Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Preparation
2.1.1. Preparation of Live Cell Cultures
2.1.2. Fluorescent Staining of HeLa Cells
2.2. High-Resolution Bright-Field Light Microscope
- 1.
- A phase contrast microscope Nikon BioStation IMQ with a high-sensitivity 1.3 Mpx cooled monochrome camera, 40× objective magnification (Nikon, N.A. 0.8);
- 2.
- A bright-field microscope Olympus IX51, 40× objective magnification (objective LUC Plan FLN 40× 0.60 Ph2, ∞/0-2/NF 22), camera Infinity 1 giving raw image data;
- 3.
- A fluorescent microscope Nikon Eclipse 80i, 40× objective magnification (Nikon Plan Fluor 40×/0.75, Ph2 DLL, ∞/0.17, WD 0.66), mercury discharge tube, cell sample was stained by fluorescein.
2.3. Microscope System Calibration and Image Correction
- 1.
- Focus position determination:
- (a)
- During image acquisition, a light is passing through the microscope optical path and sample. The resulting signal was then captured by a camera sensor. Filters of ND 0.1, 0.2, 0.3, 0.4, 0.5, and 0.8 were scanned through their depths with a step of 130.9 nm. In this way, six sets of images were obtained. The results for neutral densities ND <0.1 and ND 0 were acquired for a path of ray with the blank support UV fused silica and for a clear path of ray without any light absorber, respectively;
- (b)
- The sets of images for ND <0.1–0.8 were processed by Image Info Extractor Professional software (ICS FFPW) (chosen Rényi parameter ) [28]. The point information gain entropy (PIE) gives a total change of the image information after iterative removing of one pixel from an individual image. The highest value of this parameter allows one to pick an in-focus image taken from the center of the coatings (Figure 3). This reduced the diffraction-induced aberrations from the boundaries of the calibration sample.
- 2.
- Acquisition of calibration images and transmission spectra of linear step filters:
- (a)
- A set of calibration data (40 images for each neutral density) was taken at the determined focus position (see item 1b). The calibration images were calculated as a pixel-wise mean through the image sets corresponding to each neutral density. In this way, we reduced a random noise in the measured data;
- (b)
- An optical waveguide P400-1-UV-VIS was housed inside a 3D-printed mechanical adapter at the determined focus position and connected to a spectrometer Ocean Optics USB 4000 VIS-NIR-ES. The spectral responses ND <0.1–0.8 in Figure 4a correspond to electroluminescences of relevant calibration sample filters. The spectral response ND 0 reflects electroluminescence captured without any absorber by an optical spectrophotometer during calibration sample illumination.
- 3.
- Calculation of the radiant fluxes reaching each microscope camera pixel after passing the calibration filters.
- (a)
- (b)
- After data smoothing and interpolation, the light spectra reaching each pixel of the camera sensor (Figure 4c) were obtained as multiplication of the incoming spectra (measured by the calibrated spectrometer; see item 3a and Figure 4b) by the respective (red, green, blue) quantum efficiency profile of the Kodak KAI-16000 image sensor (Figure 2c);
- (c)
- For each neutral density of the filter coating, the radiant flux (Figure 4d) reaching each pixel during exposure was calculated as an integral (trapezoidal rule) of the area below the respective incident spectrum.
- 4.
- Calibration curves and image correction:
- (a)
- Calibration curves were obtained for each image pixel position and respective extensions of values of camera channel radiant flux (see the range of filter coatings in items 1–2). The calibration curves map the pixel intensities to the real spectra responses (e.g., Figure 5). Each pair of consecutive points was interpolated linearly;
- (b)
- In order to extend the range of experimental (useful) intensities, the initial calibration curves for each pixel were extrapolated linearly about 80% (Figure 5, magenta line). Statistics of the pixels’ intensity values for each calibration filter was evaluated (Figure 6) in order to see the deviations of the sensor pixels’ responses from idealities;
- (c)
- Hereafter, the calibrated image format (as double precision floating point type) showing the spatial distribution of radiant fluxes was transformed into a png format. The calibration curve associates the value of each calibration intensity with the respective experimental measured photon energy.
2.4. Least Information Loss (LIL) Image Conversion
3. Results and Discussion
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
bpc | bits per channel |
PBS | fetal bovine serum |
FFPW | Faculty of Fisheries and Protection of Waters |
ICS | Institute of Complex Systems |
ILCX | x-position read out by a microscope incremental linear sensor |
ILCY | y-position read out by a microscope incremental linear sensor |
LED | light-emitting diode |
LIL | Least Information Loss algorithm |
LWD | microscope objective low working distance |
N.A. | Numerical Aperture |
NAMC | Numerical Aperture Modulation Contrast iris |
NIST | National Institute of Standards and Technology |
ND | Neutral Density |
OD | Optical Density at 633 nm, OD T, where T is a light transmission |
at 633 nm | |
PBS | phosphate buffer saline |
PIE | Point Information Gain Entropy |
PIG | Point Information Gain |
PNG | portable network graphics |
rH | relative humidity |
rpm | rotations per minute |
UV | ultraviolet |
VIS-NIR-ES | visible and near-infrared with enhanced sensitivity |
WD | microscope objective working distance |
Appendix A. Optical Fiber Spectrophotometer Calibration
Appendix B. Least Information Loss Matlab Algorithm and Pseudocode
Algorithm A1: The Least Information Loss algorithm for a grayscale image. |
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Platonova, G.; Štys, D.; Souček, P.; Lonhus, K.; Valenta, J.; Rychtáriková, R. Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data. Photonics 2021, 8, 333. https://doi.org/10.3390/photonics8080333
Platonova G, Štys D, Souček P, Lonhus K, Valenta J, Rychtáriková R. Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data. Photonics. 2021; 8(8):333. https://doi.org/10.3390/photonics8080333
Chicago/Turabian StylePlatonova, Ganna, Dalibor Štys, Pavel Souček, Kirill Lonhus, Jan Valenta, and Renata Rychtáriková. 2021. "Spectroscopic Approach to Correction and Visualisation of Bright-Field Light Transmission Microscopy Biological Data" Photonics 8, no. 8: 333. https://doi.org/10.3390/photonics8080333