The overall goal of the following experiment is to characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells by directly measuring the local elastic properties of fresh mirroring lung tissue using atomic force microscopy micro indentation. This is achieved by cutting the agarro inflated mouse lung tissue with a razor or scalpel blade to prepare lung parenchyma strips of five by five millimeters in length and width, and 400 micrometers in thickness. Next unfixed un perme lung tissue strips are immuno stain, which identifies areas of interest for a FM micro indentation.
Then a FM micro indentation is performed on tissue strips in PBS at room temperature. In order to directly measure the local elastic properties of the fresh lung tissue results are obtained. That show striking differences in the range and distribution of tissue stiffness in normal and fibrotic lung parenchyma and large spatial variations in stiffness, particularly within the fibrotic lung sample.
Based on stiffness maps extracted from forced displacement curves obtained using a FM micro indentation. The main advantage of this technique over existing measure like tissue strip stretching is that it offers unprecedented spatial resolution providing a unique perspective on microscale variation in tissue stiffness. This measure can help answer key questions such as how stiffness varies spatially within the tissue and what the scope and the spatial scale of the stiffness changes are in the disease.
Processes that result in tissue remodel To prepare strips of lung tissue begin by stabilizing the lung structure for cutting by inflating isolated mouse lungs. Intratracheally with 50 milliliters per kilogram body weight of warm, 2%low gel point aros prepared in PBS tie off the trachea and cool the inflated lungs in a bath of PBS at four degrees Celsius for 60 minutes. The aros will gel and stiffen in the air spaces to gently stabilize the lung structure During this interval, using a razor or scalpel, cut the aros stabilized mouse lung tissue into strips of approximately five by five millimeters in length and width, and 400 micrometers in thickness.
Then wash the strips in PBS at 37 degrees Celsius for five minutes to remove residual aros to exclude large airways and vessels. Cut strips from subpleural regions distant from main stem bronchi if airways en large vessels are to be imaged cut strips from lung tissue more proximal to main stem bronchi to isolate areas of interest for a FM micro indentation. Visualize the tissue by face contrast microscopy or immunostain and visualize by fluorescence microscopy.
Please see the written portion of this protocol for the procedure. Immediately before a FM characterization, attach the tissue strips to poly L lysine coated 15 millimeter cover slips by raising the cover slip from below the floating tissue, making sure the tissue strip spreads evenly on the cover slip surface. If necessary, sandwich the strip with a second clean uncoated cover slip and apply mild pressure to assist with tissue attachment to the poly L lysine coated cover slip.
Calibrate the A FM system following the manufacturer's instructions immediately before each round of micro indentation experiments. For this, determine two critical parameters. The cantilever spring constant using the thermal fluctuation method in air and the cantilever deflection sensitivity, which is a parameter used to scale the photo diode output signal to the actual cantilever deflection distance.
Calibrate the deflection sensitivity by obtaining a standard forced displacement curve in PBS on a clean glass slide. Then calculate the slope of the forced displacement curve in the forced displacement curve measurement. The A FM tip is extended towards and retracted from the sample surface at a single location with the deflection of the cantilever delta D monitored as a function of tip displacement delta Z.It is recommended to use a silicon nitride triangle cantilever with a five micrometer diameter bo silicate spherical tip using a FM probes with a spring constant of 0.06 newton's per meter.
We have mechanically characterized soft materials with sheer modulized spanning 100 to 50, 000 pascals. Wipe the bottom surface of the sample cover slip with tissue paper, then fasten it to a standard glass slide with vacuum grease. Mount the glass slide on the A FM sample stage and cover the tissue with 500 microliters of room temperature PBS.
Then place the a FM scanning head over the sample. Adjust the microscope sample stage to set the microscope for eyepiece viewing to align the A FM tip in the center of the field of view. Move the A FM sample stage to choose an area of interest on the tissue.
Then switch to CCD camera viewing to record phase contrast image and or fluorescence images of the tissue as desired. Move the A FM tip slowly downwards until it is in contact with the sample accurate. A FM micro indentation characterization of soft samples requires small indentation depths to avoid large local strains, which invalidate the hertz model used for elastic modulus calculation to avoid large strains, perform indentation in trigger mode by setting the cantilever maximum deflection to 500 nanometers.
This deflection limit will restrain the maximum indentation force to less than 30 nano newtons. The indentation velocity should be selected to be sufficiently slow to explore elastic rather than viscoelastic properties of the soft sample. Select a velocity range of two to 20 micrometers per second for lung tissue for a single measurement from an area of interest.
Move the A FM probe to the location of interest and perform a single indentation to collect a standard force displacement curve. The probe will move only in the Z direction. For automated mapping of a region of interest, switch to force map mode, select the scan size and sampling points within the selected area.
The A FM tip RAs across the sample surface between indentation movements and collects individual forced displacement curves at each point within an inde defined sample grid. We found it practical to use a 16 by 16 sample grid to map an 80 micrometer by 80 micrometer area at an indentation velocity of 20 micrometers per second, which can be completed in about 10 minutes. To calculate the Young's modulus fit the force displacement curve to the hertz spherical indentation model using lee square's non-linear curve fitting as shown here where F equals K sub C times delta D is the force to bend the cantilever.
K sub C is the cantilever spring constant. R is the sphere tip radius delta equals delta Z minus delta D is indentation, and new is the samples poisson ratio, which equals 0.4 for lung tissue. To assess the quality of the fit, calculate the SSR value or the sum of squares of the difference between the data and the fit values during the non-linear curve fitting.
Eliminate unreliable or uninterpretable measurements by discarding data from bad curves with large SSR values. If desired, convert elastic modulus or E to sheer modulus. G.Using the relationship E equals two times the sum of one plus new times G.To visualize spatial patterns of stiffness collected in force map mode, plot modulus data and contour maps, for example, in a 16 by 16 grid covering an 80 micrometer by 80 micrometer area.
To process a large amount of force curve data, a custom algorithm may be written to automatically fit force displacement curves, extract modular and or plot contour maps or ELA graft. Using the same procedures and parameters just described, this figure shows that Im properly cut and stain tissue. The alveolar micro of lung parenchyma is well preserved as observed by immunofluorescent staining for the basement membrane component, laminin without fixation or permeation.
These panels show immunofluorescent staining for collagen, one in fresh unfixed lungs harvested from mice previously treated with PBS or treated with blio mycin to induce fibrosis. Sample force displacement plots obtained from a FM micro indentation are shown here with the same applied force. The A FM tip generates a large indentation on a soft region resulting in a relatively flat force displacement curve shown in blue versus a small indentation and a steep force displacement curve for a stiffer region shown in red.
In order to obtain clean force curves after each indentation, the A FM tip needs to be retracted completely from the sample surface and free of contact before the next indentation. This contact free state corresponds to the flat region of the curve where the tip translates without cantilever deflection. This figure shows a typical false curve without a flat region, which occurs when the tip is not completely retracted from the sample surface, such as when soft sample attaches to the tip, making it impossible to determine the contact point if the tip is completely trapped in soft sample.
The curve may look like this without clear deflection, but small noise. This figure shows stiffness. Data extracted from forced displacement curves displayed spatially as stiffness maps referred to as ELAs graft where color scales with stiffness ELAs graft demonstrate striking differences in the range and distribution of tissue stiffness in normal and fibrotic lung parenchyma and large spatial variations in stiffness, particularly within the fibrotic lung sample.
After watching this video, you should have a good understanding of how to characterize the local mechanical environment of lung parenchyma by directly measuring the local elastic properties of fresh lung tissue using Atomic Force microscopic copy.
