Transcript
Guide to Confocal Microscopy on the Zeiss LSM 510 META NLO
Criss II Rm 405 Heather Jensen-Smith PhD
[email protected] 402-280-2248
SWITCHING ON THE ZEISS LSM 510 CONFOCAL MICROSCOPE
You should go through these steps (even if you are using the microscope without the confocal): 1) Turn on the remote control switch 2) Turn on the computer, log in, start the software (“LSM510 META”) 3) Turn the key on the Chameleon to the ON position, if needed (DAPI, 2 photon ex)
Manual operation of the Axioplan 2 microscope
1) HAL – halogen source, 3200K button 2) Field stop, aperture stop, neutral density filters 3) Lens change and filter change buttons on side, behind focus knobs 4) Focus buttons, left side, at the front 5) FL – epi fluorescence source 6) Field stop, aperture stop 7) Epi path block 8) F1, F2 buttons – do not use
LASER SCANNING CONFOCAL OPERATION
Four Steps to Confocal Image Acquisition: 1. Turn on the laser or lasers 2. Use ‘vis’ mode on microscope to locate area of interest for confocal microscopy switch to LSM mode 3. Select a confocal configuration from the Configuration window 4. Adjust the zoom, orientation, and detector gain using “Fast X-Y” 5. Acquire the image using “Single”
MICROSCOPE EYEPIECE VIEWING
Set slider to “VIS” (in): eyepiece viewing of transmitted light and fluorescence
LASER SCANNING CONFOCAL OPERATION
For laser scanning image acquisition: “LSM” - slider out
Starting the LSM 510 software
1) Double click the LSM 510 icon 2) Select “Scan New Images” 3) Select “Start Expert Mode” Note 1: Attempting to acquire new images in ‘use existing’ mode will result in a scanned image window with a ‘rainbow pattern,’ exit program then select New Images to correct Note 2: Rapid microscope initialization (1-2 sec) suggests the confocal was turned on prior to the computer, restart system and/or check microscope power switch to correct
1) Select Acquire
2) Select laser or lasers 3) Switch required laser/s to Standby (if required) then On Ar laser tube current should be 5.7 to 5.9. Output [%] should be about 45-50%
LASER CONTROL WINDOW
List of general fluorochrome colors excited with a given laser: Argon = green HeNe1= red HeNe2= far reds Cham= DAPI, 2photon ex
LASER SELECTION AND ATTENUATION SETTINGS
CONFIGURATION WINDOW
100 3-5%
Use of any laser above these settings may damage or destroy your sample and possibly the lasers! Please attempt to adjust the intensity of your acquired by modifying the sensitivity of the detectors instead.
CONFIGURATION WINDOW
2) Shows the optical configuration (light path to photodetector used to generate image) 3) Modify by selecting each optical element or load a specific configuration Your account is pre-loaded with a small number of basic green (Alexa 488,Cy2, FITC), red (Cy3, Rhodamine, Alexa 568) and blue (DAPI) configurations. You may add to and/or modify these to generate a user-specific list of configurations.
CHOOSING THE CONFIGURATION SINGLE TRACK Use for single, double labeling (must be spectrally
MULTI TRACK Use for double or triple labelling
distinct fluorophores)
Simultaneous scanning only ADVANTAGES Faster image acquisition
DISADVANTAGES Cross talk between channels
Sequential scanning, line by line or frame by frame ADVANTAGES When one track is active, only one detector and one laser is switched on. This dramatically reduces crosstalk.
DISADVANTAGES Slower image acquisition
CONFIGURATION WINDOW
1) Select single or multitract 2) Press the Configuration button 3) Select a stored configuration from this list, press Apply 3) Store any new configuration under a new name if desired
SINGLE TRACK – laser/s scan simultaneously
CONFIGURATION WINDOW
3) Load single track specfic configuration 1) Select Config 2) Select Track
Transmitted light images can be generated during Fl imaging. Optical path to ChD detector located at the back of the microscope (at the base) must be switched to permit TL images to be Note no image is visible through the eyepiece when the light path is open to generated. ChD.
(configuration-selected laser/s and attenuation displayed in Ex. Window)
APPLYING THE CONFIGURATION AND CHECKING THE SETTINGS IF YOU SELECT Store BY MISTAKE, IT WILL ASK YOU IF YOU WANT TO OVERWRITE THE CONFIGURATION. ANSWER NO! 5) Select Apply 6) To check for correct settings, click the Spectra button
The spectra button opens a window to display the activated laser lines for excitation (colored vertical lines) and channels (colored horizontal bars)
Each new login loads a predefined set of “correct” configurations.
SCAN CONTROL WINDOW
1) Select Scan 2) Select Mode
3) Select the Frame Size as predefined number of pixels or enter your own values (e.g. 300 x 600). Use Optimal for calculation of appropriate number of pixels depending on N.A. and λ. The number of pixels influences the scanning resolution!
ENTERING SCAN SPEED Enter the scan speed - a higher speed with averaging gives the best signal to noise ratio. Scan speed 8 usually produces good results. Use 6 or 7 for superior images. The slower the scan, the greater the bleaching.
SETTING UP THE DYNAMIC RANGE (8/12 Bit per pixel)
Select the dynamic range - 8 bit will give 256 grey levels, 12 bit will give 4096 levels. Photoshop will import 12 and 16 Bit images. Publication quality images should be acquired using 12 bit.
CONFOCAL MICROSCOPE
Features above and below the plane of focus fall outside the pinhole and appear black producing a true optical section.
The depth of the optical section is dependant on: 1) Pinhole diameter (greater pinhole thicker section) 2) Wavelength (longer wavelength thicker section) 3) NA of objective (higher NA thinner section)
CHANNEL SETTINGS - ADJUSTING PINHOLE
Pinhole size = 1 Airy unit
1 “Airy units” produces the best signal : noise ratio Pinhole adjustment changes the Optical slice. When collecting multi channel images, adjust the pinholes so that each channel has the same Optical Slice. This is important for co-localization studies.
MINIMUM PIXEL SIZE DETERMINED BY NYQUIST SAMPLING THEOREM
×
NA
PIXEL SIZE
10
0.3
0.51 µm
20
0.5
0.31 µm Values are for scan zoom = 1.0
40
1.3 (oil)
0.12 µm
60
1.4 (oil)
0.11 µm
Adjusting the field size (“XY”) to 56 µm with the 60× lens would produce a pixel size of 0.1 µm Brightness of image = magnification2/NA2
Field size can be adjusted by changing the objective magnification, or by optical zooming. Changing from 40 × to 60 × will reduce the field size, but will also reduce the amount of light available.
ACQUISITION OF IMAGES
Select “Single”
ADJUSTING THE ACQUISITION OF IMAGES 1) “Find” automatically preadjusts detector sensitivity (always saturated though)
2) Select “Fast XY” for continuous fast scanning - useful for finding/changing the focus, setting gain and intensity
3) Stop
SELECTING GAIN AND OFFSET - CHOOSING A LOOKUP TABLE 1) Select Palette 2) Select Range Indicator
Red = Saturation (maximum) Blue = Zero (minimum)
SCAN CONTROL - SETTING GAIN AND OFFSET “Detector gain” determines the sensitivity of the detector by setting the maximum limit “Ampl. Offset” determines the minimum intensity limit “Ampl. Gain” determines signal amplification Gain Saturation at the maximum →reduce “Gain” Saturation at the minimum increase “Offset” Gain set correctly
Offset set correctly
Offset Amplifier Gain increases the whole signal, and the offset will need to be decreased.
ADJUSTING GAIN AND OFFSET Both Gain and Offset saturated
1) Increase the Offset until all blue pixels disappear, and then make it slightly positive. 2) Reduce the Gain until the red pixels only just disappear.
OPTICAL ZOOMING The level of zoom can be changed either by using the zoom control under “microscope”, or by selecting “Crop” on the image menu
The image can also be rotated by selecting and dragging the bars (Only works with overlapped image)
MULTI TRACK CONFIGURATION 1) Select “Multi Track” for sequential scanning
2) Select “2P-DAPI/Cy2/Cy3”
2P-DAPI/Cy2/Cy3 (MULTI TRACK)
Two configurations used sequentially
TRACK 1 488/543 nm, Ar laser
2P-DAPI/Cy2/Cy3 (MULTI TRACK)
Two configurations used sequentially
TRACK 2 760 nm Chameleon laser
ADJUSTING THE LASER, GAIN AND OFFSET MULTI-TRACK CONFIGURATION Each channel is selected independently, and the laser power and other parameters are optimised as described in the previous slides. For accurate co-localization, adjust the Pinholes so that each channel has the same Optical Slice. 0.8 “Airy units” gives the best signal:noise ratio for normal confocal. Open the pinhole when using the Chameleon – twophoton excitation is inherently confocal and does not require a pinhole.
SETTING UP GAIN AND OFFSET - MULTI TRACK 1) Select Split 2) In Palette, select Range Indicator 3) Select each channel separately under Channels and adjust the Laser, Gain, and Offset as described previously. ***Switching btwn tracks takes time (fast xy is slower), optimize one track at a time (fast xy faster), then view both at same time
Split
LINE AVERAGING Averaging improves the image by increasing the signal : noise ratio Averaging can be achieved line by line, or frame by frame 1) Select Line or Frame 2) Select number for averaging. In general, the more the better. In this case, each line will be scanned 4 times (Be careful photobleaching increases with every scan so averaging more than 4 times will likely start to bleach your sample)
FRAME AVERAGING 1) Select “Frame” 2) Select the number for averaging - The more the better (max 16) BUT repeated scans increase bleaching. Continuous averaging is also possible in this mode
Frame averaging helps reduce photobleaching, but does not give quite such a smooth image. There is also a longer delay between each track when using “Multi Track”. Continuous averaging has a “Finish” button which allows the scan currently in progress to be completed before stopping
COLLECTING AN AVERAGED IMAGE 1) Return to Mode, and under Scan Average select the number for the average.
Under “Channels” select single”. An averaged image will be collected.
Range indicator switched off
SCANNING A Z SERIES USING “MARK FIRST/LAST” 1) Select “Z Stack” 2) Start scanning using “Fast XY” or “XY Cont” 3) Keep your eye on the image and move the focus to the beginning of the Z series - select “Mark First” 4) Move the focus back in the opposite direction to the end of the Z series, and select “Mark Last” 5) X:Y:Z sets the Z-interval so that the voxel has identical dimensions in X, Y, Z. 6) with Auto Z Corr., Detector Gain, AOTF, Ampl. Offset and Ampl. Gain can be varied between two (A, B) freely selectable slices of a stack NOTE Focusing can be achieved manually (preferred), or using “Stage” on the LSM menu Focus Increment
CONFOCAL Z-SECTIONING NUMBER OF SECTIONS FOR CORRECT SAMPLING Optical thickness d depends on: • • • •
wavelenght λ objective lens, N.A. refractive index n pinhole diameter P d ~ P n λ / (N.A)2
~ 0.5 µm @ 63x1.4
Optimal: (no missing information @ minimal number of sections) slices overlap by the half of their thickness
Nyquist- or Sampling- Theorem
Z STACK - NUMBER OF SLICES AND INCREMENT 1) Select Z slice - the window Optical Slice will appear 2) Select Optimal interval the computer will calculate the optimum number of sections 3) Select “Start”
For more or less sections adjust Num Slices
Z - SERIES USING “Z SECTIONING” 1) Select Z Stack 2) Select Z Sectioning 3) Select Line Sel 4) Select the large arrow button and position the XZ cut line
Z SECTIONING - SETTING RANGE
1) Decide whether to Keep Interval or Keep Slices 2) Select “Range” and position bars to decide where the Z - series begins and ends
Set limits for Z-Series
3) Select “Start”
Vertical section of sample
VIEWING A Z - SERIES
1) Select “xy” 2) Select “Slice” 3) Use scroll bar to view individual sections
VIEWING A Z - SERIES USING GALLERY
1) Select Gallery 2) Select Data for scale Use Subset to extract sections
VIEWING Z- SERIES USING ORTHOGONAL SECTIONS 1) select Ortho 2) Select mouse ( Select) 3) Using the mouse, position the cut lines. To save orthogonal sections, select Export and save as contents of image window.
SELECTING AND SAVING A REGION OF INTEREST 1) Select Overlay and define shape for ROI 2) Select “Extract region” 3) Save data
USING “EDIT ROI” FOR FASTER IMAGE ACQUISITION AND DATA SAVING 1) Select “EditROI” from the LSM menu bar 2) Select “Fit Frame Size to bounding Rectangle” 3) Choose ROI shape 4) position and size with mouse 5) Scan To remove ROI select blue bin
MULTIPLE REGIONS OF INTEREST 1) Un-select “Fit Frame Size to bounding Rectangle” Choose ROI shapes 2) Position and size with mouse 3) Scan To remove ROI select blue bin
TIME SERIES 1) Set up scanning parameters (ZSeries) 2) Select “Time series” from the LSM menu 3) Select “min,” “sec” or “ms” 4) Enter the number of cycles 5) Select “Start T”
VIEWING A TIME SERIES Z Sections for any time
Time points for any Z Section
Both Z sections and time series
TIME SERIES - PHYSIOLOGY EXPERIMENTS 1) If required, use multiple regions of interest 2) Set up time series as before 3) Instead of using “TimeSeries”, select “MeanROI” to start scanning
View and save data by selecting
IMAGING A LARGE AREA USING TILING 1) Select “Stage” on LSM menu 2) Enter the “Tile Numbers” 3) Select “Start”
3
3
Any position can then be marked and a single image acquired by selecting “Move to” and then single
TILED IMAGE
SAVING DATA - USING DATABASE 1) Select “Save or “Save as” on image window or LSM menu bar 2) Select an existing or generate (and name) a new database to save your images to. Please save to the ‘Big Disk’ 3) Then generate a file name for your image and notes if required 3) Select “OK” 4) Save subsequent images to this DB or a generate a new one Remember images do not exist without a database so be sure to transfer an entire database when saving files to DVD’s for future use. You must open the entire .mbd database to select individual images for export, ect.
Creating a database for acquired images
1) Select “New” database 2) Select drive Big Disk H: from pull down menu 3) Save to your folder on the H drive
SAVING INDIVIDUAL IMAGE FILES - USING “EXPORT” 1) Select “File”from LSM menu 2) Select “Export” 3) Select “Image type” 4) Select “Single image with raw data,” “Series with raw data,” or “Contents of image window” 5) Select “Save as type” “Tif - Tagged Image File” is OK for 8 bit - use “Tiff -16 bit” for 12 bit acquired images. Most other software will not recognize 12 bit.
SHUT DOWN PROCEDURE (cont’d) 1. Acquire - Laser - Switch off lasers
•Allow the Ar & HeNe lasers time to cool (light turns off)
•Turn the key on the Chameleon to Standby •log out •shut down the computer IF it’s the weekend or after 5pm (i.e., you don’t anticipate anyone else using the scope in the next hour)
SHUT DOWN PROCEDURE (cont’d)
2. File - Exit LSM 510 program. 3. Go to Windows START, shut down computer operating system. 4. Turn off the remote control switch.
• Please sign a terms of use agreement before using the microscope – Include contact info for the person responsible for billing, your fund/org numbers (CU only) and a billing address – Non-Cu researchers will receive monthly invoices (PO # are accepted with prior approval), CU researchers will be automatically billed, statements are sent out at the beginning of the month
• Please site the CUIBIF in all your publications: “This research was conducted at the Integrative Biological Imaging Facility at Creighton University, Omaha, NE. This facility, supported by the C.U. Medical School, was constructed with support from C06 Grant RR17417-01 from the NCRR, NIH.”
Questions? Contact : Heather Jensen-Smith 402-280-2248
[email protected]
CUIBIF Billing Address/ Remit payment to: Mary Anne Keefe Department of Biomedical Sciences, Criss II Rm 313 2500 California Plaza Omaha, NE 68178 402-280-2015
