Advertisement
Quick Links
LMW Microscopy Lab : Leica SP2
Enter you name, the date, the time, and the account number in the user log book.
Things to check before start‐up.
Make sure that your sample slides are clean and sealed. Use Windex and cotton balls or Kimwipes to clean
coverslips. Fixed samples need to be sealed with nail polish.
Check the objectives. The 10x and 20x objectives are DRY objectives, and should NEVER have oil on them!
There are 40x and 63x OIL immersion objectives, and a 63x WATER immersion objective also on this
microscope. If oil immersion objectives have oil on them, wipe the lens gently only with lens paper.
System Start‐up.
1. Turn on the microscope controller
Scanner Laser HeNe
switches.
2. Turn on the computer
desktop.
3. Turn on the Mercury Arc Lamp power supplier,
if you look at fluorescently labeled samples.
4. Start the LCS program by
double‐clicking the LCS icon on
the desktop.
On the pop‐up starting window panel, select
the Personal that contains your setting for
acquisition parameters you have created or
Company for a default setting.
Manual for Leica SP2 Confocal Microscope
b
and the
c
d
and PC/Stand
e
and log on to the
Page 1
`6/26/2012
b
d
c
f
e
Advertisement
Subscribe to Our Youtube Channel
Related Manuals for Leica SP2
Summary of Contents for Leica SP2
- Page 1 LMW Microscopy Lab : Leica SP2 Page 1 `6/26/2012 Manual for Leica SP2 Confocal Microscope Enter you name, the date, the time, and the account number in the user log book. Things to check before start‐up. Make sure that your sample slides are clean and sealed. Use Windex and cotton balls or Kimwipes to clean coverslips. Fixed samples need to be sealed with nail polish. Check the objectives. The 10x and 20x objectives are DRY objectives, and should NEVER have oil on them! There are 40x and 63x OIL immersion objectives, and a 63x WATER immersion objective also on this microscope. If oil immersion objectives have oil on them, wipe the lens gently only with lens paper. System Start‐up. 1. Turn on the microscope controller and the Scanner Laser HeNe and PC/Stand switches. 2. Turn on the computer and log on to the desktop. 3. Turn on the Mercury Arc Lamp power supplier, if you look at fluorescently labeled samples. 4. Start the LCS program by ...
- Page 2 LMW Microscopy Lab : Leica SP2 Page 2 `6/26/2012 Oveview of Leica Confocal Software (LCS) Upon LCS program start, the basic image acquisition menu will appear. The Acquire button should appear pressed. Click the Beam button to display the Beam Path Setting as appeared. Click the Z‐Scan button and select z‐Wide (this is required to focus to sample on the regular slide holder by the Remote Control Knobs; see below). With the Obj button , you can select the objective you want to use. (A message window may pop up, and click OK) With MicCtrl button , you can switch between Scan mode (for laser scanning) and Visual mode (for viewing through the eyepiece). ...
- Page 3 LMW Microscopy Lab : Leica SP2 Page 3 `6/26/2012 Basic Operation of Leica DM IRE2 Microsocpe. Bright‐field viewing: 1. Place your sample slide with coverslip (preferably #1.5 thickness) facing down to the objective on the stage. 2. For bright‐field viewing, turn on the microscope halogen lamp light by turning the wheel toward you and turn the VIS‐SCAN switch to VIS position (Note: switch back to Scan position for confocal scanning, see below) 3. Start with low magnification objectives (10X or 20X) first to focus onto your specimen. You can place the desired objective in position by turning the lens turret by hand or by clicking Obj button and selecting an objective you want to use (recommended; see below). The information of the selected objective and their z‐position can be read on the readout panel at the front of the scope. 4. Focus on to the sample by moving the objective turret with the Coarse Focus Buttons (upper and lower) and the Fine Focus Knob . Push the ...
- Page 4 LMW Microscopy Lab : Leica SP2 Page 4 `6/26/2012 Fluorescent viewing: You can check your sample to see if your fluorescence labeling works by using the mercury lamp illumination and the appropriate filter sets. The microscope has filter sets for viewing DAPI (A), green fluorescence (I3) and red fluorescence (N2.1). 1. Press either the left or right horizontal arrow button on the front of the scope to choose the filter (FLUO) set. “Scan” position has no filter set in place and is used for laser confocal scanning. The fluorescent light can be blocked (closed) or emitted by pressing the Shutter button (The “closed” light should be off for fluorescent viewing.). 2. Turn the VIS‐SCAN switch to Scan position to block the transmitted bright‐field light, if necessary. 3. Examine the sample through the eyepieces. (Make sure that PORTS is in VIS and MAG is at 1x. If not, change it selecting Visual from MicCtrl button in LCS program window.) 4. When you are ready to scan your sample with laser, close the SHUTTER to prevent any unnecessary bleaching of the sample with mercury arc lamp light. Setting up for Confocal Laser Scanning. To turn on the laser(s); ...
- Page 5 LMW Microscopy Lab : Leica SP2 Page 5 `6/26/2012 LCS Imaging program setting 1. First, select the appropriate laser lines and set up detectors for the fluorescent labels you used on your sample from the drop‐down L list (Leica Factory setting). This will automatically set up the emission detector band‐width , activate the proper detector (PMTs) , dichroic beamsplitter , and excitation laser wavelength and power . You can adjust the range and position of detector bandwidth and the laser power level as necessary. 2. You can also change the color scheme of individual images into any color or grey scale by clicking the pseudo‐color selector associated with each PMT on the window screen (Note: the color information will be saved with image files when saved). 3. Press Mode button to select Scan Mode (default is XYZ). ...
- Page 6 LMW Microscopy Lab : Leica SP2 Page 6 `6/26/2012 4. Press Format , and Speed buttons to select these parameters (defaults are 512x512 format and 400Hz scan speed). 5. Press Z‐scan button and select z‐Wide in order to use Z POS knob to move the objective for focusing (Default is z‐Galvo, which is used with Galvo stage adaptor). 6. Click MicCtrl button to select Scan mode for laser scanning (it will automatically shut off visual output through the eyepieces). 7. Press Continuous button to start scanning (it will become Stop button). An image or images, depending on the number of active detectors, will appear on the right monitor. Remote Control Knobs 8. Optimize the images by adjusting parameters including; 1. The z‐position (focusing) within the specimen (Z‐POS knob 2. Zoom factor (ZOOM knob ): Default is 1; increasing zoom will magnify the image and improve the resolution, but also will bleach the sample faster! : Turn PMT knobs to increase the detector sensitivity so to ...
- Page 7 LMW Microscopy Lab : Leica SP2 Page 7 `6/26/2012 Click the Stop button again to stop scanning. (You can save the current parameter setting with Save button and put a specific name for later use). Viewing Images. Single button– puts up full screen of selected fluorescent channel. Ch 1, 2, 3, 4 buttons– activate the channel on the screen. Tiled button– views up to 3 channels simultaneously. Ovl button – overlays (merge) multiple channels into one. Display button‐ change the ...
- Page 8 LMW Microscopy Lab : Leica SP2 Page 8 `6/26/2012 After scanning, the image will be saved in the directory (temporarily in the RAM). Save the data to the hard drive with the Save button on the top menu icon panel , and type the file name. (Note: All the images (as *.tiff) will be saved under the saved Leica file (as *.lei)). Computer crash or power outage will result in loss of unsaved data! SAVE FREQUENTLY!! If you want to create a new folder for a new set of images, click New icon on the menu panel. ...
- Page 9 LMW Microscopy Lab : Leica SP2 Page 9 `6/26/2012 Sequential Scanning Mode This confocal microscopy can detect up to four channels (3 fluorescence and 1 transmission) simultaneously as long as all the excitation and emission spectra of fluorophores are well separated. Imaging of samples stained with different dyes thus simply requires manipulation of laser power and adjustment of the detector bandwidth. However, often excitation of one fluorophore may cause the emission to appear into the range of another (bleed‐ through) or can be induced by neighboring laser lines (cross‐talk), both of which produce false signals. To avoid these, the sample should be scanned sequentially by collecting one fluorophore signal at any given time. There are two ways to set up sequential scan mode depending on fluorescence of your sample. To scan sequentially the sample without need to use 405 nm UV laser (no DAPI staining) : 1. Choose the beam setting parameter for your fluorophores (for example, GFP and DsRED) by selecting the Leica FITC‐TRITC from the preset list). It will set laser lines, dichroic mirror, and PMTs for simultaneous imaging of the two colors. 2. Set up a condition for one ...
- Page 10 LMW Microscopy Lab : Leica SP2 Page 10 `6/26/2012 5. Click the Seq button in the Beam Path Setting window. The Sequential scan settings window will appear. Add the fluorophore acquisition setting one at a time by selecting it from the preset list menu and then click Add button in the Sequential scan settings window. 6. Keep the scan mode at “between lines”. Do not close the Sequential scan settings window. You can save this sequential acquisition settings by Save button in the Sequential scan settings window. 7. Start the acquisition by clicking Single Scan or Series button depending on your imaging condition. ...
- Page 11 LMW Microscopy Lab : Leica SP2 Page 11 `6/26/2012 To scan sequentially the sample with need to use 405 nm UV laser (DAPI staining): Due to the hardware for UV laser, sequential scanning of a specimen containing UV dye (i.e. DAPI or Hoechst) needs a different configuration. Here is an example of specimen stained with DAPI and FITC. 1. For setting up DAPI, double click L‐ 405 only from the preset list. It will make UV laser and PMT1 active. Start scanning by clicking Continous button. 2. While scanning, adjust the image with PMT1 knob and Offset knob (and laser power if necessary). 3. Stop scanning and select the number of line averaging with the Li.A button. Save the setting as described above (for example “training blue”). 4. Next, double click on L‐488DD from the preset list and it will make 488 nm laser and PMT2 active and turn off UV laser and PMT1. Start scanning and adjust the image quality with PMT2 knob and Offset knob (and 488 nm laser power if necessary). 5. Stop scanning and select the number ...
- Page 12 LMW Microscopy Lab : Leica SP2 Page 12 `6/26/2012 Acquiring Z‐series images. Z‐series allows acquisition of optical sections through the certain volume of your sample that can be used for making 3D images. 1. To set the z range, press Continuous button, turn Z‐POS knob in counterclockwise to focus onto a specific z position of your sample and click End button (it will appear pressed). 2. Turn Z‐POS in opposite direction (clockwise) to focus on a deeper region of the specimen and click Begin button (pressed). 3. Click Stop button to stop scanning. 4. (optional) Click Series button to check the thickness of the z‐series and determine the number of optical sections. 5. Click Sect button to select the predetermined number of optical section, or to select Others… 6. Selecting Others brings up a z‐configuration window to determine the number of sections and spacing (step size) between sections. Enter a desired step size (~1 µm for 63x objective) and click Calculate button next to the # Section. It will automatically calculate the # of sections according the Step size. Press OK. 7. Choose the number of averaging using Li. A. button and click “Series” button to begin ...
- Page 13 LMW Microscopy Lab : Leica SP2 Page 13 `6/26/2012 Acquiring Time Series images. Time series allows time‐lapse imaging of live samples to study the changes and dynamics of your object of interest. The time‐lapse images can be saved as multiple Tiff files and AVI files for playing in movie programs. 1. To set obtain time‐ series images, select scan Mode as xyt. It will activate Time buttons . 2. Click Time button to open the time‐lapse setting window and set the parameters such as the time interval between frames and the number of frames. Set time 3. Check one of three parameters and interval enter the desired numbers into the other two parameters Hit Enter key Set the number on keyboard, which will calculate of frames the value for the checked ...
- Page 14 LMW Microscopy Lab : Leica SP2 Page 14 `6/26/2012 Processing and exporting images: The original images are saved as single tiff files; for example a two‐color channel image is saved in two tiff files ‐‐ one for each channel. You can save a current screen image after image processing such as brightness‐contrast enhancement and merging channels (useful for powerpoint presentation). 1. Adjust images and put annotations you want, such as scale bar. 2. Click the Display button and select 1:1 to display image in exact pixel dimension. 3. Click on the image you want to export and right‐click on the mouse to bring the context menu; Send To>Experiment>Selection (raw)/Selection (snapshot)/All (snaphot). Select one of these options and it will export to a new image file. Selection (raw): export the current selected image only (no annotations included). Selection (snapshot): export the current selected screen image with all annotations. All (snapshot): export all the screen images with annotations. 4. For time series, you can go through or play the time lapse movie with sequence buttons (First, Next, Play, Prev, Last). For Z‐series, Max or Avg buttons projects the different sections into a projected 2D image. You can also visualize the cross‐section of the specimen with Sect. button. 5. Save the *.lei folder file whenever you add new images. ...
- Page 15 LMW Microscopy Lab : Leica SP2 Page 15 `6/26/2012 Saving Images. This is a very important for your effort and data. Computer crash or power outage will result in loss of unsaved data! SAVE FREQUENTLY!! To save the imaging data files, click “Save” button at the top of the window. Select D drive and users folder. Create your folder, rename the Experiment# and save. Since the storage memory in the computer will be filled up quickly and this lack of hard drive space prevents image acquisition, all user files will be deleted after two weeks. Be sure to copy your files to a flash memory or CD/DVD disks. Shut‐down Procedure Please enter your time of use in the log book and note any problems and suggestions during your time. 1. Clean the oil from the objectives only with lens paper (not Kimwipe). Clean the microscope and stage. 2. Close the LCS program. 3. Copy your data files to a flash drive or CD. Users are responsible for their own files, as there is no automatic backup at present. 4. Log off the computer (not turning off the computer) and turn off the microscope control box. 5. If there is someone who wants to use, leave the instrument ON. 6. If you are the last user of the day: 1) Turn off the mercury lamp . 2) Minimize the power of the Ar/ArKr level for >3min before turning it off. 3) Around 3 min after minimizing the Ar/ArKr ...
Need help?
Do you have a question about the SP2 and is the answer not in the manual?
Questions and answers