Automated Holotomographic Microscopy
to 100 tiles, organized as squares, providing access to large fields of view of up to 1 mm. Te user must specify the time-lapse frequency and length of the acquisition. Tanks to the absence of phototoxicity [2] and to state-of-the-art environment control, the length of the time-lapse experiment is limited only by biologi- cal factors such as cell crowding, as the device can maintain cells in tis- sue culture incubator conditions. At the end of the acquisition, the user can proceed with image analysis, and image data can be exported in a vari- ety of formats. Focus stability is essential when
Figure 1: Design of the CX-A. (a) The CX-A holotomographic optical setup allows for hologram recording at 360° around the sample using a 45° incident laser. This setup is combined with epifluorescence. (b) and (c) The CX-A consists of a 3D Cell Explorer-fluo microscope unit mounted on an automated stage platform.
temperature of 37°C and humidity saturation, as well as 5% CO2
, were achieved throughout the image acquisitions. Image processing. Acquired 3D volumes were all sub-
jected to maximum projections along the z-axis, from -2 μm to +6 μm with respect to the focal plane, in order to obtain the 2D images displayed in Figures 4–6. Tis processing was provided by the export function of the EVE soſtware. Quantitative cell analyses provided in Figure 6 were made using EA.
Results and Discussion EVE supports three major functions: i) setting up acquisi-
tion protocols, ii) monitoring of data acquisition, and iii) quan- titative image analysis. Te acquisition process is compatible with single- or multiple-well plates. For each well, it is possible to perform single field of view or grid-scan acquisitions of up
imaging multiple positions, such as screening experiments in a multi- well setup. An autofocus method was created to fit the specific optical con- straints of HTM. Te strategy relies
on the identification of the plane along the z-axis that contains the sharpest content based on the digital processing of holo- gram acquisitions (Figure 2a). If this position is different com- pared to the current z position, the stage is adjusted accordingly (Figure 2b). Figure 3 shows the HTM imaging of specific drug effects
applied at various concentrations (Figure 3a), which allows for a new type of screening approach, where complex phenotypic variations can be detected with high resolution and contrast (Figures 3a and 3b). Tese features are unique but qualitative, and, so, to fully unleash the potential of HTM, quantitative image analysis is essential. Te strength of HTM is the ability to catch multiple bio-
logical structures [2] at once (Figure 3c). Tis strength, how- ever, becomes a weakness when it comes to segmenting cells. Ease of segmentation and quality of images are exclusive, as it is easier to segment a blob with no internal sig- nal variations than a very complex cellular object displaying a struc- tured, heterogeneous signal [9]. How can unstained cells with complex texture and a signal that is orders of magnitude more heterogenous than a cellular fluorescent signal be ana- lyzed? EA is the answer. Segmenting cells in microscopic
Figure 2: The CX-A maintains perfect focus with a proprietary autofocus strategy. a. Autofocus setup sequence. b. The autofocus strategy is to seek the sharpest signal by digital hologram processing and to adjust the stage position accordingly.
26
images relies on one simple concept: observable cells must display a sig- nal that is higher than background. Creating such contrast has always been the challenge of microscopy, and current cell detection strategies have oſten been developed based on fluorescent nuclear
stains such as
DAPI [13], Hoechst [14], and others [15]. Fixed cells stained with DAPI
www.microscopy-today.com • 2021 September
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