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zeiss_axiovert_200m [2020/01/01 21:05] bioimaging [Microscope overview] |
zeiss_axiovert_200m [2020/03/01 17:24] bioimaging [Microscope overview] |
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===== Microscope overview ===== | ===== Microscope overview ===== | ||
- | The Zeiss Axiovert 200M is a fully motorized inverted //widefield fluorescence microscope// ideal for live-cell imaging applications. It is equipped with a small stage incubator for temperature control and CO2 supply. Using the Metamorph software you can control the motorized filters, shutters and stage to simultaneously set up multi-color time-lapse imaging experiments in multiple stage positions. Its sensitive cooled CCD camera (Photometrics Coolsnap HQ CCD) allows you to capture weak fluorescent signals and minimize photobleaching/photodamage in light sensitive samples. You can also perform Z-stack acquisition and use [[https://huygens.i3s.up.pt/login.php|PPBI Huygens RM]] [[http://micro.magnet.fsu.edu/primer/digitalimaging/deconvolution/deconvolutionhome.html|deconvolution]] server to significantly improve image resolution even in thick samples. If you are performing long time-lapse experiments in live samples (aqueous media) and need hardware focus control, check the [[zeiss_cell_observer|Zeiss Cell Observer]] with Definite Focus. If don't want to use deconvolution, check out one of the confocal systems such as the point scanners [[zeiss_lsm_710|Zeiss LSM 710]] and [[zeiss_lsm_880|Zeiss LSM 880]] or the spinning disks [[3i_marianas_sdc|3i Marianas SDC]] and [[zeiss_cell_observer_sd|Zeiss Cell Observer SD]]. If your personal computer cannot handle all the data you collected, check out the [[big_guy|Big Guy]] or [[colossus|Colossus]]. If you have FISH or other fixed fluorescence slides try the [[leica_dm5000b|Leica DM5000B]] instead. | + | {{ :widefield_lightpath.png?0x300}}The Zeiss Axiovert 200M is a fully motorized inverted //widefield fluorescence microscope// ideal for live-cell imaging applications. Using the Metamorph software you can control the motorized filters, shutters and stage to simultaneously set up multi-color time-lapse imaging experiments in multiple stage positions. Its sensitive cooled CCD camera (Photometrics CoolSNAP HQ CCD) allows you to capture weak fluorescent signals and minimize photobleaching/photodamage in light sensitive samples. You can also perform Z-stack acquisition and use [[https://huygens.i3s.up.pt/login.php|PPBI Huygens RM]] [[http://micro.magnet.fsu.edu/primer/digitalimaging/deconvolution/deconvolutionhome.html|deconvolution]] server to significantly improve image resolution even in thick samples. If you are performing long time-lapse experiments in live samples (aqueous media) and need hardware focus control, check the [[zeiss_cell_observer|Zeiss Cell Observer]] with Definite Focus or the [[nikon_eclipse_ti|Nikon Eclipse Ti]]. If don't want to use deconvolution but need optical sectioning, check out one of the confocal systems such as the point scanners [[zeiss_lsm_710|Zeiss LSM 710]] and [[zeiss_lsm_880|Zeiss LSM 880]] or the spinning disks [[3i_marianas_sdc|3i Marianas SDC]] and [[zeiss_cell_observer_sd|Zeiss Cell Observer SD]]. If your personal computer cannot handle all the data you collected, check out the [[big_guy|Big Guy]] or [[colossus|Colossus]]. If you have FISH or other fixed fluorescence slides try the [[leica_dm5000b|Leica DM5000B]] instead. |
* **Microscope**: Zeiss Axiovert 200 | * **Microscope**: Zeiss Axiovert 200 | ||
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==== Objectives ==== | ==== Objectives ==== | ||
^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ pixel size* (μm) ^ 10 μm = ^ (color) 10 μm = ^ Reference ^ | ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ pixel size* (μm) ^ 10 μm = ^ (color) 10 μm = ^ Reference ^ | ||
- | | 10x | EC Plan-Neofluar | Air | 0.30 | 5.2 | 1.00 | 10 pixels | 23.3 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420340-9901-000/Objective-EC-Plan-Neofluar-10x-0.30-M27|420340-9901-000]] | | + | | 10x | EC Plan-Neofluar | Air | 0.30 | 5.20 | 1.00 | 10 pixels | 23.3 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420340-9900-000/Objective-EC-Plan-Neofluar-10x-0.30-M27|420340-9900-000]] | |
- | | 20x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=420650-9901-000|EC Plan-Neofluar]] | Dry | 0.50 | 2 | 0.48 | 21 pixels | 46.7 pixels | | + | | 20x | EC Plan-Neofluar | Air | 0.50 | 2.00 | 0.48 | 21 pixels | 46.7 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/440340-9904-000/Objective-EC-Plan-Neofluar-20x-0.5|440340-9904-000]] | |
- | | 40x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=440350-9903-000|EC Plan-NeoFluar]] | Dry | 0.75 | 0.71 | 0.24 | 41 pixels | 93.7 pixels | | + | | 40x | EC Plan-NeoFluar | Air | 0.75 | 0.71 | 0.24 | 41 pixels | 93.7 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/440350-9903-000/Objective-EC-Plan-Neofluar-40x-0.75|440350-9903-000]] | |
- | | 63x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=420782-9900-000|Plan-Apochromat]] | Oil | 1.40 | 0.19 | 0.15 | 65 pixels | - | | + | | 63x | Plan-Apochromat DIC | Oil | 1.40 | 0.19 | 0.15 | 65 pixels | - | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420782-9900-000/Objective-Plan-Apochromat-63x-1.4-Oil-DIC-M27|420782-9900-000]] | |
**Upon request:** | **Upon request:** | ||
- | ^ Magnification ^ Model ^ Type ^ NA ^ WD (mm) ^ pixel size* (μm) ^ 10 μm = ^ | + | ^ Magnification ^ Model ^ Immersion ^ NA ^ WD (mm) ^ pixel size* (μm) ^ 10 μm = ^ Reference ^ |
- | | 1.25x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=422310-9900-000|EC Epiplan-Neofluar]] | Dry | 0.035 | 4 | 7.58 | 1.3 pixels | | + | | 1.25x | EC Epiplan-Neofluar | Air | 0.035 | 4 | 7.58 | 1.3 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/422310-9900-000/Objective-EC-Epiplan-Neofluar-1.25x-0.03-M27|422310-9900-000]] | |
- | | 5x | [[https://www.micro-shop.zeiss.com/?s=571974962233f6&l=en&p=uk&f=o&a=v&m=s&id=420330-9901-000|EC Plan-NeoFluar]] | Dry | 0.16 | 18.5 | 2.00 | 5 pixels | | + | | 5x | EC Plan-NeoFluar | Air | 0.16 | 18.5 | 2.00 | 5 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420330-9901-000/Objective-EC-Plan-Neofluar-5x-0.16-M27|420330-9901-000]] | |
- | | 20x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=440341-9904-000|EC Plan-Neofluar Ph 2]] | Dry | 0.50 | 2 | 0.48 | 21 pixels | | + | | 40x | Plan-Neofluar Ph3 | Oil | 1.30 | 0.20 | 0.24 | 41 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/440451-9903-000/Objective-EC-Plan-Neofluar-40x-1.30-Oil-Ph3|440451-9903-000]] | |
- | | 40x | [[https://www.micro-shop.zeiss.com/?s=3407786617d462&l=en&p=uk&f=o&a=v&m=s&id=440451-9903-000|Plan-Neofluar Ph3]] | Oil | 1.30 | 0.20 | 0.24 | 41 pixels | | + | | 63x | Plan-Apochromat Ph3 | Oil | 1.4 | 0.19 | 0.15 | 65 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420781-9910-000/Objective-Plan-Apochromat-63-1.4-Oil-Ph3-M27|420781-9910-000]] | |
- | | 63x | [[https://www.micro-shop.zeiss.com/?s=571858182d6d9d&l=en&p=uk&f=o&a=v&m=s&id=420781-9910-000|Plan-Apochromat Ph3]] | Oil | 1.4 | 0.19 | 0.15 | 65 pixels | | + | | 100x | α Plan-Apochromat DIC | Oil | 1.46 | 0.11 | 0.09 | 105 pixels | [[https://www.micro-shop.zeiss.com/en/de/shop/objectives/420792-9800-000/Objective-alpha-Plan-Apochromat-100x-1.46-Oil-DIC-M27|420792-9800-000]] | |
*** pixel sizes for binning = 1** | *** pixel sizes for binning = 1** | ||
+ | |||
+ | ==== Cameras ==== | ||
+ | ^ Model ^ Type ^ Frame Size ^ Pixel Size (µm) ^ | ||
+ | | [[https://labs.pbrc.edu/cellbiology/documents/CoolSNAPHQ.pdf|Photometrics Coolsnap HQ]] | CCD | 1392 x 1040 | 6.45 x 6.45 | | ||
+ | | [[https://www.leica-microsystems.com/products/microscope-cameras/p/leica-dfc450-c/|Leica DFC450]] | color CCD | 2560 × 1920 | 3.4 x 3.4 | | ||
==== Stage ==== | ==== Stage ==== | ||
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===== System Turn On Procedures ===== | ===== System Turn On Procedures ===== | ||
- | * Turn the fluorescence light power source on. | + | * Turn on the fluorescent light power source |
{{halide_710.jpg?80|}} | {{halide_710.jpg?80|}} | ||
- | * Turn on the Uniblitz shutter controller (on the back). | + | * Turn on the Prior stage controller |
- | {{ax_uniblitz1.jpg?100|}}{{ax_uniblitz2.jpg?100|}} | + | |
- | * Turn the Prior microscope stage controller on. | + | |
{{ax_prior_on.jpg?100|}} | {{ax_prior_on.jpg?100|}} | ||
- | * Wait 10s then turn the microscope on. | + | * Turn on the microscope |
{{ax_mic_on.jpg?100|}} | {{ax_mic_on.jpg?100|}} | ||
- | * Turn the camera on. | + | * Turn on the CCD camera |
{{ax_cam_on.jpg?100|}} | {{ax_cam_on.jpg?100|}} | ||
- | * Turn the computer on. | + | * Turn on the computer |
- | * Login to the computer (Axiovert 200M user). | + | * Log in to Windows (Bioimaging User) |
- | * Start the Metamorph software. | + | * Start the Metamorph software |
- | * If you need temperature control, turn on the control box | + | * If the software doesn't detect the camera, power cycle the camera and try again |
- | {{heating2.jpg?100|}} | + | |
- | * If you need CO2, open the valve and turn on CO2 supply on the control box | + | |
- | {{co2.jpg?100|}} | + | |
===== Microscope Turn Off procedures ===== | ===== Microscope Turn Off procedures ===== | ||
- | * Turn off the computer. | + | |
- | * Turn off the camera. | + | If there is another user for this microscope in the __next hour__: |
- | * If you used any __immersion oil in the objectives, clean them up__. | + | * Leave the fluorescent lamp on |
- | * Turn off the microscope stage controller. | + | * Clean up immersion objectives |
- | * Turn off the shutter controller. | + | Else: |
- | * Turn off the microscope. | + | * Clean up immersion objectives |
- | * If there's __another user__ for this microscope in the __next hour__ leave the mercury __lamp power source on, else turn it off__. | + | * Shutdown the computer |
- | * Be extra careful in weekends and at the end of the day to make sure that the mercury lamp is not going to be turned on for an excessive time. | + | * Turn off the camera |
+ | * Turn off the Prior stage controller | ||
+ | * Turn off the microscope | ||
+ | * Turn off the fluorescent lamp | ||
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[[resources|Back to the Resources page]] | [[resources|Back to the Resources page]] |