Fig 1.
Cheek cell image using brightfield and darkfield microscopy.
(a) Schematic diagrams of a darkfield condenser, (b) brightfield microscopy image, and (c) darkfield microscopy image of a cheek cell. Scale bars in the Figs represent 5 μm. Images were taken with UplanSapo 60× oil (NA 1.35). All images (b, c) were taken using the color camera.
Fig 2.
Schematic of the whole slide imaging (WSI) system with darkfield internal reflection illumination (DIRI).
DIRI was incorporated into the WSI system’s motorized stage. Three color light-emitting diodes (LEDs) illuminate the slide glass from the side, and the specimen scatters this light. The scattered light is then incident on the objective lens above the stage. The dichromatic mirror on the motorized turret of the microscope can be removed from the light path when acquiring darkfield images. A tube lens above the dichromatic mirror focuses the sample image onto the imaging device. A charge-coupled device camera then captures the image. A sharp cut-off filter is placed between the field diaphragm and the mirror.
Fig 3.
DIRI with three-color-LED array.
(a) The three-color-LED array and controller, (b) a sample illuminated with green-LED array, (c) a schematic diagram of the three-color LEDs and a slide glass sample, and (d) a sample illuminated with red (left), green (middle), and blue (right) LEDs in the E gracilis experiment.
Fig 4.
Brightfield image and red, green, and blue darkfield internal reflection illumination (DIRI) images of tissue microarray (TMA) sample.
(a) Illuminated by brightfield, (b) red, (c) green, and (d) blue. All images were taken with UplanSapo 40× oil (NA 0.95) using the VC50 color camera. The exposure times are; (a) 8.7 ms, (b-d) 700 ms. The sample was skin tissue. Scale bars in the Figs represent 200 μm.
Fig 5.
Maximum intensity illumination of color DIRI and color balance modification.
(a1) Image taken with the maximum intensity of color DIRI with red 255, green 255, and blue 255. (a2) Photoshop color chart of a1 showing the location in the color balance. (b1) Image taken with the modified color DIRI with red 150, green 255, and blue 100. (b2) Photoshop color chart of b1 showing the location in the color balance. All images (a1, b1) were taken with UplanSapo 40× oil (NA 0.95) using the VC50 color camera. The exposure time is 400 ms. The sample was skin tissue. Scale bars in the Figs represent 200 μm.
Fig 6.
Diaminobenzidine (DAB)-stained brain slices.
(a) Brightfield image. (b) Color DIRI image with red 150, green 225, and blue 100. Images were taken with PlanApon 2× (NA 0.08) (a), and UPlanSapo20× (NA 0.75) (b). The exposure times are (a) 4 ms, and (b) 150 ms. Both are using the VC50 color camera. Scale bars indicate 0.5 mm.
Fig 7.
Brain slice images obtained by color DIRI.
(a) Brightfield image taken with PlanApon 2× (NA 0.08) using the VC50 color camera. The exposure time is 4.6 ms. (b) Darkfield image taken with DIRI with Orca R2 B/W camera. The exposure time is 100 ms. (c) LED-illuminated fluorescence image of area Z1 taken with UPlanSapo40× (NA 0.95) with Orca R2 B/W camera. The exposure time is 100 ms. (d) Epi-fluorescence image of area Z1 taken with UPlanSapo40× (NA 0.95) with Orca R2 B/W camera. The exposure time is 100 ms. (e) LED-illuminated fluorescence image of area Z2 taken with the UPLSAPO 40× silicon immersion lens (NA 1.25) with Orca R2 B/W camera. The exposure time is 100 ms. (f) Epi-fluorescence image of area Z2 taken with the UPLSAPO 40× silicon immersion lens (NA 1.25) with Orca R2 B/W camera. The exposure time is 100 ms. Scale bars in the Figs represent (a–d) 2 mm and (e, f) 0.1 mm.
Fig 8.
Photo responses of E gracilis cells stimulated by green LED.
(a) Initial distribution of cells when the LED was off. Small black dots in the figure indicate cells. (b) The distribution of cells 10 min after loading when the LED was off. (c) The distribution of cells 30 min after turning on the green LED with an intensity of 5. Arrow indicates the position and direction of illumination of the LED. (d) Distribution of cells 10 min after increasing the light intensity to green 255. All images were taken with UPlanSapo10× (NA 0.4), and the scale bars in the figure represent 2 mm. All images were taken using Orca R2 color camera. The exposure time is 100 ms.
Table 1.
Comparison of Hausmann’s darkfield, white-LED DIRI, and three-color LED DIRI.