Leica SP8

Confocal · Leica

A flexible confocal platform with 6 laser lines and 4 independent detectors. The SP8’s highly configurable detection system makes it well suited for experiments requiring broad spectral coverage and simultaneous multi-channel acquisition.

Instrument Features

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6 Laser Lines

Broad excitation coverage with 6 laser lines spanning UV to far-red (405, 458, 488, 514, 561, 633 nm). AOTF (Acousto-Optical Tunable Filter) provides precise, electronically controlled laser power adjustment for each line independently.

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4 Independent Detectors

Combination of PMT and HyD (Hybrid Detector) detectors allow simultaneous 4-channel acquisition. HyD detectors offer superior sensitivity and signal-to-noise for dim samples, with photon counting capability.

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Spectral Detection Flexibility

Continuously tunable detection windows on each detector allow precise spectral band selection without changing filters. Minimizes crosstalk between channels and maximizes signal collection for each fluorophore.

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Simultaneous Multi-Channel Acquisition

All 4 channels can be acquired simultaneously, enabling fast multi-color imaging with minimal photobleaching and no time delay between channel acquisitions — critical for co-localization and dynamic studies.

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FRET Capability

Sensitized emission and acceptor photobleaching FRET protocols are well supported. The precise spectral control and multiple laser lines make the SP8 a strong platform for protein-protein interaction studies.

🖥️

LAS X Software

Leica Application Suite X provides an intuitive workflow for experiment design, acquisition, and analysis. Includes automated wizards for Z-stacks, tile scans, FRET, and time series with straightforward channel configuration.

Startup Guide

1

Power On the System

The computer needs no special login.

  1. Follow the startup poster at the microscope.
  2. Turn on buttons 1 to 5 in the order shown on the poster.
  3. Open the LAS X software on the computer.
2

Load your sample and find it

  1. Place the dish on the stage with the 10x objective in position.
  2. Get the sample in focus using the controls on the microscope stand interface.
  3. Find and focus your sample with the 10x.
  4. Move to your final objective (20x, 40x or 63x oil) and add immersion oil as needed.
  5. Refocus on the sample.

Use oil only on the oil objectives. Clean oil objectives with lens tissue only and clean oil off sample and objectives when going from oil to 10x and back

ObjectiveImmersion
10xAir (the only air objective)
20xOil
40xOil
63xOil
3

Set up imaging parameters

Reusing the saved parameters is the quickest route. Use Dye Assistant only when your dyes differ.

Reuse saved parameters

  1. Go to IPS and Load the pre established parameter file. It is a file on the desktop, and it only needs to be loaded.
  2. Open the Projects tab and open any image.
  3. Click Apply so the imaging parameters are loaded into the acquisition settings [3].
  4. If the lasers are off, answer yes when LAS X asks to turn them on [3].

Or use Smart Setup

  1. GO to “DYE assistant” and select all the fluorophores you in your sample
  2. It is optimal to manually select which detector to use (HyD or PMT). Ask Adeela for setting this up
  3. Line switching will be the fastest way to image
4

Acquire an image

  1. Click Live and focus the sample. Use a lower format and fewer averages while searching, then return to your final settings before capturing.
  2. Keep gain in the low 700s. Adjust laser power rather than raising gain above 800.
  3. Do not saturate pixels. Use the glow over lookup table, where saturated pixels show in blue, and lower laser power or gain until they disappear.
  4. Set the scan speed to about 6.
  5. Turn on bidirectional scanning. If the image looks doubled or blurry, adjust the phase until a vertical structure no longer appears duplicated.
  6. Average 2 to 4x as needed for noisy images.
  7. Keep the pinhole at 1 Airy unit for confocal imaging.
  8. Use the format optimisation button to set pixel size to Nyquist.
  9. Z stack: Move to one end of the sample and click Begin, then to the other end and click End. Choose the system optimized step for Nyquist spacing. *Use z compensation for thick samples where signal fades with depth
  10. Click Capture Image for a single image, or Start for the full series with sequences, z stack and time lapse
  11. Save the project to the folder your trainer gives you. Images sit in a temporary location until you save the project
5

Tile scans and multiple positions

Open Sample Navigator when you want to set up a tile scan or multiple positions.

  1. Open Navigator and select the region of interest [4].
  2. Turn on automatic stitching for tile scans. If you have a z stack, each tile gets one [4].
  3. For large tile scans, set focus points so every tile stays in focus [4].
  4. For several positions or multi position time lapses, use mark and find [4].
6

Shutdown

  1. Clean oil off objectives and save data for processing
  2. Exit out of LASX
  3. Check iLabs schedule in and if anyone is coming in within an hour leave system on
  4. If no one is coming in for more than an hour turn follow instructions on poster to shutdown system

💡 HyD detectors provide the best sensitivity for dim samples — use them for your weakest channels and reserve PMTs for bright channels to get the most out of simultaneous 4-channel acquisition.

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