Transcript
The Fluorescence Microscope Instructions for Zeiss Axiovert 200M Microscope By Jon Ekman
Basic operation instructions as of 4/3/2009
Start-up . . . . . . . . . . . . . 1 . . . . . . . . . Turn ON Master Switch 2. . . . . . . . . Turn ON X-Cite Hg Lamp 3. . . . . . . . . . . Log on to Computer 4. . . . . . . . . . . . . Load Software
Align Optical Components . . . . 1. . . . . . . . . . . Koehler Illumination
Acquire Image. . . . . . . . . .
Start-up --Turn on all Hardware 1. Turn MASTER switch ON. This will turn on the stage controller, the Unblitz transmitted light shutter and the Cascade 512B B/W camera and the AxioCam HRC color camera. The Axiocam MRm is on by default.
Using the Camera dialogues: (below) Roper, Axio MONO & Axio COLOR buttons on the taskbar will help to quickly align the optics to the camera you plan to use.
2. Turn ON X-Cite 120 Hg lamp. Must stay ON a minimum of 20 minutes. If you turn it off the internal logic chip will not let the system arc until it is cooled down.
The “Eyes” button switches the light for viewing the sample through the microscope binoculars. FL Closed will shutter the light coming from the Excite 120 lamp.
3. Log in to the computer.
BF = Bright Field
4. Load the Axiovision 4.7.1 software. (Shortcut on desktop)
All other buttons guide light via filter sets to specific cameras.
5. Ensure the all cameras are on and answer yes (y) to DOS window question. Once the software is up: 1. Set save location to E:\workspace\ (your user name) by going to Tools > Options > Storage > Image Storage > Folder for “Auto save” 2. Set the default name for your experiment images. Go to: Workarea > Multidimensional Acquisition > Experiment > Name --type a name for the experiment.
1. . . . . . . . . . . . . Select Camera 2. . . . Multiple Label Fluorescent Images 3. . . . . . . . . Fluorescent Image Tiles 3. . . . . . . . . Transmitted Light Tiles
Save Data. . . . . . . . . . . . . 1. . . . . . . . . . Save to Local machine 2. . . . . . . . . . . . Save to Network
Shut-down . . . . . . . . . . . . 1 . . . . . . . . . . . . . . Quit Software 2. . . . . . . . . . . Log OFF Computer 2. . . . . . . . Turn OFF Master Switch 3. . . . . . . . Turn OFF X-Cite Hg Lamp
Resources . . . . . . . . . . . . 1. . . . . . . . . . . Standard Suppliers 2. . . . . . . . . . . . Fluorescent Dyes 3. . . . . . . . . . Microscope Reference
Transmitted Light Imaging
Turn ON X-Cite 120 UV lamp
The green switch located on the right side of the microscope base indicates both Microscope power and halogen power.
Note: For Fluorescence imaging: Check the Reflector turret is set to desired excitation/emission
OPEN the Halogen lamp shutter through software by selecting Extra Shutter 1 to allow light to the sample.
• Zeiss Reflector Turret set to: (1) 350EX/460EM DAPI (2) 470EX/515EM FITC (3) 560EX/620EM Rhodamine (4) C5 640EX/680EM Cy5
Set the Zeiss Reflector Turret to position (5) DIC.
Fluorescent Imaging
• Cycle Fluor Key to allow light to sample.
The microscope is configured so the condenser will change to DIC/Phase contrast depending on the objective chosen. This can be overridden in the software. Specific settings can be saved using the Tools > Settings Editor.
Load a sample and find focus...
Align Optical Components lumination (see next page).
1. Load a sample. Tilt the lamp carrier back to allow access to the stage. Lower the objective lens by pressing the Focus Load Key (7) on lower right of microscope stand. Rotate the preferred objective lens into position beneath the stage using the Objective Keys Forward - Backward (8). Load your sample. Select the Focus Work Key (9) and the scope will raise the objective back to the last highest position it was in before it was lowered.
• The contrast or “shear” may be adjusted by manipulating the small knob on the Wollaston Prism located just below the objective lens. For phase contrast microscopy [currently only the 10x or 20x objectives]: • Rotate the condenser wheel to the appropriate setting : 10x = Ph 1; 40x =Ph 2. • Swing OUT the polarizer located between the condenser and the lamp.
2. Select eyes from the Toolbar to direct the light path to the eyepieces. Also, make sure that the Black Rotary Knob under the Binocular Tubes is set to 100% vis.
• Align the microscope for Koehler Illumination.
3. Select Imaging Method: For widefield fluorescence viewing: • Choose the appropriate excitation and emission filters for sample. • Adjust the Incident Iris Stop Slider until light Intensity is adequate. • Find Focus. For conventional brightfield viewing: • Rotate the condenser wheel (4) to the BF position as indicated on the LCD on the scope. (Right side buttons)
Optical Components
Zeiss Axiovert 200M For Nomarski differential-interference contrast microscopy (DIC): • DIC objectives automatically change the condenser wheel to the appropriate DIC setting for their numerical aperture. (The appropriate condenser setting is determined by the numerical aperture of the objective —see the quick notes.)
• Swing OUT the polarizer located between the condenser and the lamp.
• The analyzer is built into the No Filter (DIC) filter cube in the Zeiss Reflector Turret.
• Align the microscope for Koehler Illumination.
• Swing the polarizer into place between the condenser and the lamp.
(6) Halogen Voltage Up - Down (7) Focus Load Key (8) Objective Keys Forward - Backward (9) Focus Work Key (10) Course Focus Knob (11) Fine Focus Knob
Benefits of Aligning Optics: • Evenly illuminated image. • Brilliant image without reflection or glare. • Minimum heating of specimen.
• Align the microscope for Koehler Il-
Koehler Illumination PROCEDURE: Transmitted Light 1. Focus on specimen with the objective to be used for data collection. 2. Close down lamp field stop [diaphragm at top (1) ] while viewing. 3. Lower condenser slightly (2) until diaphragm image is in focus. 4. Center image using condenser centering screws (3). 5. Open diaphragm (1) to edge of field, fine focus and open further to just clear field.
6. Adjust contrast using condenser diaphragm (4). 7. Remove eyepiece (5) and check to see that 75%-80% of visible aperture is filled with light (more light=better resolution but less contrast).
Extra Shutter The system has a shutter (Uniblitz) between the transmitted light halogen and the microscope. In the software this is under Microscope > Extra Components > Extra Shutter
Acquire Image
Objective List
Select Camera
2.5x . . . . . . . . Plan-Neofluar1 NA. . . . . . . . . . . . . . . . . 0.075 WD(mm) . . . . . . . . . . . . . . . . 9.5 Coverslip . . . . . . . . . . . . . . . 0.17 Part #. . . . . . . . . . . . . . . 440310
1. Select camera PvCam, AxioCam MR, Axiocam HR or Apotome Mode from Acquisition > Camera >
10x . . . . . . . . . Plan-Neofluar1
2. Use correct camera dialogue box to align optics to camera chosen. Button are on taskbar: Roper, Axio-MONO & Axio-COLOR.
NA. . . . . . . . . . . . . . . . . . 0.30 WD(mm) . . . . . . . . . . . . . . . . 5.6 Coverslip . . . . . . . . . . . . . . . 0.17 Phase. . . . . . . . . . . . . . . . . Ph-1 DIC. . . . . . . . . . . . . . . . . DIC II Part #. . . . . . . . . . . . . . . 440331
3. Select Live from Tooolbar this will open up the “Live” Preview window. Normally the Live Properties for the camera selected will pop-up too.
20x. . . . . . . Plan-Apochromat3
4. Set exposure using the Adjust Tab and the Measure button to help visualize the live image and then focus live image with coarse or fine focus on the microscope stand.
NA. . . . . . . . . . . . . . . . . . 0.80 WD(mm) . . . . . . . . . . . . . . . 0.55 Coverslip . . . . . . . . . . . . . . . 0.17 DIC. . . . . . . . . . . . . . . . . DIC II Part #. . . . . . . . . . . . . . . 440640
Note: In the live image window footer you can select a suitable scaling for the objective you are using from the List of available scalings. By default, software will select the right scaling for the camera and objective being used.
40x LWD . . . . . . LD Achroplan2 NA. . . . . . . . . . . . . . . . . . 0.60 WD(mm) . . . . . . . . . . . . . . . . 1.8 Coverslip . . . . . . . . . . . . . . . 0.17 DIC. . . . . . . . . . . . . . . . . DIC III Part #. . . . . . . . . . . . . . . 440865
5. Check settings on Live Properties and optimize for the camera and experiment. Make changes for the camera settings on the following controls. NOTE: Adjust and General are the two most important tabs.
63x OIL. . . . . Plan-Apochromat3 NA. . . . . . . . . . . . . . . . . . . 1.4 WD(mm) . . . . . . . . . . . . . . . 0.19 Coverslip . . . . . . . . . . . . . . . 0.17 DIC. . . . . . . . . . . . . . . . . DIC III Part #. . . . . . . . . . . . . . . 440762
• Adjust Tab: The Measure button can be used to determine an optimum exposure time. The percent value under exposure represents how much of the CCD’s dynamic range in Intensity will be used by the measure button. (For example: 95% of 255 for 8bit would give a max pixel intensity of 242 when the measure button is used) The Roper camera’s EM gain can only be controlled here (when camera is selected). Be sure to optimize it for the most fluorescent channel.
100x OIL . . . . Plan-Apochromat3 NA. . . . . . . . . . . . . . . . . . . 1.4 WD(mm) . . . . . . . . . . . . . . . 0.17 Coverslip . . . . . . . . . . . . . . . 0.17 DIC. . . . . . . . . . . . . . . . . DIC III Part #. . . . . . . . . . . . . . . 440780
• The arrow keys and sliders can be used for fine adjustment of the exposure time.
fluorescence imaging, hit 3200K to reset the camera for best color reproduction.
• If you are using a color camera, you need to perform a white balance for the image. If you select the automatic balance (Automatic button), the camera tries to determine an optimum value itself. If you are using the color camera for
• General Tab: Key points For PVCAM (Roper Cascade 512B, use a Gain of 2 or 3 for normal imaging; Gain of 1 is for Binning. Set your black reference and capture a Shading Correction to remove artifacts
Objective Notes: 1.) Plan-Neofluar: very good quality-corrected for spherical aberration and corrected for chromatic aberration in red and blue wavelengths. 2.) Achroplan: good quality-corrected for adequate field flatness (spherical aberration) and chromatic aberration in red and blue wavelengths to a lesser degree than the Neofluar. A compromise for special applications like long working distance. 3.) Plan-Apochromat: excellent quality-corrected for spherical aberration and corrected for chromatic aberration in red, blue and green wavelengths.
Imaging Continued
cence module
General Properties for all cameras. For MosaiX Tiling to work correctly, keep the camera orientation settings as displayed here.
With the Multichannel Fluorescence module you can define up to 8 channels to be used for acquisition. (Our system has 5) By default 5 channels were added during training covering (1) DAPI, (2)FITC, (3)Rhodamine, (4)Cy5 and (5)DIC (for brightfield) The colored tabs can be activated/deactivated with a mouse right-click • Select dye channel (1-5) 4. Take Exposure Readings/Channel with Measure button. Select Measure Adjust exposure and % weight to get best image possible without over exposure of ROI.
from the images caused by electronic noise and dust on the optics.
MDA Multidimensional Acquisition
7. Select Snap to acquire a basic image. 8. Use Multidimensional Acquisition to acquire multi-channel images, Z-stacks, Time Series Saved stage positions and for MosiaX with multiple channels. 9. Basics are select Channel Tab (C), set exposure with “Measure” button and choose “Start” button to acquire image.
Multiple Label Fluorescence Imaging 1. Setup cameras & exposure using Live 2. Name/Load Experiment Select Multidimensional Acquisition Select the Experiment Tab Check modules to use: To activate, place a check mark in the Work area activating the specific property pages under Multidimensional Acquisition: • Multichannel Fluorescence “C”
Once all the channels and exposures are set, Setup Z-Stack or Time Lapse or Mark&Find or MosaiX each property page has setup options that can be added to an experiment. 6. Save Settings for future reference. Select Experiment tab
• Mark&Find -- Saved positions
7. Start
• MosaiX -- Tiling
Click on the Start button at bottom of the workspace. During acquisition a progress bar informs you about the procedure.
Select “C “ Tab: Multichannel Fluores-
File Formats
Image export: bmp, jpg, tif, tga, png, psd, cmp, avi, lsm, mov, j2k, jp2, pcx, tga, wmf, pcf
5. Setup extra Modules if needed
• Time Lapse -- Time series
3. Select Hardware Settings
Image import: bmp, tif, jpg, gif, tga, png, j2k, jp2, mac, msp, ras, pct, eps, wmf, psd, img, cmp, zvi, lsm, czi
Select next Channel and repeat to max of 8 channels. (Duplicate will add a new channel, Remove will remove a channel)
Save the settings you have made as a new experiment (see above), so that you can use them again in the future.
• Z-Stack -- For Z-stacking
Image Formats:
Select OK to write new settings to “Extended parameters”
In most cases it is best to save all files in *.zvi format and convert or batch convert to *.tif format later.
Files are named and saved as they are acquired. Unsaved images in software are saved with a ~ symbol. If the software crashes, files will be saved like this; you can rename as needed without reopening the AxioVision software.
MosaiX Tiling Setup Camera 1. Select camera PvCam, AxioCam MR, Axiocam HR or Apotome Mode from Acquisition > Camera > 2. Use correct camera dialogue box to align optics to camera chosen. Button are on taskbar: Roper, Axio-MONO & Axio-COLOR. 3. Select Live from Tooolbar this will open up the “Live” Preview window. Normally the Live Properties for the camera selected will pop-up too. 4. Check Image Orientation
Example of Flat Fielding using the BS script for the Roper camera.
• Select Live > Live Properties > General Tab 5. Set Exposure/Channel • Select Measure -Measure will auto expose using settings in “extended parameters” area in Multidimensional Acquisition. Adjust “Exposure” and “% weight” to get best image possible
dow appears
Select OK to write new settings to “Extended parameters”
Setup MosaiX
Select next Channel and repeat to max of 8 channels. (Duplicate will add a new channel, Remove will remove a channel)
MosaiX can be run independent of Multidimensional acquisition or from within it, either way the setup is the same.
Ensure to “Flat Field” cameras to remove all artifacts present in the optic train. For the Roper camera there is a script to apply shading correction to images. This script needs to be edited individually and saved to be accessed via “extended parameters” “user interaction” submenu to get shading correction applied while running MosaiX.
1. Set Acquisition Region
The AxioCams can all have shading correction applied easily under the General Tab.
MosaiX mode choices: Circular Setting In MosaiX “Circular” mode, Columns and Rows are centered around the live image position at the time of setup or on Mark & Find position lists. Overlap (%) can be a low as 2% to as high as 25% or more depending on sample quality and contrast. After choosing Rows, Columns & Overlap(%) you can acquire image 2. Set Acquisition Region continued. MosaiX choices: Rectangular Setting • Rectangular allows for large regions to be acquired with focus plain adjustments, keeping the sample in focus from one end of the sample to the other. • Select Live and ensure the light path is set to camera and the scalings are correct • Select Rectangular • Select -Setup Acquisition Setup win-
• Select the Red X to clear previous Columns and Rows Select the Four Arrows to “Mark” starting point Move Joystick/Stage X-Y while viewing live image “Mark” ending point. The software will then calculate number of Tiles based on Overlap(%) selected. 3. Set Focus Correction Choose a minimum of three “infocus” tiles to create an image plain. Check focus correction box and select OK. 4. Setup Extra Modes (optional) Any other parameters like Zstack and Time series can be set now if needed,
Quick Notes Fluorescent Tiling: Setup Camera . . . . . . . . . . . 1 . . . . . . . . . . . . . . Select Camera 2 . . . . . . . . . Check Image Orientation 3 . . . . . . . . . . . . . . . . Flat Field 4 . . . . . . . . . . Set Exposure/Channel
Setup MosaiX. . . . . . . . . . . 1 . . . . . . . .
Set Acquisition Region 2 . . . . . . . . . Set Focus Correction 3 . . . . . . . . . . . Set Extra Modes 3 . . . . . . . . . . . . . Start MosaiX
Stitch Files Together. . . . . . . . under Multidimensional Acquisition.
together.
5. Start MosaiX
Key parameters are Search Depth and Sobel Size
Click on the Start button at bottom of the workspace. During acquisition a progress bar informs you about the procedure. Files are named and saved as they are acquired. Unsave images in software are saved with a ~ symbol. Note: Tools > Options > (Display & Acquisition --multiple choices) can help tweak performance of the software while acquiring MosaiX tile. See Large Area Tiling. Stitch Files Together 1. Under MosaiX in Workspace select Stitching In most cases the default settings for the software will stitch most images
1 . . . . . . . . . . . Adjust Parameters
2. Click on the Start button at bottom of the workspace. During acquisition a progress bar informs you about the procedure. If images do not stitch well, change the display to Tile View Then Right click and select edit mode you can then adjust each tile individually.
2 . . . . . . . . . . . . . . Select Start
Convert Files. . . . . . . . . . . . 1 . . . . . . . . . Set Region of Interest
2 . . . . . . . . . Set Focus Correction 3 . . . . . . . . . . . Set Extra Modes
Save Files. . . . . . . . . . . . . 1 . . . . . . . . . Save to local machine
2 . . . . . . . . . . Export Files as tiffs 3 . . . . . . Save to Files Server (Zeus)
Large Area Tiling:
In many cases subtle tweaks in edit mode and then another stitch run will correct the alignment of the sample.
Setup Camera . . . . . . . . . . .
Reverse a stitch by:
Tweak Options Settings. . . . . .
Setup MosaiX. . . . . . . . . . .
Image = Image to be reset select and choose image.
1 . . . . . . . . . Turn Off Live Window
Stitching Mode = Original positions instead of Stitching positions.
3 . . . . . Acquisition Prevent overview 4 . . . . Display No Fit Image to Window 5 . . . . . . . . . . Display No Update
Convert Tiled image Convert Tiled image to Single file using Convert Tile Images. Default is the whole image. Use this interest.
to select a region of
2 . . . . . . Acquisition Convert to 8bit
Max File Size. . . . . . . . . . . . The maximum file size is 21,000 pixels. The software will crash at acquisitions larger than that. The Default for the software is 4,000 pixels but is set by ITG to 10,000 pixels. The image will be converted this maximum size using the MosaiX Convert. Dropping the camera resolution to 8bit helps to acquire larger areas with higher magnification objectives.
Saving files
Quick Notes Present Spatial Calibration:
Data is saved by selecting the ‘Save As’ button at the top left corner of the AxioVision interface, or by selecting File > Save or File > Save As. Zeiss organizes files two ways: either as a single “Experiment-name file” with a “*.zvi” extension or as an experimentname folder with grouped “Experiment-name *.zvi files” inside. Files created with MosaiX and Time Series can get quite large so default saves and acquisitions are set to save to the local machine. 1. Save to Local Machine. --Default You may save files to Workspace (E:\Workspace\”username”). A folder was created there at login for each user. 2. Before leaving, save to Network Share -- “username” on ‘ITG File Server (zeus.itg. uiuc.edu)’(Z:)
Roper Cascade 512B Base Port. . . 2.5x Bin 1 . . . . . . . . . . 5.94 mm/pixel 10x Bin 1 . . . . . . . . . . 1.56 mm/pixel 20x Bin 1 . . . . . . . . . . 0.78 mm/pixel 40x Bin 1. . . . . . . . . . 0.40 mm/pixel 63x Bin 1 . . . . . . . . . . 0.25 mm/pixel 100x Bin 1. . . . . . . . . . .0.16 mm/pixel
Zeiss AxioCam MRm Left Port. . . 2.5x Bin 1 . . . . . . . . . . 2.39 mm/pixel 10x Bin 1 . . . . . . . . . . 0.63 mm/pixel 20x Bin 1 . . . . . . . . . . 0.31 mm/pixel 40x Bin 1. . . . . . . . . . . 0.16 mm/pixel 63x Bin 1 . . . . . . . . . . 0.10 mm/pixel 100x Bin 1. . . . . . . . . . 0.06 mm/pixel
Zeiss AxioCam HRC Front Port. . . Most users opt to save the data to their network share on our central server (named Zeus). The server share is represented as the ‘Z:\’ drive in the windows environment. ITG is equipped with gigabit ethernet, and saving to the Z:\ drive is relatively fast. Also, this permits easy access to the data from any computer in the world using secure file transfer protocol (sftp). Instructions on accessing your network share remotely are available at http://www.itg.uiuc.edu/help/datahandling/userhelp.htm.
2.5x Bin 1 . . . . . . . . . . 3.91 mm/pixel 10x Bin 1 . . . . . . . . . . 1.02 mm/pixel 20x Bin 1 . . . . . . . . . . 0.51 mm/pixel 40x Bin 1. . . . . . . . . . 0.26 mm/pixel 63x Bin 1 . . . . . . . . . . . 0.16 mm/pixel 100x Bin 1. . . . . . . . . . 0.10 mm/pixel
Saving as TIFFs (.tif ) Single images, z-scans, multi-channel images will be saved as a series of TIFF images that can be viewed in other programs (Adobe Photoshop, ImageJ, Analyze, Image Pro Plus etc…). The position in the dataset to which an image belongs is encoded into the name, thus there will be a separate TIFF image for each channel of each zposition of each time point of a dataset (assuming xyzt mode is used). You may save Overlays, like micron bars, by selecting Burn-in annotations during Save As or Export. A text file which contains the instrument parameter settings for every image in an experiment is saved along with the individual TIFF files making up the dataset in the experiment directory. Large files can be Batch converted without opening them up using the File > Export command Before exporting check the Save In: location is set to either your folder in E: workspace or to Zeus
Default file format for AxioVision is *.ZVI Files can also be saved as *.tif Other options are: Image import: bmp, tif, jpg, gif, tga, png, j2k, jp2, mac, msp, ras, pct, eps, wmf, psd, img, cmp, zvi, lsm, czi Image export: bmp, jpg, tif, tga, png, psd, cmp, avi, lsm, mov, j2k, jp2, pcx, tga, wmf, pcf
References
B&W Camera Quantum Efficiency
Calibration Standards Calibration Grid Slide from Micro Brightfield: http://www.mbfbioscience.com Fluorescent Reference Slides from Microscopy Education: http://www.microscopyeducation.com Zeiss http://www.zeiss.de/us/micro/home.nsf --AxioCam HRC color CCD • Complete color information of every image detail • 1388 x 1040 standard size, 4164x3124 Co Site Sampling • 6.45 µm x 6.45 µm Pixel Size • 3x14 Bit color depth and 14 Bit grey value coverage • Large 2/3“ CCD sensor • Dynamic range of 2200:1 • Spectral Sensitivity approx. 400 - 700 nm
--AxioCam MRm Monochrome CCD • 1388 x 1040 CCD basic resolution • 6.45 μm x 6.45 μm Pixel size 12 bit • Dynamic range of 2200:1 • Spectral Sensitivity approx. 350 nm-1000 nm
Photometrics http://www.photomet.com/
Cascade II 512B Black & White EMCCD • Back Illuminated >90% Quatum efficiency • 512x512 CCD array • 16µmx16µm Pixel Size • Frame transfer CCD • Spectral Sensitivity approx. 350 - 1000 nm
Commercial anti-fade mounting media • ProLong Gold Antifade Mountant:
http://www.invitrogen.co.jp/products/pdf/ mp36930.pdf • Vectashield Antifade Mounting Medium:
http://www.vectorlabs.com/VECTASHIELD/ VECTASHIELD.html • VECTASHIELD HardSet Mounting Medium:
http://www.vectorlabs.com/products.details. asp?prodID=1483
Fluorescence Microscopy Books/Papers Allan, V., Ed., Protein Localization by Fluorescence Microscopy: A Practical Approach, Oxford University Press (1999). Andreeff, M. and Pinkel, D., Eds., Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, John Wiley and Sons (1999). Conn, P.M., Ed., Confocal Microscopy (Methods in Enzymology, Volume 307), Academic Press (1999). Goldman, R.D. and Spector, D.L., Eds., Live Cell Imaging: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2004). Herman, B., Fluorescence Microscopy, Second Edition, BIOS Scientific Publishers (1998). Hibbs, A.R., Confocal Microscopy for Biologists, Springer (2004). Matsumoto, B., Ed., Cell Biological Applications of Confocal Microscopy, Second Edition (Methods in Cell Biology, Volume 70), Academic Press (2003). Michalet, X., Kapanidis, A.N., Laurence, T., Pinaud, F., Doose, S., Pflughoefft, M. and Weiss S., “The power and prospects of fluorescence microscopies and spectroscopies,” Annu Rev Biophys Biomolec Struct 32, 161–182 (2003). Murphy, D.B., Fundamentals of Light Microscopy and Electronic Imaging, John Wiley and Sons (2001). Molecular Probes Ntziachristos, V., “Fluorescence molecular imaging,” Annu Rev Biomed Eng 8, 1–32 (2006). Pawley, J.B., Ed., Handbook of Biological Confocal Microscopy, Third Edition, Springer (2006). Periasamy, A., Ed., Methods in Cellular Imaging, Oxford University Press (2001). Rizzuto, R., and Fasolato, C., Eds., Imaging Living Cells, Springer-Verlag (1999).
Spector, D.L. and Goldman, R.D., Basic Methods in Microscopy, Cold Spring Harbor Laboratory Press (2005). Stephens, D., Ed., Cell Imaging (Method Express Series), Scion Publishing (2005). Yuste, R. and Konnerth, A., Eds., Imaging in Neuroscience and Development: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2004).
Turret Position 1
Chroma Filter Sets for Axiovert 200M Starndard set in Axiovert 200M Default Reflector Turret set to: (1) 350EX/460EM DAPI Chroma Set 31000V2 (2) 470EX/515EM FITC Chroma Set 41025 (3) 560EX/620EM Rhodamine Chroma Set 41039 (4) C5 640EX/680EM Cy5 Chroma Set 31023 (5) Transmitted DIC cube
Alternative set for Axiovert 200M
Turret Position 2
Second Reflector Turret set to: (1) Custum 350EX/565EM Chroma Set 31000V2 with 565 Emitter (2) Qdot 605nm Chroma Set 32007 (3) Qdot 655nm Chroma Set 32012 (4) Reflected Darkfield cube (5) Reflected Brightfield cube
Filter Cube Box for Axiovert 200M (1) Custum Zeiss Apotome Calibration Cube (2) 425EX/540EM Lucifer Yellow Chroma Set 31010 (3) 780EX/800EM Li-Cor Chroma Set 41037 (4) Custum UV blocking 400DCLP & GG400LP EM (5) (6)
Turret Position 3
Turret Position 4
Imaging Technology Group Beckman Institute for Advanced Science and Technology University of Illinois at Urbana-Champaign 405 North Mathews, Urbana IL 61801 USA Phone: 217.244.0170 FAX: 217.244.6219
