Laser scanning confocal microscope can: (1) light section scanning, 3D image processing, time series shooting imaging; (2) cell, green fluorescent protein, biofluorescence sample observation and analysis; (3) fluorescence in situ hybridization analysis.
Operation method
Confocal microscope laser scanning method
Principle
Confocal laser scanning microscope (CLSM) with a laser as a scanning light source, point by point, line by line, surface by surface rapid scanning imaging, scanning laser and fluorescence collection share an objective lens, the focal point of the objective lens that is the focus of the scanning laser is the focus point, but also the instantaneous imaging of the object point. Due to the shorter wavelength of the laser beam, the beam is very fine, so the confocal laser scanning microscope has a high resolution, about 3 times that of an ordinary optical microscope. The system is focused once and the scanning is limited to one plane of the sample. When the focusing depth is different, the images of different depth levels of the sample can be obtained, and these image information are stored in the computer, which can show the three-dimensional structure of the cell sample through computer analysis and simulation.
Materials and Instruments
Cells Move I. Observation and instrumentation 1. Turn on the instrument power and light source Caveat 1. the instrument is surrounded by areas away from sources of electromagnetic radiation;2. the environment is free from vibration and strong air disturbance;3. the room has a shading system to ensure that the fluorescent samples will not be bleached by exogenous light;4. the environment is clean;5. control the working temperature in 5 ~ 25 ℃. Common Problems There are many types of samples that can be measured by laser scanning confocal microscopy. These include biological materials, tissues (sections), cellular (subcellular) structures and so on. The sources of fluorescence in the samples are as follows: autofluorescence, fluorescence staining, immunofluorescence, fluorescent proteins, induced fluorescence and enzymatic produced fluorescence. The excitation and emission wavelengths of most of these fluoresceins can be found in the dye information library of the instrument's own software. For more product details, please visit Aladdin Scientific website.
Fluorescent dye Water Culture medium
Laser Scanning Confocal Microscope Slides Wash Bottles Droppers Test Tubes
Generally turn on the microscope and laser, then start the computer, and then start the operation software, set the excitation light wavelength of the fluorescent sample, select the appropriate filter block. So that the photomultiplier tube (photo multiplier tube, PMT) detector can get enough signal results. The precautions for using mercury lamp are the same as those for ordinary fluorescence microscope.2. Set up the corresponding scanning mode
In visual mode, adjust the magnification of the objective lens used to find the cells to be detected under the fluorescence microscope. Switch to scanning mode and adjust the parameters of double-aperture needle and laser intensity to get a clear confocal image.3. Acquire image
Select the appropriate image resolution, scan the sample completely and save the image result.4. Turn off the instrument
When the instrument is finished measuring the sample, turn off the laser part first, and the computer can still continue image and data processing. If you want to exit the entire laser scanning confocal microscope system. The computer and the main switch should be turned off after the laser has been turned off and allowed to cool for at least 10 minutes.Acquiring three-dimensional images
The laser scanning confocal microscope has a cell “CT” function, so it can obtain a series of optical section images without damaging the cells. Select & ldquo;Z-Stack" mode to realize this function.1. Turn on the "Z-Stack" option.2. Determine the location and number of layers of the optical section.3. Start "Start" to obtain a 3D image.Acquiring Time-Series Images
The "Time-Series" function of the confocal microscope can automatically acquire images at set intervals within the time specified by the experimenter. Simply set the desired time interval and the number of images required, turn on the "Start T" function key, and the experiment can proceed. The "Time-Series" function greatly reduces the labor intensity of the experimenter and is useful for experiments such as fluorescence bleaching recovery and calcium ion imaging.