- High sensitivity point scanning confocal imaging
- Spectral array GaAsP detectors
- Upright motorized stage microscope
- In vivo or in vitro samples
- Turn on the X-Cite (if you need to see fluorescence down the eyepieces)
- Turn on the computer (#2), log on (Select the "LMCFuser" account and enter the password), wait until Windows starts.
- Turn on Main switch (#3)
- Turn on System switch (#4)
- Turn on the Components switch (#5)
- Turn on the Lasos Argon laser switch (large black box) by turning key to position 1 (#6)
Open the Software
Double-click the ZEN icon on the desktop and choose "Start System" when the boot status window pops up
The system is based around an Axio Examiner Z1 upright fixed stage microscope that is designed to have highly configurable sample holders with a lot of space under the objectives.
The motorized stage is usually in place which is good for slides and small samples. The height of this stage can be crudely adjusted (with the objectives raised) by loosening the front legs, lowering the condenser and loosening the thumb screw on the back right side. Now you can carefully move the stage up and down to the appropriate height and re-tighten the screw. The line on the stand indicates a good position for slides.
If you need to use a custom stage the motorized plate stage and condenser can be entirely removed.
Choose an objective and lower it to the imaging (i.e. the lowest) position. Be careful when you lower the objective that you don't hit the sample with the objective. If the objective is too close to the sample, turn the focus knob to separate them. If you need to swap an objective, go to the "Maintain" tab and select the lens so the system computes correctly spatial measurements and pinhole settings.
Go to the Ocular tab, click online and choose the means of viewing - Brightfield, Blue, Green or Red fluorescence
Focus carefully - the objective moves up and down and the stage is fixed so be careful not to hit your sample with the objective (not good for either party).
Click "RL Illumination Off" on the touch pad or the shutter off button to turn off fluorescence (if you just use BF leave it on so the system automatically turns that on each time you go to ocular)
Click the "Acquisition" tab so you see all the settings for confocal imaging. If the "Show manual tools" box is unchecked some of the less common adjustments are hidden (the Show all check marks on each blue bar gives a similar option).
LMCF has made common multi-track (sequential) between-line scanning configurations and saved them in the system. We strongly suggest that you load one of the configurations as a starting point. You may save the configuration in your name if you like.
If you follow the smart setup path . . .
However, the smart setup path does NOT necessarily assign the fluorophores chosen to the optimal detector for the emission wavelength of the chosen fluors. Specifically, fluors with wavelengths in the middle of the visible light spectrum may NOT be assigned to the higher sensitivity GaAsP detector. In addition, smart setup tends to run a multi-track scanning to switch between frames, which involves constant movements of the parts. That slows down the acquisition and causes more mechanical failure of the system.
Or you can manually change the excitation lasers, emission bands, dichroics etc in the Imaging Setup and Light Path tabs.
- The MBS (Main Beam Splitter = dichroic) needs to match the lasers used in the visible and 405 light paths)
- One track for simultaneous imaging of the channels, more than one track for sequential scanning
- For multi-track: Frame switching should be used only if different dichroics, pinholes or overlapping emission bands are required. Line switching is preferred and more convenient and possible when only electronic things change between tracks (eg AOTF control of excitation, which detector is active)
- Ch1 and Ch2 are standard alkali PMTs, ChS1 is an array of 32 high-sensitivity GaAsP PMTs. This can be used as up to 8 channels in Channel mode (but they have to have the same gain at any time, but different digital gain is allowed).
- If you have created a configuration you can save this for easy reloading in the future
- Please ask if you need some help to design an optimal setup for your particular fluorophores and experiment.
Pressing Auto-Exposure is a good starting point to get an image on the screen to optimize. Auto-exposure changes the detector gain values so something is visible.
Live (this was called FastXY in the 510 software) constantly scans quite quickly at a fairly low resolution to allow you to make adjustments. (If you need to have the scan parameters you will subsequently adjust to be in effect you can press continuous).
Adjust the z-position with the coarse/fine focus knob (notice these are on the touch screen pad as well as the microscope) to choose the plane of interest. If you are taking a z-stack generally choose the brightest plane to adjust the settings.
Zoom - you can do this with the "Crop" button underneath the image - resize the box that appears - or using the scan area controls in the Acquisition Mode panel.
Laser powers - adjust the sliders (note the are non-linear) so you have reasonable excitation. The lowest value you can get away is best to minimize fluorophore saturation and photodamage.
Pinhole - adjust so a suitable optical slice is obtained. A trade-off between z-axis resolution and brightness. A pinhole of one Airy unit essentially gives you the best resolution, opening it slightly from there will allow you to collect more signal. For multi-track scanning, set up the pinhole in the channel with the longest wavelength. The other channels will automatically match the section thickness.
Gain/offset optimization - adjust the gain (detector sensitivity) and offset (background level) for each channel. Selecting range indicator is helpful when doing this. In this mode saturated pixels are highlighted red and 0 intensity blue, a few red pixels and the background 50% blue is often a good point to aim for. VERY IMPORTANT: the gain levels for the GaAsP detectors (ChS1, ChS2,...) should be set between the range of 600-800 volts, the linear range of the detector. If you have a good image with a gain below 600 volts, then adjust the laser power down to minimize your photobleaching/phototoxicity to your sample. Conversely, if you need to adjust the gain on these detectors above 800 volts, reduce the gain back into the range and raise the digital gain to increase the image intensity.
Averaging and scan speed - Averaging and slower scan speed improve signal to noise ration (SNR), choose a good balance for quality and speed.
Sampling rate - The number of pixels in the image can be selected. The optimal button selects the correct number for a particular objective, wavelength and zoom. This will give you a digital sampling rate adequate to fully sample the optical resolution.
The acquisition Mode panel also allows you to adjust between unidirectional and bidirectional scanning (faster but sometimes causes image degradation), and the bit depth of the images (8 bit is fine for standard imaging, 12 bit may help with some quantification).
Press the Snap button to acquire an image with the settings you have specified above.
Before you have a new scanning, click the disk icon beneath the open image icon to save the selected image as .zvi files. Otherwise, the image will be overwritten with the new image. Generally it is best to save to the D:/ drive and move the data to a server, USB drive etc after you have closed the software. Our policy allows us to delete all old data without prior warning.
The software creates a new tab in most but not all acquisition cases so save as you go to avoid loosing anything.
The ZVI image format can be open by the free offline viewer, FIJI, or ImageJ. You can export images as TIFF from the File\Export menu.
- Select the Time Series box underneath the Z-series box of above
- Choose the number of time points and the interval between them
- Press start experiment
And of course you can do a time series of a stack by having both things active. You will end up with a 4D dataset that can be saved as a single .LSM file.
- Retract the objective to the up position and clean any immersion objectives that you used
- Move your saved data as appropriate
If the next user is scheduled within 2 hr, please leave the system on
If nobody is scheduled to use the confocal after you in 2 hr, please power down the system
- Turn key on Lasos black box (#6) to '0' position and wait for about 4 min until fan shuts off
- Turn off System PC and components switches (#5 & #4)
- Turn off Main Switch (#3, only when the cooling fan from #6 is quiet)
- Exit Zen software and shut down PC (#2) from the Windows Start Menu
- Turn off X-Cite (#1)
- Cover the microscope