Start the System
- Turn the Key on position.
- Switch on the Main Switch.
- Switch on System/PC.
- Switch on Components.
- Turn on the PC.
- Switch on X-Cite.
- Turn the Key on to run Fan of Argon laser. Switch on Argon Laser.
- Turn Toggle Switch on position.
- Start LSM System on the software.
Find Your Sample
- Pull back the illumination pillar of the microscope carefully.
- Click Load position on touch pad.
- Place your sample (slide faced down). If 63x is going to be used, put a small drop of immersion oil onto objective lens.
- Bring down the illumination pillar back to viewing position.
- Click Work position on touch pad.
- On the software, select Ocular, click Online, select a Filter to view fluorescent image, find your samples and focus on them.
- Select Acquisition and open Smart Setup. Input data of Dyes used in the sample. Choose best signal for tracking. Filters and lasers to be used will be automatically assigned by software.
- Click Auto Exposure (automatic pre-adjustment of gain and detector).
- Click Live and optimize your image. Open Range indicator and check if there is over exposure (seen as red pixels). If there are red pixels, decrease gain until they are barely visible. Apply it for each track individually. Digital offset sets gray level of a selected background to zero and adjusts the darkest features in the image to black (default is 0). Amplified gain default is 1. Set Pinhole to 1 Airy Unit for each track.
- Choose all the tracks and click on Optimal in Acquisition mode
- Set Scan Speed 7, Bit Depth 16 Bit and Average Number 4.
- Click on Snap and get an image. If the image is taken slowly, S/N is expected to be high. Noise on the screen is seen as pixels on empty fields supposed to look black. On Display menu when white line is pulled towards left, noise will be seen as bright dots on background. When the image is screened slower it will be noticed that noise is decreased. However, it may lead bleaching sample. Increasing the gain may increase the noise. A good image may be recognized by smooth transition of colors.
- After image is taken, it can be optimized by adjusting Graphics.
- Select New when you want to save previous image.
- Mode describes the way of screening. When Frame is selected, laser screens the specimen from top to bottom as a frame.
- Choosing 16 averaging with lower laser intensity may prevent bleaching and still provide good image quality.
- Choosing Sum collects images multiple times and overlaps them. It can be wise to use when there is low signal however noise accumulation accompanies.
- When double directed screening is selected, the laser starts to screen the next line continually as soon as the previous line is screened.
- When Crop is selected, a specific area on image may be focused, direction of screener might be changed.
- When Display is selected, gamma value is assigned as default 1 and should not be changed.
- When Stage is selected, using + allows moving on the specimen.
- Time Series is used to view live cells. Here, cycle refers to number of screening and interval refers to time difference between each screening.
- When bleaching is applied, 1 cycle is assigned for bleaching while other cycles screen for imaging before and after bleaching. Iterations determines number of laser shooting. Laser intensity and iteration number may be adjusted during bleaching.
- Tile Scan screens a wider area on motorized table.
- When Position is selected, cells on the screen might be chosen. Clicking on Start Experiment initiates screening of selected positions. When this combined with cycle, selected positions are screened with time intervals.
- Linear unmixing discriminates overlapping emission values.
- When Z-Stack is selected, 3D images of specimen might be obtained. Assign the borders of your specimen on z-axis by selecting Set First and Set Last. Chose optimal interval and start experiment.