BRIGHTFIELD Olympus TH4-200 Olympus TH4-200 Please keep objectives off touching to avoid their loosening or damage.

Transcription

1 BRIGHTFIELD 1. Remove the microscope cover. 2. Dial the brightness adjustment knob (1) of Olympus TH4-200 halogen light source down to MIN. 3. Turn on the Olympus TH4-200 halogen light switch for transmitted light (2) to I (ON). 4. Check the light path. The light path selector knob (3) should be put to upper ( eye ) position. 5. Remove the ND5 and ND25 neutral density filters of filter pocket (4) from the light path of illumination turning them upwards. 6. While looking through eyepieces adjust the light intensity using the brightness adjustment knob (1). Try to avoid working at maximum of power of the lamp as that shortens life span of the lamp. 7. Adjust the interpupillary distance. While looking through the eyepieces, adjust the distance between the eyepieces (5) until the left and right fields of view coincide completely. 8. Make sure the 10x objective (6) is in place. 9. Place the slide with your specimen on the stage (7). 10. Find your specimen using the stage controls (8). 11. Focus specimen using fine/course focusing knobs (9). 12. Adjust the diopter: Close your right eye and focus on the specimen using the fine focus knob. Close your left eye and focus on the specimen using the diopter ring (10) on the right ocular. Open both eyes and confirm that the focus is comfortable. 13. If desired switch to the next objective by rotating the nosepiece (11) holding the nosepiece rim only and focus in the sample. Continue until you reach the desired magnification. Please keep objectives off touching to avoid their loosening or damage. 14. Establish Koehler illumination: Close field iris diaphragm (12) completely. Keep stage with sample on it fixed and avoid moving it in either of directions including focusing of the sample. Focus the image of the edge of the field diaphragm by raising or lowering the condenser using

2 the condenser focusing knob (13). Please note that focusing of sample and focusing of condenser is performed with different mechanisms. Check if the circle of light is centered in the field of view. Open field diaphragm till it start touching the round edge of field of view by its corners. If the diaphragm is not centered, use the two condenser centering screws (14) to move the field iris diaphragm image to the center of the field of view ( corners /edge gap is uniform). Open the field iris diaphragm until its image circumscribes the field of view. Match the opening of the condenser aperture iris diaphragm (15) with the N.A. of the objective in use in order to achieve the optimum objective performance. Start with the aperture diaphragm fully open. Start closing the aperture diaphragm and stop at the position of abrupt change in brightness of the image. This is the position of condenser objective aperture match. NOTE: Most specimen are usually low in contrast, reducing the diaphragm opening to 70%-80% of the N.A. value of the respective objective will generally provide an image of acceptable quality. That will correspond in roughly 20-30% decrease in brightness of the image. To check the opening (not recommended as it leads to contamination of optics especially if done by inexperienced users); a) remove one ocular and look down the tube, b) adjust the condenser iris so that 80% of the field is light, c) replace the ocular, or a) set condenser aperture iris scale to about 80% of the N.A. value of objective, b) example: N.A. of objective is 0.75, set the scale to 0.75 x 0.8 = Examine specimen and document image if necessary: To obtain still digitized images, use manual for Hamamatsu Digital Camera. 16. When finished: Remove the sample from the table and wipe it from oil with Whatman or Kimwhipes tissues if needed. Put 100x objective in upper position. Touch the lens carefully (no dragging through the lens!!) 3 or 4 times with dry and clean part of Whatman or Kimwhipes tissue first to remove oil from objective till no oil marks observed left on the tissue. Touch the lens carefully once with Whatman or Kimwhipes soaked with 70% alcohol.

3 Repeat the cleaning for 60x oil objective. Always use clean part of tissue or a new tissue to avoid cross-contamination between objectives. Turn the nosepiece (11) back until the empty position in turret is into place. Turn the light intensity down using the brightness adjustment knob (1). Turn off the halogen light switch (2) to 0 (OFF). Cover the microscope. Leave the room CLEAN and TIDY! FLUORESCENCE 1. Remove the microscope cover. 2. Put a record in Log Book indicating Life Time of the mercury bulb readings from OLYMPUS U-RFL-T Power Supply Unit (16). 3. Switch the UNIBLITZ VMM-3 Three Channel Shutter Driver ON. The switch is at far right corner of the device. Power and Driver active indicators for channels 2 and 3 (CH.# 2 and CH.# 3) are turned read. LED CH.#1 stays dark. Control over Channel 1 is performed through MetaMorph software interface. 4. In the case of using digital camera for acquisition switch HAMAMATSU camera driver ON. Find the switch at the lower left front corner of the controller. Power indicator of the controller will turn Green. 5. To turn on the mercury bulb, set the main switch (17) on the Supply Unit to I (ON). The light indicator (19) on the Power Supply Unit should go ON. Wait 5 to 10 minutes for the mercury bulb to stabilize. For safety reasons, the mercury bulb should stay ON for at least 30 minutes before turning OFF. 6. Setup microscope for brightfield imaging (see instructions on Brightfield # 2-14). 7. Engage the fluorescence mirror unit (18) until the proper filter cube is in place:

4 Number Excitation Emission Dichromatic in turret Mirror Unit filter filter mirror 1 Bright Field 2 U-MWU Applicable Fluorochrome 4, 6-diamidino-2-phenyl-indole HCI (DAPI), 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 8-Anilino-1-naphthalene sulfonic acid Mg salt (ANS), Alexa Fluor 350, Aniline blue, bis-aminophenyl-oxadizole (BAO) -Feulgen, Calcein Blue, Calcofluor white, Cascade Blue, Coumarin maleimide (CPM), Diphenylhexatriene (DPH), Dansyl chloraide, Dansyl hydrazine, Dimethylaminonaphthalene-5-sulfonic acid (DANS), EBFP, ERTracker Blue-White DPX, Euchrysine (Acridine Orange), Fast Blue, Filipin, Fluorescamine, Granular Blue, Hoechst 33258, Hoechst 33342, Monochlorobimane, o-phtalaldehyde (OPT), SITS, Tetracycline, True Blue 3 U-MWIBA Applicable Fluorochrome Alexa Fluor 488, Alexa Fluor 488-Phalloidin, Alexa Fluor 532, BCECF/AM, BODIPY FL, BODIPY FL Ceramide, Calcein/AM, Carboxyfluorescein-diacetate (CFDA), Cy2, DiO C EGFP, EYFP, Eosin-isothiocyanate, Fluoresceindiacetate (FDA), Fluorescein-isothiocyanate (FITC), FluorX, GFP (WT), GFP (S65T), JC-1, MitoFluor Green, NBD-Phallacidin, Oregon Green 488, Rhodamine 123, Spectrum Green, SYTOX Green nucleic acid stain, YO PRO 1, YOYO 1 4 U-MWIG IF 565 Applicable Fluorochrome 7-Amino Actinomycin D, Acidic Fuchsin (Pararosaniline), Alexa Fluor 546, Alexa Fluor 568, Cy3, Dil, Ethidium Bromide, Ethidium homodimer-1, Evans Blue, LysoTracker Red DND-99, Merocyanine 540, MitoTracker Red CMXRos, NanoOrange, Nile Red, Pararosaniline-Feulgen, Phycoerythrin B (PE-B), Phycoerythrin R (PE-R), Primuline O, Propidium Iodide (PI), Pyronin Y, Resorufin, RFP (DsRed), Rhodamine B-isothiocyanate (RITC), Spectrum Orange, Tetramethylrhodamine-isothyocyanate (TRITC), Xylenol Orange 5 Empty 6 Empty 8. Turn the light intensity down all the way if you want to avoid bright field component appeared in the captured image using the brightness adjustment knob (1). 9. To allow light from the mercury bulb to reach the specimen, slide the shutter knob (19) to position marked as empty circle. 10. To improve image contrast and to prevent color fading of fluorescent light in other part than observed region, pull out the field iris diaphragm knob (20) so that the image of the field iris diaphragm just circumscribes the field of view. 11. Examine specimen and document image if necessary: Double-click MetaMorph icon in the folder Meta Imaging Series 7.1 from Desktop to run the acquisition software.

5 Select Active value from Illum: pop-up list. Click Live icon from the Toolbar to have the live image window appeared on the screen. Use Set Current Shutter Stage button to control electric shutter (21) when needed. Switch the shutter ON ( Set Current Shutter Stage icon background becomes bright) Select desired objective and focus in image looking through eyepieces. Switch the Light Path Selector towards Camera icon. Image may appear on the screen at this stage at sufficiently high fluorescence from the sample. Click Configure Acquisition icon from the Toolbar. Click Autoscale check box to have image appeared on the screen at low fluorescence from the sample. Adjust the exposure slide at the right most position of Toolbar Click Snap for capturing Black and White image Save the captured image in your sub-directory at D:\Users. It is recommended to keep the objective magnification information next to the sample number included in every image name. 12. To avoid bleaching, slide the shutter knob (19) to the position marked as filled circle whenever you are not observing the specimen. 13. When finished: Slide the shutter knob (19) to the position marked as filled circle. If mercury bulb has burned for at least 30 minutes, turn off the mercury bulb by setting the main switch (17) on the Power Supply Unit to O (OFF). The light indicator (22) on the Power Supply Unit should go out. NOTE: The mercury bulb needs to cool down for 2 hours before turning back on. Remove the sample from the table and wipe it from oil with Whatman or Kimwhipes tissues if needed. Put 100x objective in upper position. Touch the lens carefully (no dragging through the lens!!) 3 or 4 times with dry and clean part of Whatman or Kimwhipes tissue first to remove oil from objective till no oil marks observed left on the tissue. Touch the lens carefully once with Whatman or Kimwhipes soaked with 70% alcohol. Repeat the cleaning for 60x oil objective. Always use clean part of tissue or a new tissue to avoid cross-contamination between objectives. Turn the nosepiece (11) back until the empty position in turret is into place.

6 Engage the fluorescence mirror unit (18) back to BF (brightfield) - #1 Cover the microscope leaving the mercury burner housing uncovered. Leave the room CLEAN and TIDY! SIMULTANEOUS FLUORESCENCE AND BRIGHT FIELD 1. Remove the microscope cover. 2. Setup the microscope for Bright Field and Fluorescence imaging above Adjust the transmitted light for the best balance of fluorescence and DIC brightness using the brightness adjustment knob (1) Examine specimen and document image if necessary: 5. See #11 at Fluorescence section To avoid bleaching, slide the shutter knob (19) to the position marked as filled circle whenever you are not observing the specimen. 7. When finished: Slide the shutter knob (19) to the position marked as filled circle. If mercury bulb has burned for at least 30 minutes, turn off the mercury bulb by setting the main switch (17) on the Power Supply Unit to O (OFF). The light indicator (22) on the Power Supply Unit should go out. NOTE: The mercury bulb needs to cool down for 2 hours before turning back on. Remove the sample from the table and wipe it from oil with Whatman or Kimwhipes tissues if needed. Put 100x objective in upper position. Touch the lens carefully (no dragging through the lens!!) 3 or 4 times with dry and clean part of Whatman or Kimwhipes tissue first to remove oil from objective till no oil marks observed left on the tissue. Touch the lens carefully once with Whatman or Kimwhipes soaked with 70% alcohol. Repeat the cleaning for 60x oil objective. Always use clean part of tissue or a new tissue to avoid cross-contamination between objectives. Turn the nosepiece (11) back until the empty position in turret is into place. Engage the fluorescence mirror unit (18) back to BF (brightfield) - #1 Turn the light intensity down using the brightness adjustment knob (1). Turn off the halogen light switch (2) to 0 (OFF). Cover the microscope leaving the mercury burner housing uncovered. Leave the room CLEAN and TIDY!

7 APPENDIX 1: Outline Olympus BX71 Microscope

8 1 halogen light brightness adjustment knob 2 halogen light power switch 3 light path selector 4 filter pocket 5 eyepieces 6 objective 7 stage 8 stage controls 9 fine/course focusing knobs 10 diopter ring of the eyepiece 11 objectives nosepiece 12 field diaphragm 13 condenser focusing knob 14 condenser centering knobs 15 aperture diaphragm slider 16 mercury burner power supply 17 main switch of the power supply of the mercury burner 18 fluorescence mirror unit 19 UV light mechanical shutter knob 20 field diaphragm of UV illumination source 21 UV light electric shutter 22 Light indicator on the mercury burner power supply