Visualization of Mitochondria within Intraepidermal Nerve Fibers

Samenvatting

Source: Hamid, H. S., et al. Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers. J. Vis. Exp. (2017)

This video demonstrates the method for visualizing nerve-specific mitochondria using three-dimensional imaging. It outlines the process of acquiring sequential scans of pre-labeled intraepidermal nerve fibers and generating 3D surface images to isolate and display mitochondria in relation to nerve structures.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Confocal Imaging

1. Perform confocal imaging:
   1. Collect images using a laser scanning confocal microscope with a 40X oil-immersion (1.25 numerical aperture (N.A.)) objective on an inverted microscope.
      1. At each focal plane, sequentially acquire fluorescent signals:
         Nuclei: excitation λ = 405 nm, spectral emission filter λ = 420 - 480 nm
         Nerve fibers: excitation λ= 488 nm, spectral emission filter λ = 505 - 560 nm
         Mitochondria: excitation λ = 543 nm, spectral emission filter λ= 606 - 670 nm
   2. Enter the following scan parameters into the microscope software: scan rate of 600 Hz with 2 frame averaging and zoom of 2.2; 12-bit intensity resolution (4096 gray levels).
      1. Set the microscope software for optimized lateral resolution (scan resolution = 1,024 x 1,024) and axial resolution/optical sectioning (confocal aperture = 1 airy unit (AU) with z-step size of 210 nm).
         NOTE: The resulting XYZ resolution is 172.2 nm x 172.2 nm x 210 nm with an image size of 176.1 µm x 176.1 µm x 30-50 µm.
   3. Activate a live scan for the nerve signal (green fluorescence) and adjust the z-focus control to find and set the upper and lower focal planes in the microscope software that encompass the nerve signal within the tissue section. The total z-range is typically 30-50 µm for a 50 µm tissue section.
   4. Rotate the scan field with the microscope software during a live scan so that the epidermis is horizontally or vertically positioned in the image.
   5. Scan each signal separately and adjust the detector (photomultiplier tube, PMT) voltage and offset to minimize/remove any over and under saturated pixels.
      NOTE: Scan times with the above parameters take approximately 20 - 40 min, depending on the number of z-slices.

2. 3D Visualization and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

1. Isolate the 3D epidermis:
   1. Duplicate the original image and use the maximum intensity projection (extended focus view) of the image to identify and isolate the epidermis.
   2. Use a region of interest tool to trace along the upper and lower edges of the epidermis to remove unwanted areas, such as the stratum corneum and dermis, that are absent of intraepidermal nerve fibers. Crop to this selection.
2. Use deconvolution on the nerve and mitochondrial fluorescent signals:
   NOTE: Deconvolution helps to restore the integrity of the fluorescent signals. The restoration used in this protocol is called blind deconvolution because it uses the fluorescent signals in the images to determine how much the signals spread from their original source (point spread function). The process improves signal resolution by reassigning the signal spread back to its origin location.
   1. Calculate a point spread function (PSF) for the green fluorescent nerve signal (green-fluorescence) with the following parameters:
      1. Set calculated PSF to confocal. Set the medium refractive index to 1.515 and the numerical aperture to 1.25. Set detector pinhole to 1 AU. Set laser excitation wavelength to 488 nm and emission wavelength to 515 nm.
   2. Calculate a PSF for the red fluorescent mitochondrial signal (red-fluorescence) with the following parameters:
      1. Set calculated PSF to confocal. Set the medium refractive index to 1.515 and the numerical aperture to 1.25. Set detector pinhole to 1 AU. Set laser excitation wavelength to 543 nm and emission wavelength to 617 nm.
   3. Optimize the nerve and mitochondria fluorescent signals by deconvolution using the corresponding PSFs listed above and iterative restoration feature set at 100% confidence and an iteration limit of 10 cycles.
3. Create nerve-specific surfaces:
   1. Use the "create surface" tool to make a solid surface of the nerves from the deconvolved green-fluorescent secondary labeling of the protein gene product 9.5 (PGP9.5) identified nerves.
   2. Uncheck the "smooth" feature and use the absolute intensity feature to set the threshold for the nerve signal, since it is significantly brighter than the background fluorescence.
   3. Use the absolute intensity feature to set the threshold for the nerve signal since it is significantly brighter than background fluorescence. Set threshold value low enough to accurately identify the nerves.
   4. Filter out small, non-nerve surfaces based on size.
      NOTE: If necessary, manually edit out additional non-nerve surfaces within the "Edit" tab by holding the CONTROL key to highlight multiple objects and then deleting them with the Delete key.
4. Isolate nerve-specific fluorescent mitochondrial signal:
   1. Select the Edit tab of the nerve surface to view the "Mask Properties" feature. The nerve surface created is used to isolate mitochondria within those nerves away from mitochondrial signals associated with the keratinocytes.
   2. As the "Mask All' button opens a "Mask Channel" window, choose the deconvolved red-fluorescent signal from the pull down menu under "Channel Selection" for the mitochondrial signal.
   3. Click in the box to put a checkmark in the "duplicate channel before applying mask" option.
   4. Click on the radio button in front of the "Constant inside/outside" option of the Mask Settings and click in the box to put a check mark in the "Set voxel outside surface to" option and type in 0.00 for the value. Click OK button to create the new channel that represents mitochondrial signals within the nerve surface.
5. Create mitochondria-specific surfaces:
   1. Use the "create surface" tool to make a solid surface of the mitochondria from the newly created fluorescent channel of the nerve-specific mitochondrial signals.
   2. Uncheck the "smooth" feature and select the "background subtraction" feature to set the threshold. This feature uses local contrast around the mitochondrial signal to identify mitochondria from the background.
   3. Set the threshold value low enough to identify mitochondria accurately. In this example, the lower threshold was set at 2,000 for a 16-bit (65,536) scale.
   4. Filter mitochondrial surfaces based on size. In this example, the voxel limit was set to 1.0 voxels, which is the lowest limit possible. If necessary, manually edit out non-mitochondria surfaces within the "Edit" tab by holding the CONTROL key to select multiple objects and then deleting them with the Delete key.
      NOTE: Occasionally, the software will create surfaces that are not associated with a distinguishable fluorescent mitochondrial signal. In these cases, it is possible to remove them with the "Edit" tab.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Leica SP5 laser scanning confocal microscope	Leica Microsystems, Buffalo Grove, IL	SP5
Volocity x64 software	Perkin Elmer, Waltham , MA	Version 4.4.0	Volocity software is used for image processing
Imaris x64 3D analysis software	Bitplane, Concord, MA	Version 7.7.1	Imaris software is used for image analysis