Sample Images

Super-resolution and Localization Microscopy

Complete figure explanations are available for this set of images. Clockwise from top left: Figure 1, Figure 2, Figure 3, Figure 4.

Figure 1. The capability of the Flash 2.8 sCMOS camera in localization microscopy.

Figure 2. Histograms of the height of peak pixel after background subtraction.

Figure 3. Original TIRF image frames in the green box were used to build a movie.

Figure 4. Dependence of spatial resolution and the number of image frames in localization microscopy.

High-speed imaging

• Top figure: Observation image
• Left graph: Temporal (Ca²⁺) dynamics (4 ROI)
• Sample: spontaneous (Ca²⁺) change of Fluo-4-loaded Ins-1 cells
• Acquisition setting: 45 fps (exposure time: 22 ms)

Comparison of resolution

• Bright field observation
• Sample: diatom test plate
• Objective lens: Plan Apo 40x

Comparison of field of view

• Fluorescence observation
• Sample: FluoCell prepared slide #2 (BODIPY)
• Objective lens: Plan APO 40x

High sensitivity, high resolution imaging

• Superimposed trichrome stain
• Sample: FluoCells prepared slide #2

Ca²⁺ and PKC dual imaging using Fluo-4 and DsRed (optical block: DM 550 nm)

The images to the left show fluorescence signal from Fluo-4 AM loaded cells expressing PKC-DsRed when excited by 480 nm light. Observable in this time sequence is the translocation of PKC-DsRed to the cell membrane concurrent with free Ca²⁺ elevation in response to a depolarizing stimulus.

• Sample: Ins-1 cell (insulin-producing cell)
  
• ORCA-D2 optical block: A11400-04 (DM 550 nm, Em1 520 nm/35 nm, Em2 593 nm/40 nm)
  
• Microscope: Olympus IX71
  
• Objective lens: Olympus UPlanAPO 60x / 1.20 W

Samples images courtesy of Hideo Mogami, Ph.D. Dept. of Physiology, Hamamatsu University School of Medicine. Dept. of Environmental Biology, Okazaki Inst. for Integrative Bioscience

Ca²⁺ measurement using YellowCameleon 3.6 (optical block: DM 510 nm)

The images to the left are an example of ratio imaging. Separated CFP and YFP (FRET) are measured with dual CCD devices. This sequence observed the YellowCameleon 3.6 (Ca²⁺ sensor based on CFP-YFP FRET) expressed Ins-1 cell response with a depolarizing stimulus.

• Sample: Ins-1 cell
  (insulin-producing cell)
  
• ORCA-D2 optical block: A11400-03 (DM 510 nm, Em1 483 nm/32 nm, Em2 542 nm/27 nm)
  
• Microscope: Olympus IX71
  
• Objective lens: Olympus LUCPlanFLN 60x, NA 0.70

Samples courtesy of Hideo Mogami, Ph.D. Dept. of Physiology, Hamamatsu University School of Medicine. Dept. of Environmental Biology, Okazaki Inst. for Integrative Bioscience

ORCA-D2 compared to conventional CCD image sensor

The low light mode image (ORCA-D2) compared to a conventional high-sensitivity CCD device (ICX285) which is used for scientific ccd cameras.

The brightness profile of the
area shown in the graph
is marked with white horizontal line
on the sample image.

Luminescence imaging of HeLa cells expressing Renilla Luciferase

This image is displayed by overlapping luminescence image (pseudocolor) and actual image

• Objective lens: 10x
• Cooling method: water cooling (-80 C)
• EM gain: 200x
• Exposure time: 5 min

Single fluorescent molecules in HeLa cells expressing H2B-GFP fusion protein

• EM gain: 1200x
• Exposure time: 30.5 ms
• Data courtesy of
  - Dr. Makio Tokunaga, National Institute of Genetics
  - Dr. Kumiko Sogawa, RIKEN RCAI
  - Dr. Hiroshi Kimura, HMRO Kyoto Univ. Faculty of Medicine