Collecting images on the Leica Confocal:

These instructions are for optimal image acquisition - which usually means fixed tissue with good labeling. Live imaging and weak signals require "compromises" but you need to understand the basics. Go through these instructions first.


Pinhole:


Start with a pinhole of 1.00 (Airy units=AE).

A pinhole of 1.00 AE gives the best signal to noise ratio. A pinhole of 0.5 AE improves resolution.

The pinhole isn't used for 2-photon imaging so it is opened to the largest aperture.











Display Parameters:



Select the parameters of the display window.

Tiled shows all active channels.

Ovl (overlay) shows the merged image when using multiple fluorophores. You may need to scan once to enable the overlay button.

Use the CLut button to toggle the image between Glow O/U and the selected color in the Beam window.




Before you scan check:

Factors that affect image quality

Lasers are on

Click on the button to start scanning.

If no light is visible at the sample, check that the switching rod on the right side of the microscope is out, the microscope head is not tilted and that the filters for the mercury lamp (#1, 2, or 3) are out of the light path.

If light is visible at the sample, then turn up the gain on each channel to get an image:


Getting an image on all channels:

Check the configuration of the panel box, indicated at the bottom of the screen and shown below. This configuration can be personalized for your profile. You should have a gain and offset for each channel in use. Smart gain or smart offset indicates the knob will affect the selected channel.



Start scanning.

Increase the gain to get an image.

If the gain PMT approaches 1000 then use a low scan speed.

Repeat with each channel in use.



No image? Go to troubleshooting.

Adjust the laser power if necessary.



Optimize the gain and offset for each channel:

Glow(O&U) should be selected as the LUT (look-up table) in the beam path parameters menu.

Adjust the offset (black level) to get a small amount of green.

Adjust the gain to show a minimal amount of blue.




Always eliminate the noise in an image before evaluating image quality (and before saving an image):

Use Aver (Average) to filter out this random noise in an image.


Select a sufficient number of scans to average to get rid of the noise. High gain requires more frames to average. Initially, use at least 10 for a medium scan speed and 6 for a low scan speed.











Evaluate the image:

GlowOvUn should only be used as a guideline.

GAIN: for fixed preparations, the brightest pixel(s) should ideally reach 256 to utilize the entire grayscale range possible in an 8-bit image. The image should not be saturated such that in a histogram there is a peak at 256.

Exceptions to this would be:
1.where label is "concentrated". Features may be more distinct with a lower gain.
2. structures of varying intensity i.e. dendrites vs a cell body. The signal from the cell body will be saturated in order to detect smaller dendrites with less label.
3. live imaging where laser exposure needs to be minimized or acquisition is optimized for speed.
4. samples where laser exposure must be minimized to reduce bleaching of the fluorophore.

OFFSET (black level): set the offset so the background (no tissue) is dark and unlabeled tissue is detectable. Do not make the background so dark that you drop out gray values. Unlabeled tissue has to be detectable in order to visualize controls. A histogram of most images should show the lowest intensity to be slightly above 0.

PINHOLE: Is the resolution sufficient? If not, use a smaller pinhole or an objective with a higher NA.



Improve the image:

Factors to increase intensity:

Raising the gain increases the intensity of the signal. Electronic noise also increases as the gain is turned up, requiring an increased # of frame averages to eliminate the noise. At some point the resulting image will not be as sharp as an image collected with lower gain.

Increasing the laser power will increase the intensity of the laser at the sample. This will increase bleaching of the fluorophore.

A larger pinhole will usually increase the intensity of the image collected. However, THE SIZE OF THE PINHOLE DETERMINES THE THICKNESS OF THE OPTICAL SECTION COLLECTED. The thicker optical section with a larger pinhole will decrease resolution. Background is often be higher with a larger pinhole.

A slower scan rate results in a brighter image due to the longer dwell time. Bleaching will occur faster though.

Other factors that affect intensity:

Format size
Objective




Multiple labels: check for cross-talk between the different channels.

Save the image. Until you save, images in the Experiments window are only in memory. If the program (or computer) crashes or is closed, then the images are lost.