This protocol describes the use of two-photon microscopy to image the dynamic behavior of hematopoietic stem cells interacting with their niche. To distinguish the eGFP expressing cells from auto-fluorescent background, we use spectral finger-printing. We include image processing steps to visualize the results and extract quantitative information.
For successful long-term imaging (up to 18 hours) it is essential to prepare the sample in an adequate way and to align the two-photon microscopy as good as possible. Thus, this protocol will focus on these two steps. Following this protocol, one can expect to generate time lapse movies showing eGFP labeled hematopoietic stem cells interacting with their niche and undergoing active cell division. Typical results are published in Xie et al.: “Detection of functional hematopoietic stem cell niche using real-time imaging”1. It is straight forward to adapt this protocol to other cell types and fluorescence markers. The whole experiment will take one to two days.
We describe a method to visualize the dynamic behavior of individual hematopoietic stem cells (HSC) in their natural environment.
We use mice with stem cells injected into their tail vein. The cells express enhanced green fluorescence proteins driven by an actin promoter (actin-eGFP). The protocol can be adapted to animals expressing eGFP labels in different cell types. Other fluorescence proteins can be used as long as the near infrared laser (NIR) and the microscope optics allow their excitation (e.g. we can excite dsRed with 1060nm, but not mRFP) and detection. It is straight forward to extend this approach to multiple colors.
Bone marrow cells are derived from actin-eGFP transgenic mice. To enrich for HSCs, we use a cocktail of specific antibodies and FACS analysis2. The 6 to 8 weeks old recipient mouse is radiated with 10 Gray. Ten to 12 hours later 50,000 HCSs are injected into her tail vein.
Four to 6 hours after transplantation, we sacrifice the mice. We extract the femur and tibias bones and cut them into transverse sections 2-3mm in height. The pieces are placed with the open end directly on the cover-slip bottom of a Petri-dish. To secure the upright position of the bone the Petri dish is filled with 2mm of agarose.
Bone structures scatter too much light to allow wide-field microscopy. That is why we use two-photon microscopy3. This allows us to image deeper into bone structures than with a confocal microscope. The infrared light used is less harmful to living cells than visible or ultraviolet exposure4,5. Collagen generates a second-harmonic signal (SHG). Differential interference contrast (DIC) highlights bone structures. We can use transmitted light DIC because of the relative high transparency of biological materials for light in the infrared range. These signals help to define the locations of HSCs within the living bone.
Typically we image eGFP expressing cells with an emission band-pass filter of 500-550nm. However, we could find several cells and cell-like structures with auto-fluorescence in this emission range, even in living bones without eGFP marked cells. To distinguish these false signals from real eGFP labeled cells, spectral imaging was employed.
We imaged the dynamic interaction of HSCs with their niche for up to 18 hours. For these long-term experiments temperature control is imperative.
We finish this protocol with image processing procedures for 3D exploration of the datasets and quantification of fluorescence signals. While these protocols were used mainly with fixed samples, they can easily be adapted for live cell images. The image processing techniques are most valuable when applied to multi-color z-stacks.
The whole experiment takes at least one day. The main steps as summarized in Figure 1 are:
• Isolate of hematopoietic stem cells (HSCs) from eGFP-transgenic mice.
• Transplant eGFP positive cells into host mouse.
• Prepare bones for imaging.
• Set-up and align two-photon microscope.
• Acquire eGFP reference spectrum with two-photon microscope.
Sample preparation and microscope alignment can be done in parallel. The time between the microscope alignment and the following imaging steps should not exceed one day. The imaging of the stem cells should be started immediately after the bone preparation is finished.
• Find bone using wide-field microscope.
• Find potential stem cells in bone structure with two-photon microscope. Set up two-photon microscope to image eGFP, SHG and transmitted light DIC signal, simultaneously.
• Confirm eGFP signal with spectral imaging.
• Start time lapse experiment.
• Repeat the last steps for next cell (or cells).
If the two-photon microscope is equipped with a motorized stage which can record positions, one can first locate and confirm multiple cells and store their positions. Most modern microscopes have software which allows switching between multiple locations during a time lapse experiment and revisit the previously stored positions.
• Process images.
Depending on the experiment, image processing might include low-pass filtering, global contrast adjustment, fluorescence quantification, and 3D reconstruction.