Transcript
/FL ZEISS AxioImager BioVis
Simple Workflow AxioImager FL Camera BioVis
Ocular Slider FL Camera Slider Color Camera
SWITCH ALL ON Color Camera Microscope (switch behind stage) Apotome Stage FL lamp
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3
Apotome 4
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Objectives Which Camera to use is chosen in Axivision sliders IN/OUT = Ocular/Camera Apotome out (as seen): inactive, Apotome in: active Apotome utilization is chosen in Axiovision
TFT screen stage with Slide holder and handle Condensor
LAST BUT NOT LEAST SWITCH ON THE COMPUTER AND LOGIN
BioVis Features of the Condensor are important for KÖHLERing, which is a prerequisite for best picture quality in Brightfield (Condensor is not used in FL) Screw for vertical movements of condensor Screws for centering the aperture
Positions of the Apotome Have OUT/IN position depending on usage. Be careful when draggin out. There are ”click”-positions accompanied by a beep for IN/OUT: NEVER drag it over the OUT position . NEVER remove the Apotome. (see further information: Apotome)
OUT
IN
(see further information KÖHLERing)
Apotome from above
BioVis Focus knob Coarse Fine
3a
Focus knob Coarse Fine
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3b 2
1. Microscope stand: left and right Stage UP/DOWN buttons 2. Microscope stand right: intensity of Halogen light 3. Focus knob left right, flat and tooth buttons: 3.a) Left knob: tooth buttons: Change filters, flat button FL light ON/OFF 3.b) Right knob: tooth buttons: change objective (avoid this), flat button Halogen light ON/OFF
BioVis Simple Imaging flow chart for AxioImager
2. and 4. 1. Open AxioVision by clicking the Icon on the desktop 2. Use TFT screen to swing in the objective you want to use: 5 Home>Microscope>Turret>Objective 3. Lower the stage by using the stage buttons, insert your sample slide and bring the stage up 4. Use TFT screen ...Turret > Reflector to swing in different filter 6. 5. Switch lights ON and use the oculars to find your sample incl. Focus 6. Use Axiovisions 6D-Acquisition Icon which opens floating window 6a) Tab ”Experiment” choose LOAD and load setting you need 6b) Tab ”Color”, check what channels are loaded and check, whether correct filter swings in ”during acquisition”. 6c) 6c) Tab ”Color” : choose name and color for channel
3. 6a)
6b) Focus knob
BioVis 7. Check the Camera to use
MRc = Color camera (Histology), MRm= monochromatic (Fluorescence) 8. On the microscope stand pull the upper rod out (light goes to camera, not ocular) 9. ”Color ” tab: choose channel, click measure 10. New Live window : focus and measure exposure time, accept by OK 10 a) check that ” %” is 60 % 10 b) change times and execute them by clicking into the % area again 10b) 11. Exposure times are fixed now 12. Check under ”Experiment” tab that no Z-stack etc is active 10a) 13. ”color” Tab: Press START (at the very bottom) to image 14. Save your image under D: Data:ZEISS:your folder... as zvi or tif
10b) 11.
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Z-STACKING BioVis Priority for Z-stacking is to have made already Exposure time calculations OR you trust the AUTO 1. 2. 3. 4. 5. 6.
activate Z tab choose Start/Stop (or Centre) Click at to see where you are focus where to start and OK it repeat with STOP decide for Optimal Distance or certain number of slides. 6a) Click on optimal distance Or set in no of slides and click then into ”slice distance” to claculate z-slice distance. Similar flow applies for ”Centre”, where you decide where the centre Z image should be, how many images should be taken in Z, and whether this should be done with optimal distance or not. START whole Z-stack under the Color Chanel Tab ”Optimal distance” calculates the Nyquist sampling distance, depending on N.A. This setting will not loose any Z-resolution. Setting a number of ”slides” as priority will create loss of Z-resolution and probably gaps between the different Z-slides
Positioning list
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BioVis Priority for positioning list is to have made already exposure time calculations OR you trust the AUTO* 1. 2. 3. 4.
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Open XY Tab and click MarkFind and LIVE view on Mark and Find window click Positions tab under Options tab check ””include focus...” navigate through your sample via LIVE view and 5. mark positions : press 6. give positions a name 7. Sort positions by ... 7. 8. 8. exchange positions if needed 9. execute by START 10. Images will appear in .zva folder
. Color code got to previous Exchange move to (marked) Delete go to next Undo/redo,
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*To compare positions side by side for intensities use ”fixed exposure” times. When intensities will vary very much depending on position use ”auto”
MosaiX Simple USE ”fixed exposure times”.
BioVis
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1. Open MosaiX Tab and click for Center if actual image should be centre of the mosaiX 2. choose no of columns and rows 3. choose overlap of adjacent images (10% usually ok) 4. execute MosaiX via START 5. MosaiX image is made of (here 2x2) 4 image tiles 6. if tiles do not fit into each other Tile View > stitching helps 7. save as zvi or 8. export to .tif , having ”merge image” clicked (see export page) . 6.
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MosaiX Extended USE ”fixed exposure times”.
BioVis 1. Open MosaiX Tab and click for Rectangle, than Setup 2. if tiles do not fit into each other Tile View > stitching helps 3. Acquisition set up window, NOTE Green cross marks light management via TFT screen starting point 4. Click Live view = blue frame *** 5. Navigate through sample , having e.g. Blue frame and green cross like image 5. 6. Press to calculate rows&columns 7. Execute by START in MosaiX 4. 8. Image is here 2x3 tiles 9. Apply stitching if needed (see MosaiX simple overview) 6. 4.
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***SEE NEXT page for further important instructions
MosaiX FOCUSPOINTS USE ”fixed exposure times”.
BioVis Get always a focused image even with uneven samples by applying focus points End (position seen in Live view)
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. marked focus
positions
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start
1. Follow steps 1-4 on previous page 2. Open Focus Correction tab 3. For e.g. each image focus to desired focus and set focus point 4. Follow steps 5 -9 on previous page
Using the Apotome BioVis
Apotome optical sectioning vs. Conventional FL
Visible grid
! Uncheck normalization When combined with mosaiX imaging!
Slide Apotome into microscope (beep!) 1. Activate ApoTome mode and setting window 2. check for 3. Do imaging as usual via Color chanel tab 3a) set % to 60 -70 % to enhances performance
Using color Cam in Live mode BioVis
Slide out the Color Cam rod 1. Activate the Color Cam and Live view 2. make your settings under ”Adjust” 2a) like ”measure” Color Cam is also 2b) white balance handable via the 2c) more/less light Color Chanel tabs 2d) DIC settings (do white balance in Live mode) Contrast enhancement via DIC Check objective and it´s DIC settings at TFT Use I, II, or III for DIC or H for none at condensor Play with the screw on the ”DIC slider” ”DIC slider”
condensor
TFT screen
I (II, III)
Using Shading correction BioVis
Prerequisite for best Brightfield images is KÖHLERing, than proceed with 1) activate appropriate camera 2) Open the camera tab ”general” 3) go for Live view and focus on specimen 4) Move to empty position (no specimen) 5) click on Shading Correction and Enable 5a) in case of message:
amplify BF light and repeat. Visualization of Shading Correction OFF ON ANY change of objective/sample/DIC usage will change shading conditions...
Shading correction BioVis Even if you are not successful with Köhlering you can perform the shading correction shadowing
Dust particles
DIC effect enhances small differences in optical properties of structures and results in ´fake´ 3D apperance DIC is used to enhance contrast in BF
Uncentered Condensor results in strong Using the shading correction removes Left to right shading with visualization these effects. Of dust particles Please note that any change in the beampath will give different shading situation. e.g. If you change DIC conditions (using the srew on the DIC slider)
KÖHLERING BioVis
Is needed only for BF images
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Closed aperture with focused 3. Specimen and ´sharp´ aperture edges
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KÖHLERING is the procedure to centre the beamof light which runs through the specimen by the positioning of the condensor. Procedure is monitored via the ocular 1) Focus your specimen 2) Close the aperture :buttons behind focusknob on the right side of the microscope 3) You should see now something like image 3 in the ocular (image here is in LIVE view) 4) move condensor up/down by using the big srew on condernsors left side until you get ´sharp´ image of the aperture opening edge 5) Use now the button of 2) to open the aperture untill the edges hit the field of view AND 6) (Re)Centre the opening if needed, so that all edges hit the field of view borders simultaneously 7) open the aperture to 100 % using buttons like in 2) The BF light beam runs now centered and parallel through the sample
