Digital Staining: Microscopy of Live Cells Without Invasive Chemicals
Lisa Pollaro* , Bastien Dalla Piazza , and Yann Cotte Nanolive SA , Chemin de la Dent d’Oche 1a , 1024 Ecublens , Switzerland
*
lisa@nanolive.ch Introduction
Until now it was impossible to look inside a living cell without damaging it, even using the most sophisticated devices. Traditional microscopy techniques rely on procedures requiring complicated time-consuming preparation (1–72 hours), which are likely to be invasive to the cells (risk of cell damage). T e 3D Cell Explorer from Nanolive overcomes many limitations of light microscopy in live cell imaging. Just like computer tomography (CT) for human bodies, the 3D Cell Explorer can make a complete tomographic data set of the living cell [ 1 ]. But unlike a CT for human bodies, it does it instantly and at very low cost. Key among the features is the new concept of digital staining, which allows users to look inside the living cell, discover its interior organelles, and “travel” through it in 3D on any screen. T is article describes the method and provides some examples.
Materials and Methods Tomography of cells . By a combination of holography
[ 2 ] and rotational scanning [ 3 ], the system detects changes to light as it propagates through the cell. The sample is positioned between a high-numerical-aperture air objective beneath the sample and a rotational illumination arm above ( Figure 1 ). This optical path forms one arm of a Mach–Zehnder interferometer setup, with the other being the reference path. Green light (520 nm) from a diode laser is split into sample and reference beams; the sample beam illuminates the sample through the rotational illumination arm at a very steep angle ( Figure 2 ). A hologram is recorded on a digital camera by combining the beam that has passed through the sample with the reference beam. The sample beam is then rotated by a small angle and the process is repeated, with one hologram recorded for each beam position. The parameter measured by the 3D Cell Explorer is not absorption nor fluorescence intensity of an exogenous molecule as with most light optical microscopes. Instead, the physical refractive index of the sample is obtained in a three- dimensional (3D) distribution with a resolution better than the diffraction limit given by the microscope objective. The output is the refractive index distribution within the cell. The result is quantitative cell tomography, in vitro without any invasive sample preparation. Improved image resolution is achieved by employing a synthetic aperture and multiple- viewpoint-holographic methods. After the holograms have been captured, high-resolution images of each plane in the sample are created by computer processing. Specimen preparation . As a general requirement for any type of sample, the buff ering medium should be optically clear and not scatter incoming light. For instance, solutions
12
Figure 1: Marker-free 3D Cell explorer sample stage setup. The rotating scanning head illuminates the sample from all directions with a 520 nm green laser light at low power.
with phenol red are not suitable for observation. Best are clear liquids like PBS or HEPES. T e observation is always made either through FluoroDishes (glass bottom culture dishes) or coverslips about 150 µm thick (standard practice for microscope objectives). Also, because of the limited working distance of our objectives, cells should be fi xed on the holders and not much more than 30 µm in height. Cleanliness . T e cleanness of the sample is crucial to assuring good quality images. Because of our rotating illumi- nation system, debris (either fl oating or out-of-focus) can be hit by the beam while not being in the fi eld of view. So optical surfaces must be as clean as possible, and cell holders should be carefully cleaned so that dead cells or other remains are not fl oating in the mounting medium.
Depending on the support used for the experiment, there are two procedures to follow before the start of data acquisition with the 3D Cell Explorer: (1) For coverslips, lens tissues are recommended to clean the surface of the coverslip
doi: 10.1017/S1551929515000590
www.microscopy-today.com • 2015 July
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84