Fig 1.
Optical layout of the multicolor fluorescence microscope developed for monitoring interaction between polystyrene micro-beads as surrogates for drug carriers and cancer cells in a microfluidic channel.
(a) Two-dimensional excitation and emission spectrum of fluorescein, CellTracker Red CMTPX, and indocyanine green measured by a spectrofluorometer in the range of 200 nm and 900 nm. The peak excitation and emission wavelengths are shown with vertical lines. (b) and (h) Illumination and emission units. (c) and (g) Spectrum of excitation and emission paths, respectively. (d) and (e) Illumination and microscope objectives that employed for focusing excitation light on the sample and collection of emitted light from the fluorophores, respectively. (f) Spectrum of light-emitting diodes.
Fig 2.
(a) Schematic of cell spheroid formation in microwell array from micropatterned agarose well. (b) After the expansion of HeLa cells in standard tissue culture plates, cells were transferred into agarose microarrays for 3D cell spheroid formation with a diameter of 200 μm. Circularity and diameter of the spheroid versus time are plotted in (b2) and (b3), respectively.
Fig 3.
Multicolor fluorescence microscope used for real-time image acquisition of microfluidic channel, HeLa cell spheroids, and micro-beads in a round-robin manner.
Scale bar: 100 μm.
Fig 4.
Multicolor fluorescence image formation.
(a1)-(a3) Contour plots represent the spectrum of fluorescein, CellTracker Red CMTPX, and indocyanine green measured by a spectrofluorometer at varying excitation and emission wavelengths, respectively. (b)-(d) Acquired raw individual fluorescence images of micro-beads, HeLa cell spheroids, and microfluidic channel from different spectrum bands, respectively. (e) Merged fluorescence image of beads and spheroids. (f) Formed multicolor fluorescence image by overlapping acquired spectrally-different individual images. (g) Magnified view of the region of interest (ROI) defined on the multicolor image for revealing further details. Scale bar: 50 μm.
Fig 5.
A microfluidic channel contains micro-beads and a HeLa cell spheroid is imaged with (a1) bright-field (Axio Vert. A1, ZEISS, Germany), (b1) fluorescence (EVOS M5000, Thermo Fisher, USA), and (c1) our multicolor fluorescence microscopes. (a2)-(c2) Magnified views of the region of interests (ROIs) cropped from the acquired microscope images. (a3)-(c3) Image gradients of the ROIs for contrast visualization. Scale bar: 50 μm.
Fig 6.
(a) Status of light-emitting diodes. (b)-(d) Acquired fluorescence images from 500–540 nm, 592.5–667.5 nm, and 817.5–875.5 nm spectrum bands, respectively. (e) Formed image by overlapping acquired images. (f) and (g) Quantitative crosstalk analysis by computing structural similarity measure index between reference images (REF) and images acquired using continuous (CON) and round-robin (RR) methods, respectively. Scale bar: 100 μm.
Fig 7.
(a) Multicolor fluorescence images at varying exposure times. (b) Magnified view of the region of interest (ROI). (c1) and (c2) Camera gain and LED power during the image acquisition, respectively. (d1)-(d3) Intensity profiles of the lines defined on the beads, spheroid, and channel, respectively. (e1) and (e2) Measured peak signal-to-noise ratio and structural similarity measure index between multicolor images in (b) and the reference (multicolor image with 66.0 ms exposure time), respectively.
Fig 8.
(a), (b) Time-lapse imaging for dynamic uptake of indocyanine green in a HeLa cell spheroid. (c) Effect of staining a spheroid using both CellTracker Red CMTPX and indocyanine green on the multicolor fluorescence images. (d) Time-lapse imaging of the flow created by temperature gradient. (b2) and (d2) Expressing effect of the flow on the fluorescence signal by plotting normalized pixel intensities over time on the defined red-lines in (b1) and (d1), respectively.
Fig 9.
The effect of photobleaching mechanism on multicolor fluorescence microscopy under constant excitation power.
(a) Time-lapse multicolor fluorescence images at varying exposure times. (b1)-(b3) Normalized average intensity profiles of micro-beads, HeLa cell spheroids, and microfluidic channel, respectively. Scale bar: 100 μm.
Fig 10.
The effect of continuous and round-robin excitation methods on photodamage mechanism.
(a1)-(c1) Acquired time-lapse fluorescence images of micro-beads, HeLa cell spheroids, and microfluidic channel, respectively. (a2)-(c2) Photobleaching curves by computing normalized average pixel intensity in each image. (a3)-(a4)—(c3)-(c4) Intensity profiles of the lines defined on beads, spheroids, and channel, respectively. Scale bar: 100 μm.
Fig 11.
Multicolor fluorescence microscopy for the attachment of polystyrene micro-beads to a HeLa cell spheroid surface utilizing electrostatic forces.
(a) Time-lapse multicolor fluorescence image sequence acquired at 15 frames per second. (b) The analyzed motion using Lukas-Kanade optical flow in ROI(1) and ROI(2) at 63 seconds and 518 seconds is represented as vector fields and overlaid on the fluorescence images. The spectrally different motion information of beads, spheroid, and channel is represented using green, red, and blue arrows, respectively. (c) Time-lapse fluorescence image sequence of beads and spheroid. (d) Neutral HeLa cell spheroid covered with micro-beads. (e) Gradients of the images in (d) for contrast visualization. Scale bar: 100 μm.