Leica SP8 Training Manual
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Leica SP8 Training Manual

Confocal/multiphoton microscope las af v3.3, optical imaging & vital microscopy core baylor college of medicine 2017
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Training Guide for Leica SP8 Confocal/Multiphoton Microscope
LAS AF v3.3
Optical Imaging & Vital Microscopy Core
Baylor College of Medicine (2017)

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Summary of Contents for Leica SP8

  • Page 1 Training Guide for Leica SP8 Confocal/Multiphoton Microscope LAS AF v3.3 Optical Imaging & Vital Microscopy Core Baylor College of Medicine (2017)

  • Page 2: Power On Routine

    Power & PC Microscope green Wait for Windows PC to boot and switches then turn Laser Emission key This lamp will be used to visualize Login to User Account. to ON state. fluorescence in the microscope eyepieces. 2 - Leica SP8 Training Guide...
  • Page 3 Power ON Routine – for NLO Only Turn Chameleon TiS laser key from Turn ON power strip for APT. Toggle Laser selector to position A - ‘Standby’ to ‘ON’. ‘Leica’. 3 - Leica SP8 Training Guide...
  • Page 4 Power ON Routine – for NLO Only Select USB hub to Position 1. Once logged in, verify that the USB Start ‘APTUser’ application from connection is active for the APT. desktop. Green is active. 4 - Leica SP8 Training Guide...
  • Page 5 Power ON Routine – for NLO Only Click ‘Home/Zero’ to home the Once homing is complete, click the Enter ‘36’ into position for Ch1 and device. current position display. press OK. Minimize APT software. 5 - Leica SP8 Training Guide...
  • Page 6 Note: If you do not require high speed scanning – leave Resonant set to OFF. If you turn it on you will not be able to use slower speeds. Click OK to initialize system. 6 - Leica SP8 Training Guide...
  • Page 7 This area is where the image will be displayed. Settings here allow the user to control how the image is viewed on screen. Scan Action Functions These functions start/stop scanning and initiate our experiment acquisition. 7 - Leica SP8 Training Guide...
  • Page 8 Getting Started with LAS 8 - Leica SP8 Training Guide...
  • Page 9 Note: For optimal conditions set the Argon laser power to 10% output power. 3. Close Available Lasers dialog. 4. To enable Visible Lasers for use – toggle the ON/OFF switch above + to ON state. 9 - Leica SP8 Training Guide...
  • Page 10 5. Via the microscope eyepieces, visualize your sample and adjust the focus (Z) and stage position (X,Y) accordingly 6. When sample is in focus and positioned correctly – switch microscope back to CS – Combi to begin imaging with the confocal. 10 - Leica SP8 Training Guide...

  • Page 11: Beam Path Configuration

    3. Turn on PMT for detection and select LUT (look up table or color) for that particular channel. 4. Define upper and lower emission band thresholds based on the spectrum for your fluorophore. 11 - Leica SP8 Training Guide...
  • Page 12 2. Once these options are selected you can click + to add ‘Seq. 2’ and repeat steps 1- 4 on the previous page to change the beam path settings to match your second fluorophore. 3. Repeat process for total number of channels required. 12 - Leica SP8 Training Guide...
  • Page 13 Note: We always recommend that you collect your longest wavelength fluorophores first in order to ensure minimal photo bleaching of lower wavelength fluorophores. Example Beam path for Seq. 1 – Alexa 568 Example Beam path for Seq. 2 – Alexa 488 13 - Leica SP8 Training Guide...

  • Page 14: Acquisition Setup

    This will be the most effective method for reducing noise in the resulting image. Pinhole – this controls the optical section thickness. To start – select ‘Airy 1’ button to set optimal pinhole diameter. This can be increased or decreased to adjust image quality. 14 - Leica SP8 Training Guide...

  • Page 15: Scanning An Image

    Note: Increasing Gain will effectively increase PMT sensitivity to signal, making your image brighter. This will also increase noise which can be reduced with scan speed/averaging adjustments. 5. Repeat for remaining channels. 6. Stop scan and move to ‘Fine Tuning Image Quality’ section. 15 - Leica SP8 Training Guide...
  • Page 16 Note: Capture Image will scan with same scan speed and frame size as LIVE, and in addition it will apply any Line or Frame Averaging you have set to control image noise in the Scan Control settings. 16 - Leica SP8 Training Guide...
  • Page 17 (green). In a properly balanced image you should see no blue green pixels with this LUT. Example of Overexposure (blue) and Example of Good Exposure Underexposure (green) Solution? Reduce Gain and increase Offset 17 - Leica SP8 Training Guide...
  • Page 18 Closing this aperture nonlinear effects such as in the system. will reduce the signal level but phototoxicity and photobleaching. increase axial resolution. 18 - Leica SP8 Training Guide...
  • Page 19 In other cases, if the gain is too high (causing excess noise) and the laser power cannot be increased due to photo effects then increasing the confocal aperture is the only option to improve image quality. 19 - Leica SP8 Training Guide...
  • Page 20 Continual increases in laser power will stop yielding more signal. That point is the practical laser power limit. 20 - Leica SP8 Training Guide...

  • Page 21: Detector Sensitivity

    The second is signal averaging which will scan the same image multiple times and average the result to produce an image that has an improved signal-to-noise ratio. 21 - Leica SP8 Training Guide...
  • Page 22 8x Averaging 16x Averaging Note: Averaging signal will increase the acquisition time by the factor of averaging. For example, a 4x average will increase the time to scan the image by a factor of 4. 22 - Leica SP8 Training Guide...
  • Page 23 ‘Z-Step Size’ and increasing the value. 6. Click Start to being Stack acquisition. 23 - Leica SP8 Training Guide...
  • Page 24 Note: The system will automatically calculate how many images it needs to collect over the interval/duration you set. You can also manually calculate this and entire the values accordingly. 4. Press ‘Start’ to initiate Time Series. 24 - Leica SP8 Training Guide...
  • Page 25 Generally, if you saving files one at a time you can click ‘No’ and save them separately. 25 - Leica SP8 Training Guide...

  • Page 26: Shutting Down System

    If NLO was used – follow these additional steps: 9. Power OFF APT power strip (see Power ON Routine – Step B). 10. Turn the key on Chameleon TiS laser to ‘Standby’ (see Power ON Routine – Step A). 26 - Leica SP8 Training Guide...

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