Images and Movies:

You are welcome to use any of these images or movies for presentations or website; please just give our laboratory the appropriate credit.  To download the high resolution images Right-Click and choose "Save This Link as..." or "Save Target as...".  On Macintosh, hold down control and click, then choose "Save This Link as...". Please provide credit to the Eisch Lab when using or showing images from this site.

Images of research interests

These images highlight research interests within my laboratory.  Below you'll find images and general descriptions of:

For more detailed information on the projects in the Eisch Laboratory, please see my research page.  If you have any questions about these images or our research, please don't hesistate to contact me.

Seeing is believing...isn't it? We rely on confocal microscopy for our research, but confocal microscopy is only as good as the microscopist.  To learn more about the pitfalls of confocal microscopy, check out this QuickTime movie.  It depicts how poorly-performed confocal microscopy can give misleading results. 
 
 

New cells in the hippocampal subgranular zone (SGZ)

Cell cycle

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Figure legend: Simplified schematic of the mammalian cell cycle (center) surrounded by images depicting each developmental stage leading to SGZ neurogenesis.  Cell cycle (center schematic): The length of the cell cycle in the SGZ of the young adult male rat is about 25 hours, with 9.5 hours consisting of the DNA synthesis, or S, phase, 4.5 hours consisting of G2 and mitosis (M), and the remainder consisting of G1 phase (Cameron and McKay, 2001).  Systemic injection of BrdU or [3H]thymidine leads to incorporation of these mitotic markers into the DNA of cells in S Phase (note red in newly synthesized strand of DNA).  This cell cycle and the surrounding images emphasize the continuum between the time points of proliferation or cytogenesis (left column), differentiation and migration (center column) and survival or neurogenesis (right column).  Proliferation or cytogenesis (left column): Two hours after injection of BrdU, cells in the SGZ appear on the border of the granule cell layer (GCL), and are small and irregularly-shaped.  BrdU-immunoreactive cells will often appear in dense clusters.  A cluster is defined by any BrdU-immunoreactive cells that make contact.  The top left panel is a light microscopic image depicting two BrdU-positive clusters (arrows).  When examined at high magnification (X100) with continual scanning through the Z-plane of section, these clusters can be identified as containing eleven (top cluster) and three (bottom cluster) individual cells.  The bottom panels depict confocal microscope images of a triple immunohistochemical stain of the SGZ from a rat given the mitotic marker BrdU 2 hours earlier.  Cells are labeled for BrdU (green), the neuronal marker NeuN (red) and the glial marker GFAP (blue).  Arrowheads indicate cells double-labeled with BrdU and GFAP, but not with NeuN.  Merged images of the three labels (last panel) show that all BrdU-positive cells in the SGZ are GFAP-positive (blue-green cells) and NeuN-negative.  Note that in contrast to the neuronal morphology of NeuN-labeled cells in the GCL, BrdU-labeled cells in the SGZ are small, clustered, and irregularly shaped. Differentiation, migration (center images): About four or more hours after injection of BrdU, some cells labeled with BrdU will have reached M phase and will divide.  Some of these cells will reenter the cell cycle (or Go, not depicted); others will exit the cell cycle and begin to express markers of postmitotic, migrating, or differentiating cells.  Two such markers are shown here.  The center left panel is a confocal microscope image of the SGZ depicting staining for Hu (green) and nissl substance (red).  Hu is a member of a family of RNA binding proteins shown to be expressed in neurons around the time of exit from the cell cycle (Marusich et al., 1994).  The center right panel is a confocal microscope image of the SGZ depicting class III b-tubulin immunoreactivity (green).  Class III b-tubulin is a cytoskeletal protein expressed in both postmitotic cells that may become neurons and in mature neurons.  Hu and class III b-tubulin are markers of differentiating and migrating cells, but the fate of these maturing cells is considered malleable at least until the cells express markers of mature neurons, such as calbindin or NeuN.  Survival or neurogenesis (right column): Two to four weeks after injection of BrdU, some BrdU-immunoreactive cells have migrated into the GCL and have achieved a neuronal morphology. The top right panel is a light microscopic image depicting four BrdU-positive cells (arrowheads).  These four cells are round, about 10 mm in diameter, and solitary.  Note that some surviving BrdU-immunoreactive cells are solid (black arrowhead) while others are speckled (blue arrowhead).  Due to the depth of penetration of the BrdU antibody and narrow focal plane at this magnification, an additional speckled cell is out of focus (light blue arrowhead).  The heterogeneous labeling reflects either time spent in S Phase or number of cycles of division, e.g. speckled cells were later in S-phase relative to solid cells when BrdU was injected, or speckled cells have gone through more cycles of division than solid cells (Miller and Nowakowski, 1988).  The bottom panels depict confocal microscope images of a triple immunohistochemical stain of the subgranular zone from a rat given BrdU 4 weeks earlier.  Cells are labeled for BrdU (green), the neuronal marker NeuN (red) and the glial marker GFAP (blue).  Arrows indicate a cell double-labeled with BrdU and NeuN but not with GFAP.  Merged images of the three labels (last panel) show that all BrdU-positive cells in the granule cell layer are NeuN-positive (yellow cell) and GFAP-negative.  Note that in contrast to the neuronal morphology of BrdU-labeled cell in the granule cell layer, BrdU-labeled cells in the hilus are small and irregularly shaped.  From (Eisch et al., 2000).  All confocal images shown here were collected on a confocal microscope at X65 magnification using multitrack scanning.  The images presented are taken from a 1 mm optical section through the Z plane of focus.  GCL, granule cell layer; H, hilus.

For more information on this figure and more about the cytogenesis versus neurogenesis debate, please see Eisch, 2002 article in Progress in Brain Research (Click here for pdf of this publication).

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Use of endogenous cell cycle markers to characterize dividing clusters in the hippocampal SGZ

This combinatorial approach of using exogenous and endogenous markers will enable us to learn about how a cluster of newly born cells matures over time.

Immunodetection of exogenous mitotic markers such as BrdU or [3H]thymidine is useful for "birthdating" a cell.  However, the immunodetection of endogenous cell cycle markers holds immense promise for characterizing newly born cells in the adult brain.  Some endogenous cell cycle proteins are relatively restricted to various phases of the cell cycle, allowing us to learn about how a cluster divides. This image depicts triple labeling immunofluorescent histochemistry for BrdU (panel b, red staining) and two endogenous cell cycle proteins.  BrdU was given i.p. two hours earlier.  Some parts of the cluster (large arrow) are labeled with all three colors.  Other parts (small arrows) are labeled with just green and blue, suggesting that this cluster was not in S phase when BrdU was injected.  Still other cells (arrowheads) are only labeled with BrdU and one of the cell cycle markers, suggesting that these cells are in a part of the cell cycle that only expressed one of the cell cycle markers.

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Pitfalls of Confocal Microscopy

Confocal microscopy is regarded as a sophisticated way of analyzing the phenotype of cells.  In the field of adult neurogenesis, use of confocal microscopy to analyze the phenotype of newly born cells is now de rigueur. However, as with every technique, confocal microscopy can be misused and therefore can provide misleading results.  The movie offered for downloading to the right is a good example of how poorly performed confocal can lead to the (false) appearance of double labeling.
In the case of the movie offered here, a BrdU-labeled cell appears to be a neuron when in fact it is likley a satellite glia cell.  The movie begins with a series of Z slices (from the top of the section to the bottom).  In this part of the movie, all BrdU-positive cells (in green) appear to be NeuN-positive(red).  Seeing is believing, right?
However, in the second part of the movie, the "Z stack" of slices is rotated to show the three-dimensional arrangement of these cells.  As the stack rotates, notice that some of the BrdU-positive cells are actually adjacent to the NeuN-positive cells (look at the bottom two BrdU-positive cells).  This shows that these particular BrdU-positive cells are not NeuN-positive.  Indeed, in this case, these BrdU-positive cells appear to be satellite glia as have been reported in other regions of the brain.
So how is it that the Z slice part of the movie suggests BrdU-labeled neurons, but the Z stack rotation part of the movie suggests BrdU-labeled satellite glia?  The answer is in the way the confocal microscopy was performed.  Such misleading information can result from a variety of source: Z slices that are too thick or too thin for objective and/or sample; Z slices that overlap too little or too much; inappropriate insertion of Z section thickness in 3D reconstruction programs; single or simultaneous laser scanning instead of multiple or sequential scanning (very common); use of a laser configuration that allows bleedthrough of fluorophores, etc.  This is just a short list of things that can provide misleading results in confocal microscopy.  In the case of the movie, the slices were taken with an airy disk greater than 1, leading to a wide pinhole and a thick section scanning.  Therefore, when looking top to bottom the cells appear double-labeled.  Only after 3D reconstruction and rotation is the relationship between the cells apparent.  This movie is good evidence that in addition to doing confocal microscopy, researchers would be wise to also make 3D reconstruction (or at least orthogonal slice analysis) a necessary part of phenotypic analysis.

In sum, confocal microscopy is only as good as the knowledge of the microscopist.  As this movie reminds us, don't trust what you see - unless you understand how the images were captured and processed. Visit our departmental confocal website to find links to good confocal sites on the web, or find a good book on the subject (Jim Pawley's Handbook of Confocal Microscopy is one good one).

 



Download the Quicktime movie (Takes 2:30 min at 450 KB/sec)

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