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Laser Scanning Confocal Systems

The major difference between a camera and a scanning system that moves a point of light (rasterizes) across the specimen is in the use of a single detector rather than an array of microdetectors on a chip. That single detector is generally a photomultiplier tube (PMT), which is used for its extraordinary sensitivity to finite numbers of photons. The PMT is used in concert with electronic amplification (gain) to further brighten signal from fluorescently labeled specimens.

The difference between a confocal and a standard microscope is the use of an aperture or pinhole (also called an iris). The pinhole restricts the angle of photon collection so that mostly in-focus photons incident to lens elements are included, and off-axis photons are rejected. Thus, fluorescence above and below what is in focus (the focal plane) will be out of focus and therefore not included.

In theory, an instrument set up with an aperture at the correct position along the light path will provide a perfectly in-focus plane (called a z plane) without contribution of out-of-focus light above or below, but in practice the z plane will include some out-of-focus light. The degree of z plane optical resolution depends almost entirely on a second aperture in the system: the rated aperture of the objective that is used. That aperture is called the Numeric Aperture (N.A.). The higher this aperture is rated the less out-of-focus light and the greater the z plane resolution.

Yet even with the highest numeric apertures and the smallest, functional pinholes, z resolution can still include out-of-focus light. To eliminate that, software programs that deconvolve image stacks can be used. These eliminate out-of-focus light based on mathematical models and system parameters.

A second solution involves the use of a laser that can be modified to strike fluorescent dyes with two photons (or more) at once instead of only one. The possibility of two photons striking simultaneously is rare, and it can only occur at the focal plane (as long as the projected laser light fills the back aperture of the objective). These are called two-photon, or multiphoton confocals.

Because of these components for confocal and like instruments, some of the parameters change for digital imaging:

  • Exposure becomes the amount of time a laser excites a single spot, which is called dwell time. The speed of the scan will affect the dwell time and so will pixel resolution.
  • Pixel resolutions can be set on confocal instruments. The greater the resolution, the longer it takes to scan the image. When bleaching (loss of fluorescence intensity due to being struck by light) interferes with acquisition of labels or when specimens move, smaller pixel resolutions can be chosen. Ideal pixel resolutions can be set according to Nyquist rates: ideal rates for the number of times elements of an image are captured for subsequent pixels to obtain the best resolution.
  • Focus becomes dependent on two parts: physically focusing the microscope to find the brightest z plane of the specimen (to find its centermost position in depth) and the aperture or iris setting. Confocal systems mated with microscopes set apertures automatically based on the N.A. of the objective that is used. Ideal apertures can also be determined based on Nyquist rates.
  • Gain is achieved in two ways: electronically through attenuation of voltages to the PMT (called High Voltage [HV]; can also be called by other references) and to a post-PMT amplifier (often called Gain). Instructions on setting both differ from lab to lab. As a general rule, when High Voltage amounts create visible noise, Gain is increased for additional amplification of signal and high voltage is decreased.
  • Laser power is at 100% on confocal instruments, and neutral density filters are placed in the light path to attenuate the power. "Laser power" is a reference to the percentage reduction of light through the use of neutral density filters or other means for reducing light levels. The confusion is in nomenclature: "Laser power" is often the denotation versus "neutral density filter."
  • Black Level achieves the same end as Contrast: Both are used for setting the black limit or the deepest significant black in the image.
  • Additional magnification (also called Zoom) can be chosen in confocal acquisition software. Up to a cutoff, the magnification is optical, not simply additional pixels. Contact the manufacturer to determine the cutoff.
  • Z plane selection is available on confocal systems. The z step is set automatically on systems mated with microscopes, or ideal z steps can be calculated according to Nyquist rates. Here Nyquist rates determine the z-step increment necessary for the best resolution.

Confocal Imaging Depending on Intent

The settings used in the "Steps for Imaging on a Confocal System" section that follows depend on the intent and are briefly provided in Table 4.3.

Table 4.3. Confocal Imaging Depending on Intent

Greater Definition in Images*

Colocalization and Thin Optical Sections

3-Dimensional Models

Greater Resolution

Thick Section Imaging

Single plane?

Yes

Yes**

No

No

No

Multiple planes?

No

No

Yes: > 50 sections

Yes

Yes

High pixel resolution?

High > 640X640

Low < 512X512

High

High or Low: depends on feature dimensions

Low: to accommodate 3D software

High: Nyquist rates

High or Low (depends on features)

Kalman averaging?

High: 8–32

Low: 2–4

High or Low***

High, or noise will obscure measurements

Low, or bleaching may result

High or Low, depending on noise levels

High

Confocal aperture at nonoptimal dimensions?

Yes

No

No

No

Possibly

There may be some exceptions to the recommendations given in the table, and it is up to each researcher to carefully investigate previous publications in his or her respective fields of study for additional information about methods.

Note that images intended for optical intensity measurements are not included. Laser fluctuations over time preclude measurement unless an internal standard exists. Those who are not dissuaded can still pursue optical intensity measurements as long as laser fluctuations are measured, bleaching rates are determined, and linear uptake of fluorescent dye into cells is proven. Be sure to Kalman average so that noise is reduced to minimal levels.

Steps for Imaging on a Confocal System

To better understand the steps involved when using a confocal system, steps are categorized into four phases. Phase 1 involves finding the brightest z plane on the microscope and the setup steps involved in seeing the image in the acquisition software. Phase 2 concerns setting the tonal values for the brightest significant features and black background within the dynamic range of the detector. Phase 3 includes the final steps, such as setting the z plane parameters, Kalman averaging, and saving. Phase 4 reviews documentation procedures.

Phase 1: Find Brightest Plane and Set Up

Start by finding the specimen on the microscope:

  1. Find the brightest z plane of the specimen by focusing up and down.
  2. Find the area of interest on the microscope and choose a field adjacent to the field of interest (to prevent bleaching of significant field during setup).
  3. Switch the instrument settings to allow light to travel to the confocal head.

On confocal acquisition software:

  1. Choose appropriate dyes from the DyeList dialog box.
  2. Set to Sequential (if available) and choose the brightest and most stable dye wavelengths only (DAPI is often used for its brightness and stability).
  3. Choose the fastest scan speed for the initial scans. Note that Focus settings may scan so that every other line is used along the y axis. This may bleach the labels of the specimen, leading to a patterned bright/dark appearance. A smaller "box" size, or pixel resolution in width and height, can be chosen for initial imaging to locate the specimen and reduce bleaching.
  4. Scan the image, and while scanning either physically rotate the focus knob on the microscope or adjust the z steps to find the brightest plane (in the event that the focus to the eye through the microscope is different from the confocal head).
  5. If the plane cannot be found because the sample is dim, increase High Voltage and Gain to higher values and attempt to image again. Increase laser power and the confocal aperture until the plane can be found.
  6. If the image appears all black, a setting is incorrect, the laser is off, or the instrument isn't communicating with its components, get help.

Phase 2: Set Dynamic Range

  1. Adjust the settings to fit the image within the dynamic range of the instrument.
  2. Set the panel to a Look Up Table (LUT) overlay to indicate levels at which the deepest black and brightest significant whites "clip" (exceed the dynamic range). Typically, when areas of the image clip in the brightest region, a red color is overlaid on the bright areas. When areas of the image clip in the darkest regions, a blue or green overlay appears. Adjust settings depending on whether the dye is bright or dim:
  3. For bright dyes:
    • Keep the laser power low to minimize bleaching.
    • Decrease Gain to 0 and increase High Voltage values until the whitest significant parts of the image show up as red (unless the whites in the image are already red colorized). Back off from High Voltage until no red appears in the significant white areas.
    • Increase or decrease the Black Level to create a blue overlay in the shadow areas of the image, and then back off until the blue or green disappears.
  4. For dim dyes:
    • Increase both High Voltage and Gain until the whitest significant parts of the image show up as red.
    • If white values are still too dim or if the presence of noise is too significant (it will be reduced by frame or Kalman averaging later on), the choices are as follows:
      • If the sample readily bleaches, the alternative is to decrease the pixel resolution or open the aperture to a larger diameter. Each presents its own challenge: Decreased pixel resolutions and larger apertures negatively affect resolution. Depending on the intent of the image, however, this may be an acceptable solution.
      • If bleaching occurs to a point and then plateaus, the alternatives are to decrease the scan speed (which increases dwell time) or increase magnification by zooming in or choosing a higher numeric aperture objective. Magnification changes will more likely increase resolution (depending on the specimen) but may not include a large enough field of view.
      • Adjust the settings individually for other dyes using the same methods.

Phase 3: Set Z Planes and Frame Averaging

A few final steps and saving your settings are required:

  1. Set the remaining parameters for all dyes and collect the images.
  2. Set the LUT overlay relevant to each dye used in the specimen. Using a gray LUT is preferred, because levels of brightness and darkness are easier to see and compare in grayscale.
  3. Set the upper and lower positions for z sectioning. This can also be done by selecting a single dye when bleaching is a concern. The caveat, however, is that one dye alone may not label features in all z planes.
  4. Set the increment of the z step. The Op setting for this acquisition software sets the optimal z step based on Nyquist rates. The number of slices from the top to the bottom of the specimen is shown.
  5. Rotate the section, if necessary, and if the tool is available.
  6. Set the Frame or Kalman value. In newer confocal systems, a scan line by scan line average can be done, or z frame by z frame. Typically, line scans are preferred.
  7. Set the pixel resolution, if necessary.
  8. Scan the specimen and save.

Phase 4: Documentation

Make certain that all parameters are written down or saved with the file as metadata. The most important settings are excitation and emissions wavelengths for the dyes used, numeric aperture of the lens, power of the lens, medium between the lens and the specimen (e.g., oil), confocal aperture, step size, pixel size (found in readouts), and zoom.

Save the image in the proprietary format of the manufacturer. The image may also be saved as a TIFF file (if available): Beware, however, that TIFF files are not "screen saves" at computer screen resolutions; the images may be at subsampled resolutions. Archive images to two CDs or DVDs.

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