Digital Staining
Figure 2 : Schematic diagram of the mechanical components. The rotating scanning head can illuminate the specimen from all directions with a 520 nm green laser beam at low power. A hologram is recorded on a digital camera by combining the beam that has passed through the sample with the reference beam that has not. The sample beam is then rotated by a small angle, and the process is repeated. One hologram is recorded for each beam position.
near the objective (for example, MC-50E from
www.thorlabs. com ). Lens tissues are ideal for removing all dust or fi nger- prints from the coverslip without leaving any trace of lint or fi bers. To clean the coverslip, wet the tissue with a few drops of ethanol and gently rub the surface. (2) When using a FluoroDish, fi rst wash the cells 3 times with PBS to remove cell debris, and then add the appropriate mounting medium. Second, using the same procedure described above for the coverslip, clean the external surface of the glass bottom dish, which has to be next to the objective.
In addition, it is important to clean the microscope objective lens. To do this, it is necessary to use a cotton tip applicator (for example, CTA-10 from
www.thorlabs.com ) soaked in ethanol. Ideally cells should have reached a low confl uence (30–40%) in order to have single cells within the fi eld of view. In this situation, tissues or cells grown on the membrane are not observable. T e membranes with micron- size pores generate speckles and forbid any observation. Because the fi eld of view of the microscope is ~90 µm and the depth of fi eld is ~30 µm, both the dimension and the thickness of the cell must be less than these values. Observation techniques . Again there are two options: (1) On coverslips the medium chamber is sealed to avoid liquid drying out or leakage. This can be done either by taping on an imaging spacer (for example, from Grace Bio-Labs SS1X9-SecureSeal Imaging, typically with an inner
2015 July •
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diameter of 9 mm and a thickness of 0.12 mm), by using the recommended method, or by sealing with nail polish. The last option is not the best choice because the liquid frequently overflows onto the coverslip. (2) On a FluoroDish, live or fixed cells may be directly observed on this specially designed culture dish (FluoroDish glass bottom culture dishes are available at
www.wpiinc.com ; ask for a 35 mm dish with a 25 mm well). In this case the amount of liquid is not important. It just should be enough so that the bottom surface of the dish is fully covered. 3D Cell Explorer . The microscope measures changes in refractive index as light propagates through the cell, providing 3D and 4D (time) cell tomography without any intrusion of the cell. Holography offers a means to probe cells in their native environment: label-free, non-invasive, manipulation-free, and interference-free. Rotational scann- ing (with a low-power 520 nm laser beam) allows low-noise 3D reconstructions and a resolution that pushes the limit for light (Δ xy of 200 nm and Δ z of 500 nm). This new technology allows measurement of cellular processes with real-time kinetics, enabling multi-parameter analysis at the single-cell and sub-cellular scale. The cells are not manipulated in any way in order to introduce a digital label, thus the user has the possibility of measuring the response of a stimulated cell in real-time. STEVE . The Nanolive 3D Cell Explorer works differ- ently from most microscopes available today. Images are acquired with proprietary control software called STEVE (software for tomographic exploration of living cells). The microscope quickly self-adjusts, and a full 3D image of the cell appears on the computer screen. STEVE software controls the automatic calibration routines, 3D data acquisition, and reconstruction. Based on the cell’s physical properties (that is, refractive index) the microscopist can decide which cell parts are of interest after the experiment and can stain these regions digitally, saving time and money on reagents. This means regions with similar refractive index can be stained differently based on how sharply (large gradient) or smoothly (small gradient) the refractive index is changing within each region. An offline version of STEVE may be obtained at
http://nanolive.ch/software . This is an alpha version, which works on 64-bit version of windows Vista/7/8. For additional support, Nanolive provides a document and a video to help the new user get started. Control panels . STEVE has an intuitive graphical user interface, shown in Figure 3 , that has been specifi cally designed for fast learning by showing all options and commands necessary for acquiring and staining images on the main screen. T e Panel Viewer shows acquisition results as two-dimensional (2D) slices and includes an interactive tool for defi ning digital stains that are applied to the 3D dataset. T e result is shown in a 3D visualization window. Below the Panel Viewer is a Control Field, which allows the user to access the diff erent modes of operation. To quickly scan a sample, the user can choose the “white light” mode to illuminate the sample using standard, incoherent wide-fi eld illumination. T e fi eld of view is shown on the screen in real time. Although the images obtained in this mode are low in contrast and limited to two dimensions,
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