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Phase and fluorescence imaging with a surprisingly simple microscope based on chromatic aberration

Ondřej Mandula, Jean-Philippe Kleman, Françoise Lacroix, Cedric Allier, Daniel Fiole, Lionel Hervé, Pierre Blandin, Dorothee C. Kraemer, and Sophie Morales

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Abstract

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 μm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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Supplementary Material (1)

NameDescription
Visualization 1       Time-lapse recording of triple stained Hela cell culture in combined phase contrast (gray) and fluorescence (color) image. Hoechst stain marking cell nucleus is shown in blue, eGFP marking alpha-tubulin structures in green and non-specific pmCherry .

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Figures (8)
Fig. 1.
Fig. 1. (a) A demonstration of a non-absorbing (phase) sample (3 DIV hippocampal neurons) at different levels of defocus. The cells are almost invisible when brought in-focus (middle). (b) Our reconstruction of the in-focus phase-contrast image from a single, $100\,\rm {\mu m}$ defocused image taken under semi-coherent illumination.

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Fig. 2.
Fig. 2. a) Measured chromatic displacement of the focus. The theoretical depth of field of the system ($10\,\rm {\mu m}$) is shown as a red band. While the system is approximately achromatic in the range $\lambda =500-600\,\rm {nm}$ (green to red), it is strongly chromatic at $\lambda =420\,\rm {nm}$ (violet). b) Multi-band fluorescent filter set with a phase (defocus) imaging filter at $\lambda =420\,\rm {nm}$ passing through the blue emission band.

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Fig. 3.
Fig. 3. System in fluorescence (a-b) and phase (c) mode. Note the defocus due to chromatic aberration in (c). L1 blue and green LEDs for fluorescence excitation, L2 blue LED for defocused imaging in transmission mode with a narrow band filter IF. OF optical fibre, CL collimator lens, EX excitation filter, D dichroic, EM emission filter, O objective, SP sample plane, CMOS camera. (d,e) Regions of interests (ROIs) of in-focus green and red fluorescence, respectively. (f) Corresponding ROI of raw out-of-focus data. Scale bar $50\,\rm {\mu m}$. (g) The system installed in an incubator.

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Fig. 4.
Fig. 4. ROI of a snapshot from 40h recording of triple stained HeLa cell culture (Blue - Hoechst staining cell nucleus, Green - GFP tubulin, Red - mCherry). Raw defocused images taken in transmission mode are numerically reconstructed to yield an in-focus phase-contrast image of the sample. This can be combined with the corresponding in-focus epi-fluorescence data (dashed box) producing a phase (gray) and fluorescence (RGB) combined image (right).

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Fig. 5.
Fig. 5. USAF absorption (a-c) and phase (d-f) resolution target imaged with our system. a) Full field-of-view of absorption resolution target imaged in-focus, b) central ROI (red box in a)). We can resolve group 8, element 5 with line width of $1.23\,\rm {\mu m}$. c) line profile over group 8 (red line in b)). d) Reconstructed phase image of phase resolution target (silica slide with $300\,\rm {nm}$ thick engraving of the USAF resolution target) taken at $100\,\rm {\mu m}$ defocus. e) Central region (red box in d)) showing groups 6, 7 and 8. f) $100\,\rm {\mu m}$ defocused image (raw data) of a region shown in e).

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Fig. 6.
Fig. 6. Hela cells culture in combined phase contrast (gray) and fluorescenceimage. Hoechst stain marking cell nucleus is shown in blue, eGFP marking $\alpha$-tubulin structures in green and non-specific pmCherry in red. Whole field of view ($3\,\rm {mm}^2$) with circular region ($1.2\,\rm {mm}^2$) with acceptable flat field correction (white circle). There is $\sim 700$ cells contained in the circular region. ROI shown with red dashed box at 5-hour intervals is shown in Fig. 7(a).

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Fig. 7.
Fig. 7. Time-lapse of triple stained HeLa cells. Images taken every 10 mins over 40 hours. a) Selected ROI from Fig. 6 shown at 5-hour intervals (see also Visualization 1). b) A small ROI of reconstructed phase image. Cells in division show phase wrapping (red arrows).

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Fig. 8.
Fig. 8. a) Phase contrast image (gray) with overlayed fluorescence (green) of Micrococcus luteus marked with SYTO9 in mixture with $2\,\rm {\mu m}$ polystyrene beads. Insets show the local displacement between phase and fluorescence image. Reconstructed phase contrast image of ROI marked with red box is shown in b), corresponding fluorescence image is shown in c) with arrows highlighting several Micrococcus clusters. The structure of the clusters, the dimers (red), tetramers (cyan) and multimers (green) of cocci, is at the resolution limit of our system. Two beads above and at the focal plane are highlighted with a red and green box, respectively.

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Equations (3)

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A z ( r ) = A 0 ( r ) ∗ h z ( r ) ,
h z ( r ) = 1 i λ z exp ⁡ ( i π r 2 λ z ) .
ϵ ( A 0 ) = c 1 ∫ R 2 d r | ∇ A 0 ( r ) | + c 2 ∫ Ω d r | A 0 ( r ) | 2 ,
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