Leica laser confocal microscope

The TCS SP8 DLS Leica laser confocal microscope combines the advantages of two imaging systems: with Leica TCS SP8 DLS (digital light sheet), you can achieve confocal microscopy using soft single plane illumination on the microscope. By developing the TwinFlect reflector, we can turn the confocal platform Leica TCS SP8 into a simple and easy-to-use multifunctional optical microscope.

Leica laser confocal microscope (such as Leica SP8, Stellaris series) is a high-resolution 3D imaging system widely used in biomedical, materials science and other fields. Its operation requires the combination of optical, laser control, and image processing technologies. The following is a detailed operation guide:

1、 Preparation before operation

1. Environmental and equipment inspection

Environmental Requirements:

Independent darkroom or shading environment to avoid external light interference with fluorescence signals.

The temperature should be controlled at 20-25 ℃ and the humidity should be ≤ 60% to prevent lens condensation or circuit failure.

Equipment inspection:

Confirm that the status indicator light of the laser (such as 405nm, 488nm, 561nm, 633nm) is normal (green indicates ready).

Check if the objective lenses (such as 10x/0.3, 20x/0.75, 63x/1.4 oil lenses) are clean and if the stage movement is smooth.

Turn on the cooling system (such as the laser water cooler) to ensure that the temperature remains stable at the set value (usually 18-22 ℃).

2. Sample preparation

Biological samples:

Live cells need to be cultured in confocal specialized culture dishes (such as Lab Tek II) to avoid difficulties in focusing caused by the use of ordinary glass slides.

Fixed tissue slices need to be sealed with anti fluorescence quenching sealing agent (such as ProLong Gold), with a thickness of ≤ 20 μ m.

Material samples:

Non transparent samples need to be polished to a surface roughness Ra<0.1 μ m, while transparent samples (such as polymers) need to avoid interference from spontaneous fluorescence.

Marking requirements:

Fluorescent dyes need to be matched with the laser wavelength (e.g. DAPI excited at 405nm, Alexa Fluor 488 excited at 488nm).

When marking with multiple colors, ensure that the emission spectra of the dyes do not overlap (signals can be separated by spectral splitting function).

2、 Basic operation process

1. Startup and initialization

Start sequence:

Turn on the main power → 2 Start the laser cooling system → 3 Turn on the microscope host → 4 Start control software (such as LAS X or Stellaris Suite).

Software self-test:

The software automatically detects modules such as the laser, scanning head, and stage, and confirms that the status is "Ready".

Run the 'Daily Check' function to calibrate the optical path and detection system sensitivity (such as PMT gain, laser power).

2. Sample positioning and focusing

Low magnification preview:

Select a 10x air mirror, turn off the laser, and use transmitted light (such as halogen lamps) to quickly locate the sample area.

Move to the target position using the XY knob on the stage or software navigation function.

Confocal focus:

Turn on the 488nm laser (low power, such as 5%) and select the "Live" mode for real-time observation.

Adjust the Z-axis stepper motor and use the "Fine Focus" tool to gradually focus until the fluorescence signal is strong.

When switching to a high magnification objective lens (such as a 63x oil lens), specialized immersion oil (such as Type F oil) needs to be dripped between the objective lens and the cover glass.

3. Image acquisition parameter settings

Laser power and PMT gain:

Laser power: gradually increase from 1% to avoid photobleaching (usually not exceeding 20%).

PMT gain: Adjust to a signal strength between 100-800 (12 bit image) to avoid saturation (>4095).

Scanning parameters:

Resolution: Choose 512 × 512 (quick preview) or 2048 × 2048 (high-precision imaging) according to your needs.

Scanning speed: Line average frequency 1-4 times (increases when noise is high), frame average frequency 1-8 times (decreases when dynamic imaging of live cells).

Pinhole size: usually set to 1 Airy unit (AU), balancing resolution and signal-to-noise ratio.

Spectral settings:

When marking with multiple colors, scan the sample emission spectrum in "Lambda Scan" mode and set the wavelength range (e.g. DAPI: 410-480nm, Alexa Fluor 488: 500-550nm).

4. 3D imaging and time series

Z-axis stacking:

Set the starting/ending position and step size (such as 0.5 μ m), and the software will automatically capture multiple layers of images and synthesize a 3D model.

Time series:

When imaging live cells, set the time interval (e.g. 1 frame every 30 seconds) and total frame rate (e.g. 100 frames) to record the dynamic process.

Enable the 'Autofocus' function to maintain focus stability and avoid defocusing caused by cell movement.

3、 Leica Laser Confocal Microscope Function Application

1. Light Sheet Lighting Mode

Suitable for rapid low light toxicity imaging of thick samples, such as zebrafish embryos.

Operation steps:

Switch to the light module and adjust the thickness of the lighting light sheet (usually 2-5 μ m).

Set up dual cameras to synchronously capture (such as transmitted light and fluorescence signals).

Use the "Multi View" function to stitch multiple angle images and reconstruct three-dimensional structures.

2. Super resolution imaging (such as STED)

Breaking through the diffraction limit, the resolution reaches 20-50nm.

Operation points:

Use specialized STED objective lenses (such as 100x/1.4 oil lenses) and depletion lasers (such as 592nm).

Adjust the depletion laser power (usually 50-200mW) and phase mask mode (such as 2D or 3D STED).

After collection, use "Huygens" or "Thunder" software for deconvolution processing.

3. Spectral splitting and linear deconvolution

Separate overlapping signals during multi-color marking:

Obtain the sample emission spectrum library in "Lambda Scan" mode.

Load spectral data in the "Spectral Unmixing" tool, and the software automatically calculates the pure signals of each channel.

Adjust the threshold to remove background noise and save the split image.

4、 Maintenance and upkeep

1. Daily cleaning

Optical components:

Use a balloon to remove dust from the surface of the objective lens and filter, and do not use cotton swabs or tissues to wipe directly.

If cleaning is required, dip a small amount of ethanol (or specialized cleaning solution) into lens paper and gently wipe it in a spiral manner.

Stage and sample compartment:

Wipe with an anti-static cloth to avoid residual oil or sealant.

2. Laser maintenance

Usage restrictions:

Work continuously for no more than 8 hours to avoid overheating and damage to the laser tube.

When shutting down, first reduce the laser power to 1%, and then turn off the laser switch.

Lifetime monitoring:

Check the remaining lifespan in the "Laser Status" interface of the software (such as 488nm laser usually ≥ 20000 hours).

When approaching the end of its lifespan, contact Leica in advance to replace the laser tube.

3. Regular calibration

Optical path calibration:

Calibrate the optical axis and pinhole alignment using standard fluorescent microspheres (such as TetraSpeck) every quarter.

Mechanical calibration:

Check the accuracy of the XY/Z axis of the stage annually (error should be<0.1 μ m), and adjust the parameters of the stepper motor if necessary.

5、 Common problem solving

Image without signal

Check if the laser is turned on, if the PMT gain is greater than 0, and if the filter matches (if the emission filter does not block the signal).

image blurring

Confirm whether the immersion of the objective lens is sufficient, whether the pinhole is offset, and whether the sample is flat (such as defocusing caused by the protrusion of live cells).

Unstable laser power

Clean the laser outlet window and check if the cooling system water temperature is within the set range (such as ± 0.5 ℃).

Software lag

Close other large programs, reduce image resolution or scanning speed, or increase computer memory (recommended ≥ 32GB).

6、 Leica Laser Confocal Microscope Safety Precautions

Laser safety:

Wear laser protective goggles during operation (wavelength must cover all laser lines used).

Do not stare directly at laser beams or reflected light, and avoid prolonged exposure of the skin to high-power lasers (such as>50mW).

Bio-Safety:

When handling live cells or pathogens, operate in a biosafety cabinet to prevent sample contamination.

Chemical safety:

When using organic solvents (such as ethanol) for cleaning, operate in a fume hood to avoid inhaling volatile gases.