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Fluorescence, phase-contrast, and bioluminescence imaging of live cells incubated in plastic bottom dishes using a 20X high numerical aperture (NA) phase contrast objective lens


Introduction
Imaging stem cells incubated in plastic bottom dishes

Glass bottom culture dishes used for the fluorescence observation of live cells are commonly marketed by many incubation chamber manufacturers; however, some types of cells cannot be successfully cultured on a glass surface and require the use of plastic bottom dishes.
Certain types of stem cells, such as iPS cells (induced pluripotent stem cells) and ES cells (embryonic stem cells), are better able to differentiate into target cells when cultured on a plastic substrate. In fact, some cell types cannot be differentiated into target cells at all when incubated in glass bottom dishes. The downside of using plastic bottom dishes, however, is that fluorescence imaging is often complicated by the autofluorescence produced by the plastic material, resulting in a high degree of background signal. When this happens, the signal-to-noise ratio is impaired and it becomes more difficult to image the fluorescent sample.
Stem cell researchers want to be able to carry out vivid fluorescence observation while using plastic bottom dishes. In response to this need, Olympus developed a 20X high NA phase-contrast objective lens (UCPLFLN20XPH) in which the numerical aperture is greatly improved compared to a conventional 20X phase-contrast objective lens. The improved numerical aperture produces a better signal-to-noise ratio enabling better fluorescence imaging with plastic dishes. As a result, researchers can more effectively observe the stem cell differentiation process with fluorescence time-lapse imaging and more easily assess the confluency or transfection rate of cells cultured in plastic bottom dishes.
In this application note, we demonstrate how Olympus’ 20X high NA phase contrast objective lens enables vivid fluorescence and phase-contrast observation of stem cells in a plastic bottom dish as well as an example of bioluminescence imaging observation using the same objective lens.

Application: Fluorescence and phase-contrast observation using Olympus’ 20X high NA phase-contrast objective lenses

Mouse embryonic stem (ES) cells with GFP-labeled histone (GFP-H2B) in the nuclei were incubated in plastic bottom dishes. These cells were imaged with a conventional 20X phase-contrast objective lens (NA=0.4) and then compared to images taken with Olympus’ 20X high NA (NA=0.7) phase contrast objective lens. 

Mouse ES cells expressing GFP-H2B

Fluorescence observation

左:ES細胞_LUCPLFLN_FL 右:ES細胞_UCPLFLN_FL

Phase-contrast observation

左:ES細胞_LUCPLFLN_PH 右:ES細胞_UCPLFLN_PH

Left:Conventional 20X phase-contrast objective lens LUCPLFLN20XPH (NA=0.45)
Right:20X high NA phase-contrast objective lens UCPLFLN20XPH (NA=0.70)

When the above images are compared, bright fluorescence images were acquired with the 20X high NA phase-contrast objective lens (UCPLFLN20XPH) and the nucleoli are easily observed. This is due to the fact that the objectives have an improved numerical aperture while retaining a long working distance (W.D.: 0.8–1.8 mm).
These images demonstrate that fluorescence images comparable to those obtained using glass bottom dishes can be acquired when the UCPLFLN20XPH objective is used. With the aid of a correction collar, the spherical aberration generated due to differences in the thickness of the plastic dish bottom can be easily corrected.

Photographic conditions:
Specimen: Mouse ES cells
Fluorescence label: GFP-H2B
Incubation chamber: Plastic bottom dish
Objective lens: High NA phase-contrast objective lens UCPLFLN20XPH
CCD camera: Digital microscope camera DP80
Microscope: Inverted research microscope IX73

Application: Increasing the fluorescence sensitivity of the CCD DP80 digital microscope camera using monochrome CCD mode

As described above, Olympus’ high NA 20X phase-contrast objective lens (UCPLFLN20XPH) enabled vivid fluorescence and phase-contrast observation of cells incubated in a plastic bottom dish. However, when a dish is repeatedly taken out from the incubator to check the fluorescence of the specimen during experiments, it is necessary to minimize the phototoxicity produced by the excitation light. In this case, using a highly sensitive CCD camera can reduce the intensity of the excitation light while by reducing the duration of time that the sample is exposed to potentially cytotoxic light.
Olympus’ DP80 digital microscope camera offers the dual functionality of color reproducibility and high-sensitivity monochrome CCD imaging. By employing the DP80’s monochrome CCD mode in conjunction with the 20X high NA phase-contrast objective lens (UCPLFLN20XPH), Nanog-GFP expressed in mouse iPS cells was efficiently detected at a lower light intensity, as illustrated by the images below.

Nanog-GFP expressed mouse iPS cells

iPS細胞_UCPLFLN_PH
Phase-contrast observation

iPS細胞_UCPLFLN_FL
Fluorescence observation

Photographic conditions:
Specimen: Mouse iPS cells
Fluorescence label: Nanog-GFP
Incubation chamber: Plastic bottom dish
Objective lens: High NA phase-contrast objective lens UCPLFLN20XPH
CCD camera: DP80 digital microscope camera
Microscope: Inverted research microscope IX73

Application: High-performance bioluminescence imaging

Olympus’ 20X high NA phase-contrast objective lens (UCPLFLN20XPH) achieves outstanding performance for demanding applications such as bioluminescence imaging with plastic bottom dishes. Bioluminescence imaging enables the user to quantitatively detect gene expression at the single-cell level. This imaging modality can easily be performed with the Olympus LV200, a microscope specifically designed for bioluminescence imaging. When the UCPLFLN20XPH objective is used with the LV200, multiple cells can be observed in a single field. As shown in the image below, phase-contrast and Nanog-Luc bioluminescence (yellow) can be easily visualized in mouse ES cells with high sensitivity at the single-cell level.

ES細胞_UCPLFLN_Luc

Photographic conditions:
Specimen: Mouse ES cells
Bioluminescence: Nanog-Luc
Incubation chamber: Plastic bottom dish
Objective lens: High NA phase-contrast objective lens UCPLFLN20XPH
Microscope: Bioluminescence imaging system LV200

Conclusion

The examples discussed illustrate that the Olympus 20X high NA phase-contrast objective (UCPLFLN20XPH) enables vivid fluorescence, phase-contrast, and bioluminescence imaging of cells cultured in plastic bottom dishes. The UCPLFLN20XPH objective lens is compatible with the IX3-ZDC2 Z-drift compensator and the IX3 series of motorized inverted research microscopes. When equipped with a motorized stage, a fully-motorized cell-based assay system for plastic bottom dishes can easily be configured.

Products used for this application

Bioluminescence imaging system

LV200

  • Completely new optical design for greater sensitivity
  • Unrivalled resolution in luminescence microscopy
  • Wide range of magnification
고급 라이브 셀 이미징을 위한 도립 현미경 시스템

IX73

  • 독특한 덱 시스템
  • 반자동 시스템
  • 시스템 솔루션
Compound Microscope System

IXplore Standard

  • Repeatability and accuracy for standard imaging tasks
  • Benefit from the same optical capabilities found in high-end IXplore systems
  • Easily upgrade to encoded functionality to boost reproducibility of experiments
  • Obtain high quality images, even with standard cell culture vessels with dedicated objectives
듀얼 CCD 컬러 및 흑백 카메라

DP80

  • 1,250만 화소의 컬러 CCD와 140만 화소의 모노 CCD
  • 컬러와 모노 센서 사이에서의 고속 전동 전환
  • 픽셀 수준의 정밀도로 정확하게 오버레이 되는 컬러와 흑백 영상
고급 대물렌즈

UCPLFLN/LUCPLFLN

  • Long working distance의 범용 대물렌즈로 우수한 명시야(Brightfield), 미분간섭(DIC)과 형광(Fluorescence) 관측을 제공.
  • 높은 투과율이 근적외선 영역대까지 적용되는 평탄한 영상 제공
  • 배양용기(Bottle and Dish) 상의 조직 배양 관찰에 특화됨. 

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