Using the Nikon TE2000 Inverted Microscope
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1 Wellcome Trust Centre for Human Genetics Molecular Cytogenetics and Microscopy Core Using the Nikon TE2000 Inverted Microscope Fluorescence image acquisition using Scanalytic s IPLab software and the B&W Hamamatsu Orca C camera INSTRUCTIONS PLEASE HANDLE CAREFULLY! Handle the microscope gently, taking care to avoid sharp impacts. 1) TO SWITCH-ON THE MICROSCOPE AND LOOK AT YOUR SAMPLE: Bright-field Microscopy: - Turn on the transmitted light by turning on the switch on the Nikon power supply box on the bench to the left side of the microscope. - Press lamp on/off button at the base of the microscope on the left. - Adjust the light intensity by rotating the control dial situated on the left side of the microscope. - Check that the D (Diffuser) and HA (Heat Absorber) filters at the top left of the microscope are pushed in. Select the optional neutral density ND filters as necessary to dim the light intensity. Push in the optional [top right] neutral density ND16 and ND2 filters to dim the light intensity - push both in for maximum light attenuation, which often the optimal setting. - The eyepiece turret dial should be set to O (open), when set to C it is closed. - The "port/optical path" dial on the right side of the base should be set to port 1. - The "reticule in/out" lever at front of the base to the right should be turned clockwise and set to a 2 o clock position. - The fluorescence filter wheel located underneath the objective wheel should be on the empty position indicated by the double arrow symbol ( ). - Place the culture vessel or glass slide on the stage. 1
2 - Move a low power objective (4x or 10x) into the light path. Set the optical zoom knob [left side] to 1.0x or 1.5x as required. - Have the phase ring set to A for stained cells or Ph1 for phase when using the 4x, 10x and 20x objectives or Ph2 for phase when using the 40x and 60x objectives. DIC H is for use with the 60x oil objective only. - A hi-resolution 60x oil objective is also available if required [for glass slides]. If you need to use this please ask a member of core staff to attach it for you - Glass microscope slides are normally viewed coverslip-side down. Focus with the coarse focusing wheel, revolve to the 40x or 60x objective as required. - Please take extra care to avoid sharp impacts when switching between objectives. You may need to adjust the height of the objective wheel and refocus. - Focus on the specimen. Adjust the condenser to the correct height for Koehler illumination. Ask core staff for help with this if necessary. - The 20x, 40x and 60x objectives have correction collars to compensate for differences in vessel wall thickness. The collars can be adjusted by rotating them, and, used in conjunction with the normal focus, an optimal image can be obtained. Please ask Core staff for help with adjusting correction collars if necessary. The 60x oil objective with DIC [Differential Interference Contrast] Microscopy: - Ask a member of core staff to attach the 60x oil objective and set up the transmitted light DIC optics [if required]. - Have the phase ring set to DIC H. - Slide the polariser filter [located above the condenser turret] to the left. - Push in the black T-A analyser slider [located under the objectives & filter wheel, to the left of the microscope base]. - Place microscope slide cover-slip side down. Move a low power objective (4x or 10x) into the light path. When you have found an area of interest carefully revolve to the 60x oil objective. - Place a drop of oil on the objective. Make sure you use the correct oil. Only Cargille oil should be used with this objective. - For correct DIC you should have set the condenser height correctly for Koehler illumination. You then rotate the polariser, analyser and slide in/out the DIC prism [under the 60x objective] to give the best contrast enhanced image. 2
3 Epi-Fluorescence Microscopy: - Note the last switch-off time in the notebook beside the microscope. The bulb must be left for at least 30 minutes to cool down before it is switched on again. - Turn the power switch on the front panel of the mercury lamp power supply box (on the bench on the right side of the microscope) and make sure the "power" monitor light (green) is on. Always switch the mercury lamp on and off with the PC, LCD display, microscope and camera switched OFF. - Press the ignition button for 2-3 seconds to light the lamp. The "lamp ready" light (orange) will come on briefly. Leave to warm up for few minutes, the orange light should then be on permanently. Leave the mercury lamp on for at least 30 minutes before switching it off. - Unless you also capturing phase contrast or transmission images as well, the height and settings of the condenser above the stage are irrelevant for epifluorescence imaging. Fluorsescence Filter Sets Optical filters are required in the microscope to ensure appropriate excitation of the fluorophore. These dichroic filter sets are moved into the fluorescent light path by rotating the fluorescence filter wheel, which is located underneath the objective wheel [nosepiece], on the right side of the microscope base. The following filter sets are available: The following filter sets are available: o DAPI/uV Hoechst - Blue emission colour [violet excitation] o FITC/GFP GFP, Alexa Green emission colour [blue excitation] o TRITC/Red Rhodamine, Alexa Red emission colour [green excitation] o CY5 CY5, Alexa Far Red emission colour [red excitation] o Transmitted light only - Fluorescence off [Phase Contrast] Note: the excitation light colour is seen at the specimen and the emission light colour is seen down the microscope via the eye-pieces. - During fluorescence microscopy, turn off the bright-field halogen light by using either the switch on the left side of the microscope main body, or by rotating the transmitted light intensity knob anti-clockwise to zero. - Rotate the fluorescence filter wheel to select the desired excitation filter set [DAPI, FITC, TRITC or CY5]. - There is a fluorescence light shutter underneath the filter wheel (at the right hand side) that needs to be opened. Set to the O position for the fluorescence light to pass to the objectives. In the C position the shutter is closed. The shutter should be kept closed [C] during transmitted light microscopy. 3
4 - Adjust the fluorescence light brightness by inserting the ND filters (ND4 and ND8) into the optical path. These are located near the rear UV lamp housing. 2) TO START THE CAMERA AND CAPTURE IMAGES: - Switch on the Hamamatsu camera by pressing the switch on the camera power supply box next the 24 computer monitor. Turn on the computer (PC on the floor under the bench) and its 24 LCD monitor (button top right edge). - If you are a Well user log on to the system and network with your usual username and password. Alternatively, log on directly to the PC as User with the password cyto02. - Double-click on the IPLab 37 icon. - Click on Camera, and then Select Camera and from the scroll down menu choose Hamamatsu 1394 ORCA Once you have found an image down the microscope that you wish to take a picture of, move the port/optical path dial (on the right side of the base of the microscope) to port 5 [all light to the Hamamatsu camera]. - Click again on Camera, and then select Acquire Focus. A live video image will appear on the screen. This configuration is intended to allow you to get the image in focus while viewing it on the computer monitor. - An Acquire Preview dialog box appears. You can improve the live image by altering the camera image acquisition parameters 1. - Adjust the camera exposure time as required. The time is listed in milliseconds. - You may adjust the image contrast by using the white and black point slider bars (White Point: the pixel value at which the image will appear white; Black Point: the image value at which the image will appear black). - By checking the Show Saturated Pixels box you will be able to see pixels that are saturated. The pixels in blue are equal to or below the saturated black point and the pixels in red are equal to or above the set white point [i.e. overexposed]. Remember to uncheck the box before taking a picture! - The Pause button will stop the preview without closing the window. - To take a picture click on Snapshot. 1 The Hamamatsu camera captures 12-bit B&W (4,096 grey levels) images by default, with a maximum resolution of 1344 x 1025 pixels [with binning set to 1x1]. This can be reduced to 672 x 512 pixels with binning set to 2x2 where every four pixels of the CCD are combined, doubling camera sensitivity and halving resolution. Camera binning is adjusted in the Acquire Preview window, under the Size tab. 4
5 3) TO CREATE AND SAVE MULTICOLOUR IMAGES: - Capture separate grey scale images for phase or DIC and for each different fluorophore [e.g. DAPI, FITC] - Click on main menu Ext, and then select Create Multiprobe Image. - Select a window, image file name and choose probe name and colour. - Select next window and repeat. - Merge images by clicking OK. - Click on Ext, and then select Enhance Multiprobe Image to adjust the contrast and positioning of images that have been merged. Min/Max: these controls allow you to manipulate the normalisation, or contrast settings, for each channel. Pixel Shift: these controls move the image of the selected channel in case the images initially produced were not perfectly registered. Blending: this affects to what degree overlapping colour channels are mixed together to create an intermediate colour (i.e. at 100% the channel with the highest intensity value will show through). - Clicking the OK button makes changes according to the dialog settings and then exit the dialog box. - Save images in your own folder in User Images in drive C: / - The Image Information button [ i ], built into the image window, displays a window of information about the image. You can go to File and then select Edit Image Info and type in additional notes (i.e. objective you used, etc.). - When any image is saved as an IPL file, you can return to the file at any time to view or edit it. IPLabs can only open images saved in this ipl format, it can t open images it has exported to TIF or jpg files. To be able to open your images with Photoshop you must create an RGB image by clicking on Ext, select Extract Multiprobe Data and then save your images (either as single channels or 24-bit colour) in a more common format - like TIFF or jpg. Alternatively, you must export 2 your images by clicking on Math, then select Convert to 8/24 bit and then Save as. You can check that the exported image looks OK using Photoshop Elements 7 on the PC. 2 Hamamatsu camera images are captured at 12-bit black and white (4,096 grey levels). Use Math, Convert to 8/24 bit to convert these 12-bit images to 8-bit [256 grey levels] B&W, if you want to view them correctly in PhotoShop or in PowerPoint. We can only distinguish up to 191 different grey levels in a B&W image. 5
6 4) TO FINISH ( and go home): - Close IPLab 3.7 before switching off the camera. In order to close IPLab 3.7 you must click the OK button in the Acquire Preview window before selecting Exit. - If using the 60x oil objective please remember to wipe off the oil and inform a member of core staff when you have finished, so the objective can be removed. Microscope oil objectives should only be cleaned with the lens tissue provided. - If used, press the halogen transmission lamp on/off button at the base of the microscope on the left and turn off the Hamamatsu camera power supply box. - Switch off the computer and its LCD monitor. - Before turning off the fluorescence mercury lamp allow the mercury bulb to run for a minimum of 30 minutes and check in the booking sheet on the door that nobody else will be using the microscope in the next few hours. - Switch off the mercury lamp power supply box and note the time off and total hours used in the notebook. If you have any microscope/imaging problems or queries, just ask a member of the Microscopy Core staff for help. There is additional help and advice on our Core web-pages: WTCHG Fluorescence image acquisition using Scanalytic s IPLab software and the B&W Hamamatsu Orca C camera. This version updated: November
7 TROUBLESHOOTING If you do not see any fluorescence, check the following: Is there a shadow in the image with fluorescence illumination? If the shadow doesn t move with the specimen, an incorrectly set filter slider or other component may be obscuring the light path. Alternatively, the mercury lamp may have a problem ask a core member for help. Is the mercury bulb turned on? Check the orange and green light on the power box. Can you see coloured light passing through the objective? If not, check that the shutter underneath the filter wheel is open. Is the filter block in place? Are you using the correct filter block? Rotate the filter wheel and see if any light of the right colour appears. Have you checked the dial below the eyepiece? It should be set to O. Is the objective correctly positioned? The objective wheel should click into position. Is the port/optical path in the correct position? 1 is for observation down the microscope, 5 is for camera use only! Is the light too faint or too bright? Remove/Add the neutral density filters. My exported images are all black in Photoshop and PowerPoint The Hamamatsu camera captures in 12-bit [4,096 grey levels]. Photoshop can only view 8-bit [256 grey level] B&W images correctly. Core staff can advise on image bit depth conversion. FOR ANY QUERIES OR DOUBTS, JUST ASK A MEMBER OF THE CORE STAFF 7
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