ph1; Ph2; Ph3 and Ph4. ● For phase contrast microscopy, be sure to use the annular diaphragm that has the same symbol as the objective, despite of the magnification of the objective. ● Fully open the aperture diaphragm. ● Bring the 10x (Ph1) objective into optical path.
● Position the Phase annular diaphragm slider to 10 -20. Set slider to 40 when using a 40x (Ph3) objective. ● Remove either eyepiece from the eyepiece tube and insert the phase centering telescope in its place. ● Rotate the eyepiece of the centering telescope to focus on both the phase plate image of the objective and the annular diaphragm image of the phase slider. ● If the objective phase plate and the annular of the slider do not coincide, use the two hexagonal screwdrivers supplied with the microscope to bring the slider annular ring to the centre of the phase plate, so that the image of the annular diaphragm is concentric with the phase plate image.
● If the slider annular ring image is moved from the phase plate image in the objective, a low phase contrast image will result. ● For phase contrast microscopy at the maximum contrast, use GIF (Green interference filter) in the optical path. ● Place the filter in the designated retaining ring above the phase annular diaphragm slider.
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VII. Photomicrographic procedure
● The optical path selector lever can be used to set the optical path to either the Binocular tube 100:0 or Binocular tube/ vertical tube 20:80 (observation: photo).
● Before starting photomicrography, check the following: ○ The condenser is centered. ○ The condenser annular diaphragm is centred. ○ The field of view diaphragm is stopped down to slightly just outside the edge of the field of view. ● For photomicrographic procedures, refer to the manual of the specific camera being used.
Filter selection Filter type Procedure For phase contrast and contrast adjustment with black and GIF (Green interference) 546nm white film
NCB (Neutral Colour Balance) Blue For general microscopy and colour photomicrography
Never attempt either of the following actions, since doing so will damage the focusing mechanism: ● Rotate the left and right knob while holding the other. ● Turning the coarse and fine focus knobs further than their limit.
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VIII. Troubleshooting Table
As you use your microscope, you may occasionally experience problems. The troubleshooting table below contains the most frequently encountered problems and their possible causes.
Optical and Operating Problems Problem Possible Cause Lamp not installed properly Filter slider in intermediate position Phase slider not in click-stop position Incorrect condenser mounting Vignetting or uneven brightness in Condenser is set too low the field of view or field of view only partially visible Condenser is not centered Field diaphragm closed too far Aperture diaphragm closed too far Revolving nosepiece not clicked into position Optical path selector lever in intermediate position Aperture diaphragm closed too far
Dust or dirt in field of view Field of view diaphragm image not focused on specimen surface
Dust or dirt on specimen’s surface Brightfield objective being used Phase annular diaphragm not in optical path Phase annular diaphragm and objective phase symbol do not Image quality: match No image under phase contrast or Slider annular ring image has moved away from the objective details cannot be viewed phase plate image Field of view diaphragm image not focused on specimen surface
Thickness of specimen holder is outside the compensating range of objective Interpupillary distance not adjusted Diopter adjustment not made Eye strain or fatigue Inadequate illumination Field of view of left and right eyepiece differ
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Electrical Power supply not plugged in Lamp does not light Lamp not installed Lamp burnt out Inadequate brightness Specified lamp not being used Lamp blows out immediately Specified lamp not being used Connectors are not securely connected Lamp flickers Lamp near end of service life Lamp not securely plugged into socket
IX. Care and maintenance
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 same area of gauze or tissue, to wipe more than once.
2. Cleaning of painted or plastic components ● Do not use organic solvents (thinners, alcohol, ether, etc.). Doing so could result in discolouration or in the peeling of paint. ● For stubborn dirt, moisten a piece of gauze with diluted detergent and wipe clean.
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 mould is not likely to form. ● Store the objectives, eyepieces and filters in a container or desiccator with drying agent.
Note: If equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired.
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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.
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Table of Contents
1. Names of Component Parts 19
2. Setting up the Instrument 20
3. Assembly 20
4. Microscopy 38
5. Troubleshooting Table 43
6. Care and Maintenance 44
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1. Names of Component Parts
Fig.1
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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
3.3 Required tools Allen hex keys: 2.5mm and 5mm (supplied with attachment).
Please consult the illustrations provided while assembling the attachment
BEFORE STARTING Turn off the microscope power switch and unplug the power cord
3.4 Protective dust covers ․ Pull out rear dust cover, located above the Microscope AC inlet.
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Fig. 2
․ Remove side dust covers located on each side of the microscope base.
Fig.3a Fig.3b
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3.5 Attaching the Epi-fluorescence attachment sleeve Attach the main body sleeve of the epi-fluorescence attachment into the microscope stand, with the notch on the sleeve facing left.
Fix the main body sleeve in this position by using the three 5mm Allen hexagonal screws.
Fig.4
3.6 Mounting the Epi-fluorescence attachment main body Align the pin on the Epi-fluorescence attachment with the notch in the Epifluorescence attachment sleeve, slide the main body of the Epi-fluorescence horizontally into the sleeve until it cannot be pushed in any further. To secure, tighten the fixing screws using a hexagonal Allen key.
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Fig. 5
3.7 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. 6
3.8 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.
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3.9 Installing the filter cassette ․ Slide the filter cassette into the opening on the right side of the microscope until the two pins slot into the two matching holes on the microscope opening surround. ․ Using an Allen hex key, secure the cassette firmly with the two 2.5mm clamp screws.
Fig.7
․ Position the filter cube cover retaining plate with the opening on the left side of the microscope. ․ Slot the two pins into the two matching holes on the microscope opening surround. ․ Using an Allen hex key, secure the filter cube cover retaining plate firmly with the two 2.5mm clamp screws.
Fig. 8
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3.10 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. • 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.
Fig.9
There are a filter cube clamp plates at each end of the filter slider. Undo the Allen hex screws holding the clamp plate on right side of the slider with the hexagonal key supplied with the fluorescence attachment and secured inside the right filter cube cover
Fig.10
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Three filter cubes can be positioned on the filter slider.
Fig. 11
• Fasten the excitation method changeover lever to a filter cube. • Insert a filter cube along the dovetail mount from the side opening (Fig. 11) and push in as far as it will go. • Remove the changeover lever from the filter cube and install the two remaining cubes in the same manner. • Remember the position of the filter cube so that you can insert the ID tag (supplied with the filter cube) for the cube added. • Install frequently used filter cubes at the end of the dovetail mount in order to make replacement easier. • If daiscopic (transmitted light) illumination is also to be used, install one diascopic cube (DIA-ILL).
Secure filter cube clamp plate with Allen hex screws (Fig. 9) Install and secure the filter cube cover on both the right and left sides with the knurledretaining screws.
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Fig. 12a Fig.12b
Fasten the excitation method changeover lever Screw the excitation method changeover lever into the filter cube through the opening in the white nylon bushing (Fig. 12b) on the right cover.
Fig.13 Insert the filter cube ID tags
Insert the ID tags for the filter cubes into the ID tag pockets on the filter block cover.
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Replacing the filter cubes • Unscrew the knurled retaining screw and remove the right filter cube cover. Undo the filter cube clamp plate. • Screw the excitation method changeover lever into the screw hole on to the side of the filter cube. • Draw the filter cube out of the filter cassette. • Replace the filter cube according to the procedure “Installing the Filter cubes”
Handling filters cubes • Always keep filter cubes in the cassette holder. • When a filter cube is not in use, store in a desiccator, or a sealed container with a drying agent as interference filter characteristics change if the filter is exposed to high humidity. • Do not allow dust to get onto the filters as this will adversely affect the image viewed. • Be careful when using air blowers, as they sometimes emit fluid when the air can is not used in an upright position. This fluid can leave difficult-to-remove spots on the filter surface.
3.11 Installing the light shielding device The light-shielding device protects your eyes against ultraviolet rays radiated from the objective to the specimen.
• Attach the light-shielding device to the eyepiece tube housing using the clamp screw on the right. • Adjust the height and angle of the light-shielding device with the knurled knob on the left.
Fig. 14
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3.12 Installing the light source
Assembly
Attaching the collector lens to the lamp house • 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.
Installing the collector lens in the lamp house: • Aligning the pin on the collector lens with the collector lens positioning groove of the lamp house.
Fig. 15
• 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.
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Attaching the lamp house to the microscope
Attach the lamp house to the microscope using the bayonet ring on the Epi-fluorescence attachment.
Fig. 16a
Align the positioning pin on the lamp house flange with the positioning groove on the bayonet mount of the fluorescence attachment Fig. 16b
Turn the bayonet ring in the direction indicated by the arrow
Secure the bayonet ring by turning it as far as possible in the direction of arrow.
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Installing the lamp Fig. 17
• 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 and remove the cover and the lamp socket assembly. (See Fig.17) • Loosen the lamp clamp screws at both ends of the lamp socket and remove “dummy lamp” installed. • 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 large and small diameter metal ends on lamp and lamp socket. • Insert the lamp into the lower flexible lamp clamp base and tighten the clamp screw. (See Fig.18a) • Insert the lamp into the upper lamp socket hole supported by the metal plate with the cooling fins and fasten the clamp screw.
Fig. 18a Fig. 18b
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• 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. Secure with the clamp screw. • The safety cut-off micro switch will “click” as you secure the lamp house cover clamp screw.
3.16 Connecting the lamp house to the power supply: Turn OFF the power switch on the power supply.
Fig.19
3.14 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. • 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.
3.15 Turning ON the lamp • Set the power supply switch to “l”. • Press the IGNITION button on the power supply unit for 5-10 seconds. • The POWER LAMP / READY LAMP will light up to indicate that the power is turned on. • The POWER LAMP / READY LAMP indicator lamp will start flashing briefly to indicate that the lamp is stabilized. • Press the RESET PAD below the RUN TIME counter on the power supply.
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• The “Run Time” counter displays the elapsed time.
Fig.19
Mercury Lamp Power Supply
3.16 Aligning the mercury arc 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.
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
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3.17 Aligning the arc lamp • Remove the stage plate insert from the stage • Take off one of the nosepiece dust caps, or remove an objective from the nosepiece and screw on the centering tool with ground glass and inscribed cross hair in its place. • Rotate it into the light path.
Fig.20
Lamp Centering Tool
• Centre the lamp while observing the window of the centering tool. • To begin alignment of the lamp, rotate the collector lens focus knob to produce a sharp image of the arc on the window of the centering tool. • Rotate the lamp horizontal and vertical centering screws until the arc image is in the centre of the window. • Using the collector lens focus knob spread the beam to achieve an evenly illuminated field. The final scattering of the beam is checked on an actual fluorescent specimen. • The size of the image of the arc can be made bigger or smaller by manipulating the collector lens focus knob.
3.18 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.
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• Cutting out the excessive light is useful in preventing contrast from being diminished. • Decreasing excessive illumination is useful in protecting the specimen from fading.
Fig. 21
Epi-fluorescence attachment main body
3.19 Centering the field diaphragm • Move the field aperture diaphragm lever to stop down the diaphragm (Raise the field aperture diaphragm lever). • If the field aperture diaphragm is off the centre of the field of view, centre with the field aperture diaphragm with self-disengaging centering keys depressed. • Open the field aperture diaphragm just slightly outside the edge of the field of view (Lower the field aperture diaphragm lever).
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Fig. 22
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=6.25%)
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.
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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.
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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.
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).
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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.
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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.
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 VWR 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.
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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.
Turn the specimen upside down with the cover glass facing the objective.
Slip on a rubber “O” ring between the outer collar and retractable lens assembly of the oil immersion objective.
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. Note: The rubber “O” ring should be discarded after each use.
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.
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.
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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.
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.
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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.
Electrical Power supply not plugged in Lamp not installed Lamp does not light Lamp burnt out Fuse is blown Inadequate brightness Specified lamp not being used Lamp blows out immediately Specified lamp not being used Connectors are not securely connected Lamp flickers Lamp near end of service life Lamp not securely plugged into socket.
Optical Shutter slider in light path. Image not visible Filter cube unsuitable for specimen. Filter cube not completely in light path. Light source is not centred ND filter in optical path Filter cube unsuitable for specimen One component of filter cube is missing Lamp on but image is unclear Objective or filters are dirty or has no contrast Specimens slide or cover glass dirty Slide glass is fluorescing Cover glass is missing Greasy residue on eyelens Room light is too brigh Revolving nosepiece not clicked into position. Shutter slider in intermediate position Filter cube not installed correctly Field of view is partially obscured Filter cube in intermediate position Field diaphragm out of centre Field diaphragm is stopped down
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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.
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 VWR 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.
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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.
Standard Fluorescence Filter Sets
MF31000DAPI and Hoechst Set: Exciter D350 / 50x Dichroic 400DCLP Emitter D460 / 50m
MF31001FITC FITC / RSGFP / Fluo 3 / DiO Acradine Orange(+ RNA) set: Exciter D480 / 30x Dichroic 505DCLP Emitter D535/40m
MF31002TRITC (Rhodamine) / DiI / Cy3 Set: Exciter D540/25x Dichroic 565DCLP Emitter D605/55m
MF31004Texas Red® / Cy3.5 Set: Exciter D560 / 40x Dichroic 595DCLP Emitter D630 / 60m
MF41008Cy5, 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.
MF31044Cyan GFP Set: Exciter D436 / 20x Dichroic 455DCLP
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Emitter D480 / 40m
This set will typically exclude the signal from YFP but not from GFP. MF41017Endow 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.
MF41028Yellow 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 coexpressed in cells. By eye, fluorescence in the 535 nm pass-band will be seen as green.
Arrangement of Filters in a Fluorescence Cube
Inverted Microscope
To objective and specimen
NOMENCLATURE
Excitation Filter - EX A filter used in fluorescence microscopy designed to pass only those wavelengths, which excite fluorescence.
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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.
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
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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: 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.
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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.
RG Red Glass - Longpass absorption glass from Schott Glassworks with cut-on wavelengths in the red and far red regions
SP
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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).
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