White Tirf Illuminator For Te2000 Instructions

Transcript

M341 E White TIRF Illuminator for TE2000 Instructions 05.3.NF.2 Preface Thank you for purchasing the Nikon products. This instruction manual is written for the users of the White TIRF Illuminator which are to be used together with Nikon’s Inverted Microscope ECLIPSE TE2000. To ensure correct usage, read this manual carefully before operating the instrument. • It is prohibited to reproduce or transmit this manual in part or whole without Nikon’s expressed permission. • The contents of this manual are subject to change without notice. • Although every effort has been made to ensure the accuracy of this manual, if you note any points that are unclear or incorrect, contact your nearest Nikon representative. • Some of the products described in this manual may not be included in the set you have purchased. • Be sure to read the manuals for any other products that you are using with this attachment (the microscope, super high-pressure mercury lamp power supply, high-intensity light source, etc.). Warning / Caution Symbols Used in This Manual Although Nikon products are designed to provide you with the utmost safety during use, incorrect usage or disregard of the instructions can cause personal injury or property damage and will lead to the forfeiture of all claims against warranty. For your safety, read the instruction manual carefully and thoroughly before using the instrument. Do not discard this manual but keep it near the product for easy reference. In this manual, safety instructions are indicated with the symbols shown below. Be sure to follow the instructions indicated with these symbols to ensure correct and safe operation. Symbol Meaning WARNING Disregarding instructions marked with this symbol may lead to death or serious injury. CAUTION Disregarding instructions marked with this symbol may lead to injury or property damage. 1 Preface WARNING 1 Intended product use This system should only be used for microscopic observation. Do not use it for any other purpose. 2 Do not disassemble Disassembling may cause electrical shock, exposure to ultraviolet light, and/or malfunction. Do not disassemble any parts other than those mentioned in this manual. If you notice any malfunction, contact your nearest Nikon representative. 3 Read the instruction manuals carefully For your safety, carefully read this manual and the manuals provided with the other products used with the system. Make certain to heed the warnings and cautions at the beginning of each manual in particular. 4 Cautions regarding the power supply Read the manual for the microscope. Cautions regarding lamp heat Read the manuals for the microscope and the light source (super highpressure mercury lamp power supply or high-intensity light source). Cautions regarding ultraviolet light produced by the lamp Read the manual for the light source (super high-pressure mercury lamp power supply or high-intensity light source). Cautions regarding lamp bursting and the gas sealed inside the lamp Read the manual for the light source (super high-pressure mercury lamp power supply or high-intensity light source). Cautions regarding the lamp specifications Read the manual for the microscope and the light source (super highpressure mercury lamp power supply or high-intensity light source). Mercury lamps The mercury lamp used with this system requires special handling. In order to use this system safely and correctly, carefully read the warnings below and beware of the dangers. Also carefully read the manual for the super high-pressure mercury lamp power supply (or high-intensity light source) and the manual (if provided) by the manufacturer of the lamp and follow their instructions. The Hazards of Mercury Lamps 1 Mercury lamps radiate ultraviolet light that is harmful to the eyes and skin when they are turned on. Direct viewing of light from these lamps may result in blindness. 2 Gas is sealed under very high pressure inside the lamps. The pressure increases when the lamp is on. If the lamp is scratched, dirty, subjected to high external pressure or physical impact, or used beyond its operational life, the sealed gas may escape or the lamp may burst. This can result in someone inhaling the gas, injuring themselves on the glass, or other accidents. 3 When the lamp is on, the lamp and its surroundings become extremely hot. Touching the lamp with bare hands could result in burns. Flammable materials placed near the lamp could ignite. 4 Using other than the specified type of lamp could result in an accident, such as a burst. Because safety is a top priority in the design of Nikon products, the hazards described above should not pose any danger as long as you heed all of the warnings and cautions in the manuals and use the system only for its intended purpose. However, the hazards described above could lead to an accident if you fail to heed all of the warnings and cautions in the manuals, if you strike the system, or if you attempt to disassemble the system. Therefore, always be sure to heed all of the warnings and cautions. 2 Preface 5 Always turn the lamp off when exchanging filter cubes or cassette holders When exchanging filter cubes or cassette holders, always be sure to turn off the lamp connected to the white TIRF illuminator. If the lamp is left on during exchange, there is a danger of exposure to ultraviolet light. 6 Use the ultraviolet light shield Do not directly see the light irradiated from the objective. Harmful excitation light will be given out from the objective depending on the excitation method used. When you see the part around the objective, be sure to see through the ultraviolet light shield. CAUTION 1 Turn off the power when assembling the equipment, connecting or disconnecting cables, or when replacing the lamp In order to prevent electrical shock and damage to the equipment, always turn off the power switch on the microscope, power supplies, etc., and unplug the power cord before assembling the equipment, connecting or disconnecting cables or replacing a lamp. 2 Do not wet the equipment If the microscope, the system or the power supply gets wet, a short circuit may result that could damage it or make it extremely hot. If you accidentally spill a liquid on the equipment, immediately turn the power switch off and unplug the power cord. Then use a dry cloth to wipe away the moisture. If any liquid gets inside the equipment, do not use it; instead, contact your nearest Nikon representative. 3 Caution concerning assembly Be careful not to pinch your hands or fingers when assembling the equipment. 4 Correctly assemble the equipment and center the lamp Be sure to correctly assemble the white TIRF illuminator and center the lamp. If this is not done correctly, light source image from the lamp may focus on structural parts (such as a filter frame) in the optical path. This may lead to an increase in temperature of the surface of the structural part, which causes deformation and smoldering. For details on assembly, see the chapter, “IV. Assembly” in this manual. For details on centering the lamp, see the instruction manual with the power supply of the lamp. 3 Notes on Handling the System 1 Handle the system gently This system is a precision optical instrument. Handle the system gently, avoiding any physical shocks. 2 Installation location In order to avoid degraded performance and to prevent malfunctions, take the following requirements into consideration when selecting a location to install the system: • Install the system in a location with little vibration. • Avoid installing the system in a location exposed to direct sunlight. • Avoid installing the system in a dusty location. • Avoid installing the system in a location subject to high temperatures (40°C or higher) or high humidity (60% or higher). (Such conditions could allow mold or condensation to form on the lenses and filters.) 3 Handling of filters • Interference filters (especially excitation filters, which are exposed to strong light) deteriorate with the passage of time. Replace them according to the number of hours they have been in use. • Filter characteristics may change if the filter is exposed to high humidity. In order to prevent changes in or deterioration of filter characteristics, avoid using or storing the filters under conditions of high humidity or high temperature, and avoid subjecting them to rapid temperature changes. When a filter is not in use, storage in a desiccator or a hermetically sealed container with a drying agent is recommended. • The filters in the eight types of filter cubes listed below offer sharp, high-resolution waveform characteristics in comparison with normal filters. However, because they have sophisticated coatings, they must be handled with extra care. Abrasion caused by cleaning is a special concern. (Follow the procedure described in “Cleaning filter and lenses” of chapter, “VI. Care and Maintenance.”) 4 Single-band filter cubes DAPI, FITC, GFP-B, GFP-L, TRITC, TxRed Multi-band filter cubes F-R, F-T, D-F, D-F-R, D-F-T Cleaning the system When cleaning the system, follow the instructions described in chapter “VI. Care and Maintenance.” Do not let dust, fingerprints, etc. get on the lenses. Dirt on the lenses, mirrors, etc. will adversely affect the view of the image. 4 Contents Preface ............................................................................................................................................................................... 1 Warning / Caution Symbols Used in This Manual .................................................................................................................... 1 Notes on Handling the System ........................................................................................................................................ 4 I. Names of Compoenent Parts .................................................................................................................................... 6 1 2 3 II. Names and functions of component parts........................................................................................................................... 6 White TIRF illuminator ......................................................................................................................................................... 7 Cassette holder.................................................................................................................................................................... 8 Microscopy ................................................................................................................................................................. 9 1 2 3 4 Setup ................................................................................................................................................................................. 10 Center the illumination lamp.......................................................................................................................................... 10 Diopter and interpupillary distance adjustment ............................................................................................................. 10 TIRF microscopy ............................................................................................................................................................... 11 Sequence of microscopy ............................................................................................................................................... 11 Epi-fl microscopy ............................................................................................................................................................... 13 Sequence of microscopy ............................................................................................................................................... 13 Combining Epi-fl microscopy with DIC microscopy....................................................................................................... 14 Combining Epi-fl microscopy with Ph microscopy ........................................................................................................ 15 General bright-field microscopy......................................................................................................................................... 16 III. Operation of Each Part............................................................................................................................................. 17 1 2 3 4 5 6 White TIRF illuminator ....................................................................................................................................................... 17 Switching between aperture diaphragm and W-TIRF diaphragm................................................................................. 17 Condenser lens operation ............................................................................................................................................. 18 Field diaphragm operation............................................................................................................................................. 19 Aperture diaphragm operation ...................................................................................................................................... 20 TIRF diaphragm operation ............................................................................................................................................ 21 Optical axis shift amount switch lever operation ........................................................................................................... 21 ND filter operation ......................................................................................................................................................... 22 Excitation filter operation ............................................................................................................................................... 22 Shutter operation........................................................................................................................................................... 23 Connector for lamp housing .......................................................................................................................................... 23 Centering tool ................................................................................................................................................................ 23 Ultraviolet light shield .................................................................................................................................................... 23 Cassette holder.................................................................................................................................................................. 24 Filter cube port and cover.............................................................................................................................................. 24 Excitation method changeover ring............................................................................................................................... 24 Address and name of the filter cube in the optical path................................................................................................ 24 Shutter lever .................................................................................................................................................................. 24 Filter cube .......................................................................................................................................................................... 25 Oil-immersion type objectives............................................................................................................................................ 27 Fluorescent photomicrography.......................................................................................................................................... 28 TV microscopy ................................................................................................................................................................... 28 IV. Assembly .................................................................................................................................................................. 29 Assembling the white TIRF illuminator.................................................................................................................................... 30 Assembling and attaching the W-TIRF slider.......................................................................................................................... 31 Attaching filters........................................................................................................................................................................ 31 Attaching the cassette holder.................................................................................................................................................. 32 Attaching filter cubes............................................................................................................................................................... 32 Microscope assembly.............................................................................................................................................................. 33 Installing the ultraviolet light shield ......................................................................................................................................... 33 Installing the light source......................................................................................................................................................... 33 V. Troubleshooting ....................................................................................................................................................... 34 Epi-fl microscopy ..................................................................................................................................................................... 34 TIRF microscopy ..................................................................................................................................................................... 35 VI. Care and Maintenance ............................................................................................................................................. 36 VII. Equipments in Combination.................................................................................................................................... 37 5 I Names of Component Parts 1 Names and functions of component parts The picture below shows all the parts needed for the TIRF microscopy mounted on the TE2000-U, which can perform bright-field microscopy. White TIRF illuminator • Main unit • W-TIRF slider • W-TIRF diaphragm x 2 (for 60 x objective and for 100 x objective) • Excitation filter slider • ND filter slider x 2 • Shutter slider • Support pillar • Centering tool • Ultraviolet light shield Light source The photograph shows the combination of super high-pressure mercury lamp housing, mercury lamp, and collector lens. T-FLC cassette holder Or T-FLC-E motorized cassette holder (The T-HUBC HUB controller is required.) Or T-FLC-HQ cassette holder Filter cube Up to six blocks can be set in the cassette holder. Three exclusive high-precision filter cubes are attached as accessories for the T-FLC-HQ cassette holder. Power supply for the light source The photograph shows the super high-pressure mercury lamp power supply. • For details on how to operate the T-FLC-E motorized cassette holder, please refer to the instruction manual supplied with the T-HUBC HUB controller. 6 I. Names of Component Parts 2 White TIRF illuminator Normal Epi-fl illumination and TIRF illumination are selectable by operating the W-TIRF slider. Field diaphragm open/close lever, Field diaphragm centering adjustment screws This lever opens/closes the field diaphragm and these screws adjust the center of the field diaphragm. ND filter slider x 2 These sliders are for two ND filters, ND4 and ND8. Connector for Push: IN (into the optical path) Pull: OUT (out of the optical path) lamp housing Condenser lens position indices Condenser lens locking screw Focal point adjustment knob This knob adjusts the position of the condenser lens. W-TIRF slider By switching the W-TIRF diaphragm and the aperture diaphragm, microscopy methods are switchable. Push: TIRF microscopy, Pull: Epi-fl microscopy And knobs on this slider are used for centering diaphragms, opening or closing the aperture diaphragm, and switching the optical axis shift amount for the TIRF microscopy. 7 Excitation filter slider This slider is used for the excitation filter (EX filter) to move into the optical path. Up to three excitation filters can be set in the slider. And at the clickstop position, each filter is placed into the optical path. At the far position, no filter is in the optical path. Shutter slider The shutter blocks the light from the light source. Push: Close, Pull: Open When the slider is pushed in, the shutter is opened. When pulled out, the shutter is closed. I. Names of Component Parts 3 Cassette holder Up to six filter cubes can be set in the cassette holder for the Epi-fl microscopy and TIRF microscopy. Filter cubes can be switched by turning the excitation method changeover ring. One of the three cassette holders below can be used with this system. T-FLC cassette holder This cassette holder is used by the manual operation. T-FLC-E motorized cassette holder This cassette holder is moved by the motor drive. The T-HUBC HUB controller is required. T-FLC-HQ cassette holder This cassette holder is used by the manual operation. Three exclusive high-precision filter cubes are attached as accessories. Excitation method changeover ring *1 (Operation part to change filter cubes) Filter cube port (and cover) Front side Cassette holder Address and name of the filter cube in the optical path *1 Shutter lever (O: open, C: close) *1 : T-FLC-E motorized cassette holder does not have these parts. Centering tool This tool is mounted on the nosepiece for operation. And it is used to center the lamp and diaphragms. 8 II Microscopy The general procedure for microscopy is described below. For details on each step, see the corresponding item in chapter “III. Operation of Each Part.” If the system is not yet assembled, see chapter “IV. Assembly” first. For details on the assembly, handling, and use of the microscope, power supply, and light source, see their respective manuals. WARNING Before using the system, be sure to read the “WARNING” and “CAUTION” at the beginning of this manual, and also the section entitled, “Notes on Handling the System.” Be certain to heed all of the warnings and cautions. Also be sure to read the manuals for any other products that you are using with this system (the microscope, power supply, super high-pressure mercury lamp power supply, high-intensity light source, and such), and heed all of the warnings and cautions in those manuals. Before starting 1 Check the cumulative “lit on” time of the lamp. If the time has exceeded the average operation life for lamps of its kind, replace the lamp. 2 Use non-fluorescent slide glass. 3 Use non-fluorescent immersion oil. 4 In order to prevent fading of the specimen, always insert the shutter into the optical path whenever you are not actually looking through the binocular eyepiece. 5 When switching the excitation method by rotating the excitation method changeover ring, always be sure the ring is set to a click-stop position. Do not operate the microscope while the ring is set to positions between click-stops. If the microscope is operated while this ring is set between click-stops, some internal parts will increase in temperature. 9 II. Microscopy 1 Setup Center the illumination lamp For an illumination system using a mercury lamp or a xenon lamp, the lamp must be centered each time lamps are exchanged. Mount the centering tool on the nosepiece, and then turn on the illumination lamp. Adjust the lamp center by observing through the window of the centering tool. For details, see the instruction manual for the light source (super high-pressure mercury lamp power supply or high-intensity light source). • Mount the centering tool on the nosepiece. Remove an objective from the nosepiece, then screw on the centering tool in its place. • The dia-illuminator can be tilted and the condenser lens can be removed so that you can completely observe through the window of the centering tool. • Set the W-TIRF slider to the aperture diaphragm side (toward the front), and then set the aperture diaphragm to fully open. Diopter and interpupillary distance adjustment Adjust the diopter and the interpulillary distance correctly by referring to the instruction manual of the microscope. 10 II. Microscopy 2 TIRF microscopy White light from the illumination system is passed through a special arc shape diaphragm, the W-TIRF diaphragm, built into the W-TIRF slider. And the light is condensed on the pupil surface of the objective by the condenser lens. As a result, the emitted illumination light from the objective becomes parallel light. As this parallel light is moved to the edge of the objective pupil, the illumination light from the objective tilts. When the optical path tilts more than the critical angle, it becomes a total internal reflection condition (TIRF illumination condition). Sequence of microscopy To perform the TIRF microscopy by using the white TIRF illuminator, operate as follows: Caution To perform the TIRF microscopy by using the white TIRF illuminator, operate as follows: • Turn off the diascopic illumination lamp of the microscope main unit. (Turn off the dia-illumination ON/OFF switch, or turn off the power switch on the power supply.) • If DIC attachments are attached to the microscope, remove the analyzer and DIC prism for the objective out of the optical path. • Check that the W-TIRF diaphragm in the W-TIRF slider is suitable for the objective. • Set the optical axis switch lever of the W-TIRF slider to match the objective. Focal point adjustment The focal point of the illumination light must be adjusted each time objectives are changed. 1 Set the aperture diaphragm into the optical path. 2 Select the excitation method. Pull the W-TIRF slider to set the aperture diaphragm for Epi-fl microscopy into the optical path. (p.17) Rotate the excitation method changeover ring to insert the filter cube of the desired excitation method into the optical path. (p.24) To use the excitation filter in the excitation filter slider, move it into the optical path. 3 Set the objective into the optical path. 4 Remove shutters from the optical path. 5 Focus on the surface of the cover glass (the contact side with the specimen) with the epi-fl microscopy. Rotate the nosepiece to set the desired objective into the optical path. (See the instruction manual for the microscope.) Two shutters are equipped on the system, one on the white TIRF illuminator and another on the cassette holder. • Pull the shutter slider of the white TIRF illuminator toward the front side. (p.23) • Move the shutter lever to the “O” (Open) position. (p.24) 11 II. Microscopy 6 Tilt the dia-illuminator of the microscope, and project the illumination light onto the ceiling of the room or the chamber. If you cannot see the illumination light, place a paper or so on above the specimen (about 20 cm to 30 cm height) and project the illumination light. 7 Adjust the focal point adjustment knob to focus the projected aperture diaphragm image. Reduce the aperture diaphragm opening. Then, adjust the focal point adjustment knob to get the best image of the aperture diaphragm edge projected on the ceiling (or on the paper). Lock the condenser lens locking screw after the adjustment to fix the condenser lens. (p.18) Centering of W-TIRF diaphragm 8 Set the W-TIRF diaphragm into the optical path. 9 Set the centering tool into the optical path. Push the W-TIRF slider toward the far side to set the W-TIRF diaphragm of the TIRF microscopy into the optical path. (p.17) Rotate the nosepiece to set the centering tool into the optical path. (See the instruction manual for the microscope.) The shape of the illumination light from the W-TIRF diaphragm is projected on the centering tool window. the W-TIRF diaphragm. 10 Center Turn the two centering adjustment knobs for the W-TIRF diaphragm on the near side of the W-TIRF slider to overlay the arc shape illumination light of the centering tool window on the index position. (p.21) the shutters to block the illumination light. 11 Close Close the shutters to block the illumination light for a while after the completion of these steps. (p.23, p.24) TIRF microscopy It is recommended that before performing TIRF microscopy, observe the specimen by Epi-fl microscopy procedure in chapter 3., find out the object, and then bring the object into focus. the objective for TIRF microscopy into the optical path. 12 Set Use oil immersion type objectives for the TIRF microscopy. It is recommended to use dedicated objectives for the TIRF microscopy. (See the instruction manual for the microscope.) the shutters from the optical path, and then perform the TIRF microscopy. 13 Remove (p.23, p.24) the angle of the illumination light to get a better image. 14 Adjust The knob on the W-TIRF slider front side of the two centering adjustment knobs for the W-TIRF diaphragm can be used to adjust the angle of the illumination light. The TIRF illumination condition can be changed. (p.21) 15 Adjust the field diaphragm if necessary. the shutters to block the illumination light. 16 Close (p.23, p.24) 12 II. Microscopy 3 Epi-fl microscopy Important When the Epi-fl microscopy is performed to bring the specimen into focus for the TIRF microscopy, the following are important. The focal point adjustment of the TIRF illumination and the centering of the TIRF diaphragm must be completed beforehand to switch the microscopy method to the TIRF microscopy after the focusing. For these adjustments, see “2. TIRF microscopy.” • Turn off the diascopic illumination lamp of the microscope main unit. (Turn off the dia-illumination ON/OFF switch, or turn off the power switch on the power supply.) • If DIC attachments are attached to the microscope, remove the analyzer and DIC prism for the objective out of the optical path. Sequence of microscopy 1 Set the aperture diaphragm into the optical path. 2 Select the excitation method. Pull the W-TIRF slider to set the aperture diaphragm for Epi-fl microscopy into the optical path. (p.17) Rotate the excitation method changeover ring to insert the filter cube of the desired excitation method into the optical path. (p.24) To use the excitation filter in the excitation filter slider, move it into the optical path. 3 Remove shutters from the optical path. Two shutters are equipped on the system, one on the white TIRF illuminator and another on the cassette holder. • Pull the shutter slider of the white TIRF illuminator toward the front side. (p.23) • Move the shutter lever to the “O” (Open) position. (p.24) 4 Focus on the specimen. 5 Center the field diaphragm. 6 Switch to any desired objective. Place the specimen on the stage and focus with the 10x objective. (See the instruction manual for the microscope.) (p.19) Switch to any desired objective and view the specimen. • Readjust the focus. • Use ND filters to adjust the brightness. (p.22) • Close the field diaphragm so that it is just outside the viewfield. (p.19) • Usually, the aperture diaphragm is left fully open. (p.20) • When using an oil-immersion type objective, apply immersion oil between the specimen and the objective. (p.27) • Place the shutters into the optical path whenever you temporarily halt Epi-fl microscopy. (p.23, p,24) 13 II. Microscopy Combining Epi-fl microscopy with DIC microscopy Even when the white TIRF illuminator is installed, DIC (differential interference contrast) microscopy can be performed by combining various accessories. DIC microscopy can be used in place of Epi-fl microscopy to find target objects in order to prevent color fading of the specimen, or Epi-fl and DIC microscopy can be used simultaneously to utilize the characteristics of both microscopies. For details on DIC microscopy procedures, see the manual for the DIC attachments. Accessories required for DIC microscopy • • • • • T-ND6 sextuple DIC nosepiece DIC prism for objective DIC objective T-A DIC analyzer TE2000 system condenser Condenser lens DIC condenser cassette Condenser turret (or condenser slider) Other required parts • T-P DIC polarizer Sequence of microscopy 1 Install the accessories. For details, see the manual for the TE2000 DIC attachment. 2 Place the shutter into the optical path to block the white TIRF illuminator optical path. 3 Rotate the excitation method changeover ring to move the empty position into the optical path. 4 Turn on the power supply, turn on the dia-illumination ON/OFF switch on the microscope main body, and light the diascopic lamp. 5 Bring the empty hole of the analyzer and that of the polarizer into the optical path. 6 Turn the condenser turret so that the “A (open)” indication is in front. When using a condenser slider, remove the condenser cassette on the optical path to open the optical path. 7 Move the DIC objective into the optical path. 8 Focus on the specimen. 9 Center and focus the condenser. 10 Place the polarizer and analyzer into the optical path. 11 Adjust the polarizer azimuth. 12 Operate the condenser to move the condenser cassette with the same indication (L, M, or H) as the objective’s DIC code (DIC L, DIC M, or DIC H) into the optical path. 13 Adjust the aperture and field diaphragms of the diascopic illumination. When performing Epi-fl and DIC microscopy simultaneously, move the desired filter cube into the optical path, and remove the episcopic shutter out of the optical path. Adjust the ND filters and aperture diaphragm on the white TIRF illuminator, and the ND filters on the microscope to balance the brightness of the fluorescent and differential interference contrast images. 14 II. Microscopy Combining Epi-fl microscopy with Ph microscopy Even when the white TIRF illuminator is installed, Ph (phase contrast) microscopy can be performed by combining various accessories. Ph microscopy can be used in place of Epi-fl microscopy to find target objects in order to prevent color fading of the specimen, or Epi-fl and Ph microscopy can be used simultaneously to utilize the characteristics of both microscopies. For details on Ph microscopy procedures, see the manual for the microscope. Accessories required for Ph microscopy • Ph objective • TE2000 system condenser housing a Ph module or ELWD-S/SLWD condenser (SLWD condenser is used only for T-DS dia-illuminator 30W.) • Centering telescope Sequence of microscopy 1 Install the accessories. For details, see the manual for the microscope. 2 Place the shutter into the optical path to block the white TIRF illuminator optical path. 3 Rotate the excitation method changeover ring to move the empty position into the optical path. 4 Turn on the power supply, turn on the dia-illumination ON/OFF switch on the microscope main body, and light the diascopic lamp. 5 Move the Ph objective into the optical path. 6 Set the optical path of the condenser to “A (open).” 7 Focus on the specimen. 8 Center and focus the condenser. 9 Set the optical path of the condenser to the same Ph code (Ph1, Ph2, or Ph3) as the objective. 10 Center the annular diaphragm of the condenser. The centering method differs according to the type of condensers. See the manual for the microscope. 11 Adjust the field diaphragm. When performing Epi-fl and Ph microscopy simultaneously, move the desired filter cube into the optical path, and remove the episcopic shutter out of the optical path. Insert and remove the ND filters on the white TIRF illuminator, and the ND filters on the microscope to balance the brightness of the fluorescent and phase contrast images. 15 II. Microscopy 4 General bright-field microscopy Even when the white TIRF illuminator is installed, general bright-field microscopy can be performed as a microscope for normal bright-field microscopy. 1 2 3 4 5 Place the shutter of the cassette holder or the white TIRF illuminator into the optical path to block the white TIRF illuminator optical path. Rotate the excitation method changeover ring of the cassette holder to move the empty position into the optical path. Turn on the power supply, turn on the dia-illumination ON/OFF switch on the microscope main body, and light the diascopic lamp. Focus on the specimen. Focus and center the condenser. Adjust the condenser aperture diaphragm and the field diaphragm on the microscope main body. For details, see the manual for the microscope. 16 III 1 Operation of Each Part White TIRF illuminator Connector for lamp housing ND filter x 2 Condenser lens position indices Condenser lens locking screw Focal point adjustment knob Field diaphragm W-TIRF open/close lever, slider field diaphragm centering adjustment screw Excitation filter slider Shutter slider Switching between aperture diaphragm and W-TIRF diaphragm A W-TIRF diaphragm for the TIRF microscopy and an aperture diaphragm for the Epi-fl microscopy are built into the W-TIRF slider. The TIRF microscopy and the Epi-fl microscopy are selectable by sliding the W-TIRF slider to and fro. W-TIRF slider operation Push: TIRF microscopy (W-TIRF diaphragm) • For TIRF microscopy: Push the W-TIRF slider toward the far side to set the W-TIRF diaphragm of the TIRF microscopy into the optical path. • For Epi-fl microscopy: Pull the W-TIRF slider to set the aperture diaphragm for Epi-fl microscopy into the optical path. Pull: Epi-fl microscopy (aperture diaphragm) Important To switch the objective between 60x and 100x for the TIRF microscopy, the W-TIRF diaphragm in the W-TIRF slider must be replaced to match the objective. For details about the replacement method, see “Assembling and attaching the W-TIRF slider” of chapter “VI. Assembly.” 17 III. Operation of Each Part Condenser lens operation The objective optical axis direction focal point of the illumination light can be changed by moving the focal point adjustment knob to and fro. This adjustment enables the Koehler illumination with the epi-fl microscopy. The illumination light is condensed on the pupil surface of the objective. As a result, the emitted illumination light from the objective becomes parallel light. But if the illumination light is not parallel, the light passes through the cover glass, even the optical axis tilts more than the critical angle, and that adversely affects the view of image and causes the damage of specimen. Condenser lens position adjustment Adjust the focal point of the illumination light again when objectives are switched. 1 Focus on the specimen with the epi-fl microscopy. To perform the TIRF microscopy, focus on the surface of the cover glass (the contact side with the specimen). 2 Condenser lens position indices and objective Indices of the focal point adjustment knob position (circumference groove) for objectives of the TIRF microscopy are located near the focal point adjustment knob. Use them as a guideline to adjust the focal point. 5 4 3 2 1 auxuliary Tilt the dia-illuminator of the microscope. Remove all ND filters. And then, project the illumination light onto the ceiling of the room or the chamber. If you cannot see the illumination light, place a paper or so on above the specimen (about 20 cm to 30 cm height) and project the illumination light. 3 Release the condenser lens locking screw by using the Allen wrench attached the microscope. 4 Reduce the aperture diaphragm opening. Then, move the focal point adjustment knob to and fro to focus the aperture diaphragm image projected on the ceiling (or on the paper). Adjust the knob to get the best image of the aperture diaphragm edge by using the condenser lens position indices as a guideline. (See the figure at right.) Focal point adjustment knob 1 100x / 1.4VC Plan Apo VC 100 x H 2 60x / 1.4VC Plan Apo VC 60 x H 3 60x / 1.45 Plan Apo TIRF 60 x H 4 100x / 1.45 & 1.4 Plan Apo TIRF100×H Plan Apo 100×H 5 60x / 1.4 Plan Apo 60 x H With this adjustment, the aperture diaphragm is imaged on the objective pupil position. 5 Lock the condenser lens locking screw after the adjustment to fix the condenser lens. WARNING Never attempt to look into the illumination light. When adjusting the focal point adjustment knob or the condenser lens locking screw, never attempt to look into the objective or the areas near the objective from above or below the stage as the lens emits a powerful illumination light including ultraviolet radiation toward the top and bottom sides of the stage. And never place or mount any reflective materials on the stage, in the room, or on the ceiling of the room or the chamber to protect your eyes and skin from exposure to illumination light reflections from the objective. 18 III. Operation of Each Part Field diaphragm operation The field diaphragm restricts illumination to the area of the specimen that is being viewed. Operating the field diaphragm open/close lever changes the size of the field diaphragm. For normal observation, the size of the diaphragm should be such that it is just outside (or inside) the edge of the viewfield. If a broader area than necessary is illuminated, stray light will enter the optical system, creating flaring, reducing the contrast of the optical image, and expanding the area of color fading of the specimen. The operation of the field diaphragm is especially important in videomicroscopy or photomicrography; generally, the best results are obtained by stopping down the field diaphragm to just slightly larger than the image area or the area that will be reproduced on the film, that is, the size of the picture composition frame. Centering the field diaphragm Field diaphragm centering screw Field diaphragm open/close lever Field diaphragm centering screw Field diaphragm image The field diaphragm of this system needs to be centered before usage. Centering the field diaphragm 1 Perform steps 1 to 4 in “3 Epi-fl microscopy” of chapter “II. Microscopy.” 2 Reduce the field diaphragm opening. (Push down the field diaphragm open/close lever.) 3 Move the center of the field diaphragm image to the center of the viewfield. (Turn the field diaphragm centering screws.) 4 Expand the field diaphragm opening as wide as the viewfield. (Pull up the field diaphragm open/close lever.) 5 Once again, move the center of the field diaphragm image to the center of the viewfield. (Turn the field diaphragm centering screws.) 19 Eyepiece viewfield III. Operation of Each Part Aperture diaphragm operation The aperture diaphragm is used to adjust the numerical aperture of an illumination optical system. When using an episcopic-fluorescent illumination optical system, the aperture diaphragm functions to adjust the brightness of the image and the size of stray light. Although the amount of stray light decreases when the aperture diaphragm is stopped down, the image of the specimen also becomes darker. Although the image becomes brighter when the aperture diaphragm is opened, the amount of stray light may also increase. It is possible to effectively eliminate stray light and secure a bright specimen image by stopping down the aperture diaphragm to a size equivalent to the diameter of the objective pupil. Centering the aperture diaphragm Front Back Aperture diaphragm open/close lever Aperture diaphragm centering screw Aperture diaphragm image Centering the aperture diaphragm 1 Perform steps 1 to 3 in “3 Epi-fl microscopy” of chapter “II. Microscopy.” 2 Reduce the aperture diaphragm opening. (Push in the aperture diaphragm open/close lever.) 3 Move the center of the aperture diaphragm to the center of the centering tool window. (Turn the aperture diaphragm centering screws.) 4 Expand the aperture diaphragm opening as wide as the centering tool window. (Pull out the aperture diaphragm open/close lever.) 5 Once again, move the center of the aperture diaphragm to the center of the centering tool window. (Turn the aperture diaphragm centering screws.) 20 Centering tool window III. Operation of Each Part TIRF diaphragm operation The W-TIRF diaphragm is used to stop down the white light of the mercury lamp at the pupil of objective and has a special arc shape slit. Centering of W-TIRF diaphragm W-TIRF diaphragm centering screw (up-and-down adjustment) Two W-TIRF diaphragms, one for a 60 x objective and another for a 100 x objective, are attached to this system as accessories. Select the appropriate diaphragm for the magnification of the objective to be used and set it into the W-TIRF slider. For the exchange of W-TIRF diaphragms, see “Assembling and attaching the W-TIRF slider” of chapter “IV. Assembly.” Centering of W-TIRF diaphragm 1 Perform steps 1 to 7 in “2 TIRF microscopy” of chapter “II. Microscopy.” After the completion of the position adjustment of the condenser lens, perform the W-TIRF diaphragm centering. 2 Move the W-TIRF diaphragm into the optical path. (Pull out the W-TIRF slider.) 3 Set the centering tool into the optical path. (Rotate the nosepiece.) 4 Adjust the position of the W-TIRF diaphragm to overlay the arc shape illumination light on the index position of the centering tool window. (Turn the W-TIRF diaphragm centering screws.) W-TIRF diaphragm centering screw (right-and-left adjustment) Illumination light Centering tool window Index Index (for a 60 x objective) (for a 100 x objective) Overlay the illumination light and the index. Optical axis shift amount switch lever operation On this system, in addition to switching the W-TIRF diaphragm to match the objective magnification enabling the TIRF microscopy for both the 60 x objective and the 100 x objective, the optical axis can be shifted to get better brightness. Optical axis shift amount switch lever operation • The optical axis shift amount switch lever is used only for the TIRF microscopy. Switching optical axis shift amount 1 Release the locking screw. 2 Slide the switch lever to the 60 x side or 100 x side to meet the objective. 3 Tighten the locking screw. If the optical axis shift amount switch lever is fixed at the halfway position, the illumination light is shifted from the normal optical axis. To use the optical axis under the shifted condition purposefully, tighten the locking screw at that condition. 21 Optical axis shift amount switch lever Locking screw III. Operation of Each Part ND filter operation An ND filter reduces illumination without changing the color balance of the light. When using strong fluorescent light, or when a specimen is badly faded, adjust the brightness by pushing in the ND filter sliders to place the ND filters into the optical path. (If the fluorescent light is too strong, the contrast may worsen.) The following chart shows how brightness is changed by different combinations of ND filters. ND4 ND8 Brightness OUT OUT 1 IN OUT 1/4 OUT IN 1/8 IN IN 1/32 IN : In optical path OUT : Not in optical path Replacing filters Pull the tab on the filter frame toward the outside and drop in the ND filter that matches the indication on the filter frame. Use a soft clean cloth to handle filters so that your fingers do not directly touch the filter frame. Excitation filter operation An excitation filter passes a certain wavelength of white light emitted from mercury lamps. This filter is used for the Epi-fl microscopy and the TIRF microscopy. Conventionally, this filter is attached in a filter cube to use. But in this system, excitation filters can be attached into the excitation filter slider to switch them by manual operation. To switch excitation filters, pull the slider to one of three click-stop positions, and then set the filter. When the slider is pushed into the far side, no filter is in the optical path. CAUTION Whenever using excitation filters on the excitation filter slider to perform microscopy, be sure to check the barrier filter settings. If the combination of the excitation filters and barrier filters are not correct, very strong light may pass through. Great care should be taken about filter combinations. Up to three excitation filters can be set in the excitation filter slider. Replacing filters 1 Stopper screws are attached on both sides of the lower surface of the excitation filter slider. Remove the far side stopper screw. And pull out the slider from the system. 2 Rotate the filter holding ring and remove it. Attach the desired filter, and fix it by screwing the filter holding ring. 3 Insert the excitation filter slider into the slit and put the stopper screw back to its original place. 22 III. Operation of Each Part Shutter operation The shutter blocks the illumination light. In order to prevent fading of the specimen, always place the shutter in the optical path when not actually performing the microscopy. Make closing the shutter a practice in order to protect important specimens. In addition, when temporarily halting the TIRF microscopy or Epi-fl microscopy and performing an observation using the diascopic light, do not forget to insert the shutter into the optical path and block the illumination light of this system. This system is equipped with two shutters, one at the shutter lever of the cassette holder and the other at the shutter slider of the white TIRF illuminator. • Shutter lever of cassette holder Moving the shutter lever clockwise places the shutter into the optical path, blocking the light. • Shutter slider of white TIRF illuminator Pushing in the shutter slider places the shutter into the optical path, blocking the light. Connector for lamp housing Turn the bayonet ring to connect the lamp housing. Super high-pressure mercury lamps are available for the light source. Zoom adapters are not available. Centering tool This tool is mounted on the nosepiece for operation. And it is used to center the white TIRF illuminator lamp, aperture diaphragm, field diaphragm, and W-TIRF diaphragm. Ultraviolet light shield The ultraviolet light shield prevents the ultraviolet light (irradiated from the objective to the specimen) from reflecting back and entering the observer’s eyes. To remove this shield, loosen the fixing screws, and then pull off the shield. 23 III. Operation of Each Part 2 Cassette holder In the cassette holder, the illumination light from the white TIRF illuminator is reflected to the objective. Up to six filter cubes with dichroic mirrors can be set in the cassette holder. And filter cubes are selectable by rotating the excitation method changeover ring. One of the three cassette holders below can be used with this system. T-FLC cassette holder This cassette holder is used by the manual operation. T-FLC-E motorized cassette holder This cassette holder is moved by the motor drive. The T-HUBC HUB controller is required. T-FLC-HQ cassette holder This cassette holder is used by the manual operation. Three exclusive high-precision filter cubes are attached as accessories. Excitation method changeover ring *1 (Operation part to change filter cubes)） Filter cube port (and cover) Front side Cassette holder Address and name of the filter cube in the optical path *1 Shutter lever (O: open, C: close) ※1 ：T-FLC-E motorized cassette holder does not have these parts. Filter cube port and cover Remove the cover to replace filter cubes. Excitation method changeover ring Filter cubes can be switched by turning the excitation method changeover ring. Address and name of the filter cube in the optical path The address and name of the filter cube in the optical path are displayed in this window. Shutter lever Moving the shutter lever clockwise places the shutter into the optical path, blocking the illumination light. 24 III. Operation of Each Part 3 Filter cube Filter cubes consist of three types of optical components: an excitation filter (EX filter), a barrier filter (BA filter), and a dichroic mirror (DM). Using the items below as a guide, select a combination of filters that is suited to your purpose and to the characteristics of the specimen and the fluorescent stain. Filter cube Barrier filter (BA filter) • You can select different combinations of excitation and barrier filters for the same excitation method. Dichroic mirror (DM) • Excitation filters, barrier filters, and dichroic mirrors can all be purchased individually. Excitation filter (EX filter) • Excitation filters will deteriorate over time since they are exposed to intense light. Replace them as necessary. • Three exclusive high-precision filter cubes are attached as accessories for the T-FLC-HQ cassette holder. Filters and dichroic mirrors are not included with the exclusive high-precision filter. Use a suitable filter and dichroic mirror. Important The exclusive high-precision filter cube, which is attached to the T-FLC-HQ cassette holder, is adjusted to match the turret address. Make sure to attach the exclusive high-precision filter cube by matching the inscribed address. 1. Selecting excitation filters (EX filters) The range of wavelengths that a given filter passes is called the “bandwidth” of the filter. An excitation filter’s bandwidth determines the brightness of the fluorescent image, the occurrence of self-fluorescence (fluorescence originating from materials other than the fluorescent stain), and the extent of fading. A wide bandwidth allows a high level of excitation light to illuminate the specimen, producing a brighter image. However, a wide bandwidth also leads to a high level of self-fluorescence and severe fading. Conversely, while a narrow bandwidth yields a dark image, since little excitation light reaches the specimen, self-fluorescence and fading are minimal. Excitation filter (EX filter) Spectral transmission Excitation filters selectively pass the light within a certain range of wavelengths needed to cause the specimen to fluoresce (excitation light) and filter out all other light. EX filter Bandwidth 0 Wavelength When self-fluorescence is pronounced, use an excitation filter with a narrow bandwidth. (The resulting fluorescent image will be darker, however.) Excitation filters are likely to deteriorate the more they are used, since they are exposed to intense light. Replace excitation filters as necessary. Excitation filter bandwidth Narrow Wide Brightness of fluorescent image Dark Wide Occurrence of self-fluorescence Minimal pronounced Pronounced Extent of fading Minimal pronounced Pronounced 25 III. Operation of Each Part 2. Selecting barrier filters (BA filters) A barrier filter allows only fluorescent light generated by the specimen to pass and blocks all other excitation light reflected from the specimen. This filter makes it possible to observe the fluorescent image without unnecessary light (that is, on a dark background). There are two types of barrier filters: LP filters (long-pass filters), which block all wavelengths that are shorter than a certain wavelength and allow all wavelengths to pass that are longer than the boundary wavelength, and BP filters (band-pass filters), which allow only light in a certain range of wavelengths to pass. Use whichever type best suits your purpose. LP filters (long-pass filters) LP filters block all wavelengths that are shorter than a certain wavelength and allow all wavelengths to pass that are longer than the boundary wavelength. The boundary wavelength is called the cut-on wavelength. When the specimen is stained with a fluorescent color for which the fluorescent light wavelength band and the excitation wavelength band (the light that is absorbed by the specimen in order to fluoresce) are extremely close together, the fluorescent light can generally be seen most effectively if a barrier filter with a “cut-on” wavelength that is comparatively short, within the range permitted by performance considerations, is selected. As a general rule, the longer the cut-on wavelength is, the more complete the separation between the excitation light and fluorescent light, and the darker the background of the fluorescent image becomes. Recently, however, because of improved filter performance, it is becoming increasingly common to use barrier filters with shorter cut-on wavelengths. 2. To view the fluorescent images of all the colors in a specimen stained in multiple colors, use an LP filter. However, when using a normal dichroic mirror, an excitation filter, and an LP filter-type barrier filter in combination, the stain that fluoresces at the longer wavelength (for example, TRITC when the specimen is stained with FITC and TRITC) may not be excited sufficiently, with the result that the fluorescent image created by the stain may appear extremely dark. In this type of situation, the use of a multi-band filter is recommended. LP filter (long-pass filter) Spectral transmission 1. FITC fluorescent wavelength band LP520 TRITC fluorescent wavelength band Wavelength Both the FITC fluorescent image and the TRITC fluorescent image are visible. BP filters (band-pass filters) This type of filter is used to view the fluorescent image created by a specific stain when a given specimen has multiple stains. (For example, in a specimen with two stains, FITC and TRITC, select BA520-560 to observe the fluorescent image created by FITC.) However, you may not be able to distinguish the self-fluorescence from the other fluorescence in the image created by the BP filter since the image will only be of one color (green, in the above example). It is best to use an LP filter if you wish to distinguish the self-fluorescence from subtle differences in hue. 26 BP filter (band-pass filter) FITC fluorescent wavelength band BA520-560 (BP type) Spectral transmission A BP filter allows only light in a certain range of wavelengths to pass. TRITC fluorescent wavelength band Wavelength Only the fluorescent image created by FITC is visible. III. Operation of Each Part 3. Excitation filter and barrier filter replacement Excitation and barrier filters can be removed from the filter cube and replaced with other filters. (The filters are screwed into the filter cube.) For the exclusive high-precision filter cube, the excitation filter is screwed into the cube and the barrier filter is fastened to the cube by screws. When changing or attaching a filter, check the direction of the filter. 4 Oil-immersion type objectives Objectives marked “oil” are oil-immersion type objectives. These objectives are used with immersion oil applied between the specimen and the tip of the objective. Always use non-fluorescent oil. (For example, Nikon Immersion Oil DF.) If other kinds of oil are used, fluorescent light from the oil may adversely affect the image. Bubbles in the oil will adversely affect the viewing of the image. Be careful to prevent the formation of air bubbles. To check for air bubbles, look at the exit pupil of the objective. (When using the T-TD B eyepiece tube D, place the Bertrand lens in the optical path by rotating the eyepiece tube turret to the “B” position. And focus on the exit pupil of the objective by rotating the Bertrand lens focusing screw. When using the T-TS B eyepiece tube S, insert the centering telescope instead of an eyepiece, and rotate the eyepiece part of the centering telescope to focus on the exit pupil of the objective.) If there are bubbles in the oil, remove them by one of the following methods: • Turn the revolving nosepiece slightly, moving the objective in question back and forth one or two times. • Add more oil. • Remove the oil and replace it with new oil. Use as little oil as possible (just enough to fill the space between the tip of the objective and the specimen). If too much oil is applied, the excess oil will flow onto the stage and around the condenser. Any oil remaining on oil-immersion type objectives or on the tip of dry-type objectives will have a discernible, negative effect on the image. After using oil, wipe all of it away, and also make sure that there is no oil on the tips of the other objectives. Oil on the condenser lens should also be wiped away carefully after use. Use petroleum benzene to wipe away immersion oil. After wiping off with petroleum benzene, wipe with absolute alcohol (ethyl or methyl) to make the surface clearer. If you cannot obtain petroleum benzene, use methyl alcohol. However, methyl alcohol does not clean as well as petroleum benzene, it will be necessary to wipe the surface repeatedly. (Usually, three or four times are sufficient to clean the lenses.) Use petroleum benzene only to remove immersion oil from the tips of objectives; do not use it for cleaning the fluorescent filters, and such. Follow the instructions provided by the manufacturer when using absolute alcohol. 27 III. Operation of Each Part 5 Fluorescent photomicrography For the basic procedures and key points of photomicrography, see the manual provided with the photomicrographic equipment. Please note, however, that when using a fluorescent specimen, the fluorescence may fade during exposure. Take the following countermeasures in order to avoid this problem: 1 Use high-sensitivity film Use “Tri-X (ISO400)” for monochrome photomicrography. For color shots, use daylight-type high-sensitivity film, such as “Kodak Ektachrome 400 (ISO400)” or “Fujichrome 400 (ISO400).” 2 Creating a bright optical system combination Even if the total magnification on the film is the same, the exposure time can vary greatly for different combinations of objectives and projection lenses. Rather than increasing the magnification of the projection lens, increasing the magnification of the objective is recommended. (This is because. in general, the numerical aperture of the objective increases as the magnification increases, and the higher the numerical aperture, the brighter the image.) 3 Adjusting the excitation light If the excitation light is too bright, the specimen will fade quickly, making it impossible to get a good shot of the fluorescent image. Therefore, adjust the brightness by inserting ND filters into the optical path. 4 Specimen If a faded portion of a specimen is shot, the exposure time increases, the color reproduction is poor, and the resulting photomicrograph will not be satisfactory. Move the specimen and shoot a more vivid portion of the specimen that has not previously been exposed to the excitation light. We recommend using the DIC method or the phase contrast method to select the portion to be shot, and then switching to the Epi-fl method for shooting the actual photomicrograph. 6 TV microscopy When performing microscopy using a high-sensitivity TV camera, it is sometimes best to insert an infrared (IR) cut filter in front of the camera receptor. Experiment, and use the IR cut filter when needed. 28 IV Assembly The procedures for assembling the system are described in this chapter. For details on the assembly, handling, and usage of the microscope, super high-pressure mercury lamp, etc., see their respective manuals. WARNING Before using the system, be sure to read the “WARNING” and “CAUTION” at the beginning of this manual, and also the section entitled, “Notes on Handling the System.” Be certain to heed all of the warnings and cautions. Also be sure to read the manuals for any other products that you are using with this system (the microscope, power supply, super high-pressure mercury lamp power supply, high-intensity light source, etc.), and heed all of the warnings and cautions in those manuals. In particular, mishandling a mercury lamp used with this system can lead to a serious accident. Exercise caution. In order to prevent electric shock, fire, accidents involving ultraviolet light, burns, and other injuries, make sure that the power switches for the system and super high-pressure mercury lamp (or high-intensity light source) power supplies are turned off before beginning assembly work. Required tools • Hexagonal screwdriver (2 mm) : 1 (provided with the microscope) • Hexagonal wrench: 1 (provided with the cassette holder) See the illustrations while assembling the system. If the microscope is already fully assembled, remove the dia-illuminator, stage, objectives, and revolving nosepiece. Scratches or fingerprints on the lenses and filters will adversely affect the image. Handle these components carefully in order to keep them free from scratches and fingerprints. 29 IV. Assembly Assembling the white TIRF illuminator 1 2 Use a hexagonal driver to remove the two screws that secure the black cover in the middle of the microscope. Removing the protective caps Remove the protective caps. Remove the two protective caps. Remove the black cover. 3 Insert the white TIRF illuminator into the guides on the back of the microscope and secure by tightening the clamp screws with a hexagonal driver. 4 Attach the protective ring to the end of the white TIRF illuminator (revolving nosepiece side). Attaching the white TIRF illuminator Clamp screw 5 Attach the support pillar into the bottom of the white TIRF illuminator. Attaching the support pillar Loosen the clamp screw securing the support pillar so that the sliding part at the end slides freely. Screw the support pillar into the tapped hole on the bottom of the white TIRF illuminator and secure the end to the installation surface by tightening the clamp screw with a hexagonal driver. Clamp screw 30 IV. Assembly Assembling and attaching the W-TIRF slider 1 Insert the TIRF diaphragm into the mounting hole on the W-TIRF slider. W-TIRF slider Pin W-TIRF diaphragm Attach the W-TIRF diaphragm suitable for the magnification of objectives (60 x or 100 x) to be used for the TIRF microscopy. When the W-TIRF diaphragm centering screw is pushed down, the W-TIRF diaphragm can be attached and detached. There is a notch on the TIRF diaphragm for positioning. Align the notch to the pin position of the mounting hole on the W-TIRF slider and insert the W-TIRF diaphragm into the mounting hole. 2 Insert the W-TIRF slider into the inserting hole of the white TIRF illuminator, and put the stopper screw on the lower far side of the slider to prevent loosening. Stopper screw (lower far side) Push down W-TIRF diaphragm Align the notch of the W-TIRF diaphragm to the pin and insert the diaphragm into the mounting hole. When the stopper screw has been attached to the T-TIRF slider already, remove it temporarily then insert the W-TIRF slider. Attaching filters Attach ND filter sliders (two pieces), an excitation filter slider, and a shutter slider. 1 To set an excitation filter to the excitation filter slider, remove the holding ring of the mounting hole, set the excitation filter, then attach the holding ring and tighten it. Up to three filters with a diameter of 25 mm and a maximum thickness of 5.5 mm can be attached to the excitation filter slider. 2 Insert all sliders into appropriate inserting holes of the white TIRF illuminator, and put stopper screws on the lower far side of the sliders. When far side stopper screws have been attached to sliders already, remove them temporarily, insert sliders, and put stopper screws to their original places. Excitation filter slider Excitation filter Stopper screw (far side) Stopper screw (near side) Slider positions ND filter sliders CAUTION Whenever using excitation filters on the excitation filter slider to perform microscopy, be sure to check the barrier filter settings. If the combinations of excitation filters and barrier filters are not correct, very strong light may pass through. Great care should be taken about filter combinations. 31 Excitation filter Shutter slider slider IV. Assembly Attaching the cassette holder Attach the cassette holder to the microscope. Cassette holder Insert the cassette holder along the attachment groove from the right of the microscope and secure with screws on the left and right. Use the provided hexagonal wrench. Note that the screws should be tightened after the filter cube port cover is removed.） Cassette holder Attaching filter cubes Up to six filter cubes can be set in the cassette holder. Before exchanging filter cubes, make sure to check that the illumination lamp has been turned off. 1 2 Remove the filter cube port cover, check the turret address, and insert the filter cube along the guide groove. Rotate the excitation method changeover ring to display the same address, and affix an ID sticker that matches the filter cube type. Attaching filter cubes Indication of address Excitation method on the optical path (position for ID sticker) changeover ring Affix “ ” or “ ” (a blank sticker that can be written on freely) for spaces in which no filter block is inserted. 3 Reinstall the filter cube port cover by placing it into position. Important • Three exclusive high-precision filter cubes are attached as accessories for the T-FLC-HQ cassette holder. These filter cubes are adjusted to match the turret address of the cassette holder. Make sure to attach the exclusive high-precision filter cube by matching the inscribed address. • When an excitation filter is used with the excitation filter slider, do not set excitation filters in the filter cube. 32 Indication of address Filter cube Excitation filter Set the filter cube with its excitation filter facing outside. (Do not set it in opposite way.) IV. Assembly Microscope assembly Follow the instructions in the microscope manual. Installing the ultraviolet light shield Attach the ultraviolet light shield to the eyepiece tube using screws. Installing the light source Install the light source on the bayonet ring of the connector for lamp housing. Be sure to attach the collector lens in between. For details see the manual provided for either the super high-pressure mercury lamp power supply or the high-intensity light source. Now the assembly is completed. 33 V Troubleshooting Improper use of the microscope may adversely affect performance, even if the microscope is not damaged. If any of the problems listed in the table below arise, take the countermeasures indicated. Also refer to the instruction manual supplied with the microscope, light source and the DIC attachment (If DIC attachment is also used). Epi-fl microscopy Problem Lamp does not light. Even though the lamp is on, the image is not visible. Cause Countermeasure The power supply is not plugged in. Plug the power cord into an outlet. The lamp connector is not connected to the power supply. Connect the lamp connector to the power supply. The lamp has reached the end of its operational life. Replace the lamp. The fuse is blown. If the fuse can be replaced, replace it. Otherwise, contact your nearest Nikon representative. The shutter is in the optical path. Remove shutters from the optical path. The filter cube has stopped at an intermediate position. Rotate the excitation method changeover ring to the clickstop position. The filter cube selection is incorrect. Use a filter block with a correct combination. The combination of excitation filter, barrier Use a filter cube with a correct filter and dichroic mirror is inappropriate, combination. or one of these components is missing. The setting is still for TIRF microscopy. Pull the W-TIRF slider to set the aperture diaphragm into the optical path. Point the optical path at the binocular The microscope’s optical path is not set to eyepiece with the optical path switch knob “Observation.” or LIGHT PATH switch. Even though the lamp is on, the image is extremely dark. The illumination light is leaking over to the observation side. The light source is not centered properly. Center the lamp. Especially when using a 100x objective, recenter the lamp while observing the fluorescent image. ND filters are in the optical path. Remove the ND filters from the optical path as necessary. The combination of excitation filter, barrier Use a filter cube with a combination suited filter and dichroic mirror is inappropriate for the specimen. for the specimen. A halogen lamp is being used with a dark specimen. Change the light source to a mercury lamp. The designated objective is not being used with UV or V excitation. Use the designated objective. The installed position of a filter cube has deviated from the prescribed position. Insert the filter cube until it touches the back of the turret. 34 V. Troubleshooting Problem Contrast is poor. Viewing is poor. Cause Countermeasure The objective or cover glass is dirty. Clean the objective or cover glass. The immersion oil is fluorescing. Use non-fluorescent immersion oil (Nikon Immersion Oil DF). The slide glass is fluorescing. Use non-fluorescent slide glass. The room is too bright. Darken the room. There is no cover glass in place. Use a cover glass. (However, no cover glass is required when using an NCG objective.) No immersion oil has been applied to the tip of an immersion-oil type objective. Apply Nikon Immersion Oil DF. The specified immersion oil is not being used. Apply Nikon Immersion Oil DF. The filter cube being used is not suited for Use a filter cube suited for the specimen. the specimen. The field diaphragm has been stopped down too far. The viewfield is vignetting. Open the field diaphragm so that it is just outside of the viewfield. The ND filter slider or shutter has stopped Pull out or push in the slider all of the at an intermediate position. way. The installed position of a filter cube has deviated from the prescribed position. Insert the filter cube until it touches the back of the turret. The filter cube has stopped at an intermediate position. Rotate the excitation method changeover ring to the clickstop position. TIRF microscopy Problem Unable to turn on system power TIRF image does not appear. Dark TIRF image TIRF image with poor contrast Cause Countermeasure The power supply is not plugged in. Connect the power cable to the outlet. The lamp is not on. Turn on the lamp. The shutter is closed. Open the shutters of the shutter slider and cassette holder. The microscope’s optical path is not set to “Observation.” Point the optical path at the binocular eyepiece with the optical path switch knob or LIGHT PATH switch. Inappropriate filter (dichroic mirror and absorption filter) wavelength settings Use filters and wavelength appropriate for the fluorescence reagent used. Illumination light is too low. Adjust ND filters of the white TIRF illuminator. Inappropriate filter cube is in the optical path. Place the filter cube appropriate to the specimen in the optical path. Illumination light focal point is not on the objective's pupil. Adjust the W-TIRF diaphragm position. Illumination light focal point is near the center of the objective's pupil. Adjust the W-TIRF diaphragm position. The illumination light emitted by the objective is not a parallel ray. Adjust the position of the condenser lens. 35 VI 1 Care and Maintenance Cleaning filters and lenses Do not let dust, fingerprint, etc. get on the lenses and filters. Dirt on the lenses, filters, etc. will adversely affect the view of image. Especially in TIRF microscopy, if the tip of the objective and the cover glass are dirty, it can substantially reduce contrast. If any of the lenses or filters gets dirty, clean it as described below. • Use an air blower to blow away dust. If that does not suffice, brush away dust with a soft brush, or else gently wipe it off with gauze. • Only if there are fingerprints or grease, dampen a piece of soft, clean cotton cloth, lens tissue, or gauze lightly with absolute alcohol (ethyl or methyl) and gently wipe off the dirt. However, do not use the same area of the cloth, etc. to wipe more than once. • Use petroleum benzene to clean off immersion oil. After wiping off with petroleum benzene, wipe with absolute alcohol (ethyl or methyl) to make the surface clearer. If you cannot obtain petroleum benzene, use methyl alcohol. However, methyl alcohol does not clean as well as petroleum benzene, it will be necessary to wipe the surface repeatedly. (Usually, three or four times are sufficient to clean the lenses.) • Use petroleum benzene only to remove immersion oil from objectives; do not use petroleum benzene for cleaning the entrance lens on the eyepiece tube, filters, etc. • Absolute alcohol and petroleum benzene are highly flammable. Be careful when handling it, when around open flames, when turning the power switch on/off, etc. • Follow the instructions provided by the manufacturer when using absolute alcohol. 2 Cleaning the product We recommend that you use a silicon cloth to clean this product. For persistent dirt, dampen a piece of gauze with neutral detergent and wipe gently. Do not use organic solvents such as alcohol, ether, or paint thinner on painted components and plastic components. Doing so could result in color-dulling or in discoloration. 3 Disinfecting the product We recommend that you use 70% medical alcohol for normal disinfection of this product. Do not use organic solvents such as alcohol, ether, or paint thinner. Using organic solvents could result in discoloration of painted components or plastic parts. In case of spillage of a sample onto this product, determine whether the sample is hazardous. If the sample is hazardous, follow your standard laboratory procedures. 4 Storage Store this product in a dry place where mold is not likely to form. Store the objectives, eyepieces, and filter cubes in a desiccator or similar container with a drying agent. Put the dust-proof cover over this product to protect it from dust. Before putting on the dust-proof cover, turn off all power switches of the microscope and the white TIRF illuminator and wait until the lamp housing gets cool sufficiently. 5 Periodical inspection (charged) Periodical inspections (expenses charged) of this product are recommended in order to maintain peak performance. Contact your nearest Nikon representative for details. 36 VII Equipments in Combination Name of equipment Microscope Model Remark TE2000-E TE2000-U TE2000-S Epi-fl cassette holder Objectives for TIRF T-FLC cassette holder T-FLC-E motorized cassette holder The T-HUBC HUB controller is required. T-FLC-HQ cassette holder Three exclusive high-precision filter cubes are attached as accessories. Plan Apo TIRF 60 x H Plan Apo TIRF 100 x H Plan Apo 60 x H Plan Apo 100 x H Plan Apo VC 60 x H Plan Apo VC 100 x H Stage ring 26 32 Stageup equipment “32” is made of glass. T-BSUK70 70 mm up kit T-FLS FL spacer TIRF2 stageup lens When using the white TIRF illuminator on the lowest step during stageup, install the stageup equipments to the white TIRF illuminator. Epi-illumination lamp and power supply unit Make sure to read the instruction manuals for the products. 37