N° 18

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TECHNICAL NOTES & APPLICATIONS FOR LABORATORY WORKCONTENT
1. Applications &Features
2. Abstract
3. Microscopy in Cell
Biology

SCREENSTAR AND CELLVIEW:MICROPLATES FORADVANCED MICROSCOPY
1/ APPLICATIONS & FEATURES
1.1/ APPLICATIONS/High-content screening/

Water or oil immersion objectives/

High-magnification objectives (40x and above)/High-resolution objectives

1.2/ FEATURES/SCREENSTAR: 190 µm cycloolefin film bottom microplate/

CELLview: 175 µm glass bottom microplate/

Recessed microplate bottom for high magnification and improved resolution/

Superior bottom quality to enable high-quality images/

Meets ANSI standards/

Universal microscope objective compatibility/

Proven cell culture surface treatment

4. Substrates in
Microscopy
5. SCREENSTAR
and CELLview:
Microplates for HighResolution Microscopy
6. Ordering Information

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N° 18

2/ ABSTRACTSCREENSTAR and CELLview are microplates for advanced
microscopic applications such as high-content screening
with water or oil immersion objectives. The special designof the two microplates allows low working distances andcombines excellent optical features with high-quality surfaces for cell culture and biochemical assays.

Typical specimen supports in microscopy for cells or
tissues are objective slides, flasks, dishes and microplates. For microscopic analysis, inverted microscopes
are commonly used. The objective is positioned below
the microscope stage that houses the substrate containing the biological specimen, causing inclusion of thebiological sample support within the optical pathway(Fig. 2) of microscopic detection.

SCREENSTAR microplates are manufactured with an optically transparent 190 µm cycloolefin film bottom.

3/ MICROSCOPY IN CELL BIOLOGY
Microscopy is a basic and highly important method in life
science frequently used in research as well as medical
diagnostics such as histological cancer classification.
Technological progression in confocal microscopy, opticalsystems and emerging technologies such as total internalreflection fluorescence microscopy (TIRFM), continues
to elevate microscopy as one of the most powerful toolsin cell biology.
Unless intentionally cultivated as suspension or spheroidal cultures, adherent cells and tissues customarily
require a surface to attach and/or grow (Fig. 1).

A

B

Figure 1:(A) HEK 293 cells growing on the surface of a black clear bottom(µClear®) tissue culture treated microplate (Item No. 655090)(B) Tumour spheroid in a well of CELLSTAR® cell-repellent
U-bottom microplate (Item No. 650970)

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Support{CELLview microplates are manufactured with a 175 µm
borosilicate glass bottom.

Specimen

Light Path

Objective

Figure 2: Specimen support, e.g. microscopic slide as part ofthe light path in an optical system

Coming from standard top microscopy, microscope objectives are typically calibrated to accommodate a 175 µm
glass surface, as the standard thickness of cover slip glass.
However, microscope slides, standard flasks, dishes andmultiwell plates or microplates generally feature an
approximate 1 mm bottom thickness (Table 1). For demanding microscopic applications at higher magnifications,a 1 mm bottom thickness can lead to a major reduction
in resolution due to focusing problems with standard
specimen supports. This effect is exacerbated especiallywith oil or water immersion objectives, as the microscope
objectives are typically calibrated to the refractive index
of glass, which differs from air or oil (Table 2), as wellas adjusted to a cover slip glass thickness of 175 µm.
For sophisticated microscopic applications, specialised
supports that feature a 175 µm bottom thickness can beadvantageous (Fig. 3). In example, for high-resolution
non-invasive fluorescence microscopy of live organisms
and cells, small petri dishes that contain an embedded
175 µm glass cover slip bottom such as the CELLview
Dish (Fig. 4) can be used to perform multiposition timelapse imaging.

N° 18FORUM

Item No.

Description

Well Bottom Thickness

Distance Well Bottom Internal

Distance Well Bottom External

664160Cell Culture Dish

1 mm

1.6 mm

0.6 mm

658175Cell Culture Flask

1.4 mm

2.5 mm

1.1 mm

6571606 Well Multiwell Plate

1.2 mm

2.5 mm

1.3 mm66518024 Well Multiwell Plate

1.2 mm

2.5 mm

1.3 mm65518096 Well Solid Bottom Microplate0.95 mm

3.5 mm2.55 mm65509096 Well µClear® Microplate0.19 mm

3.5 mm3.31 mm655866

96 Well SCREENSTAR Microplate0.19 mm

0.7 mm0.51 mm655891

96 Well CELLview Microplate

0.175 mm

0.7 mm0.525 mmTable 1:

Well bottom thickness

Media

Refractive Index

Air

1.0

Glycerine

1.47

Water

1.33

Immersion Oil

1.52

Glass

1.52

Cycloolefine

1.53

Polystyrene

1.58

Table 2:

Refractive index of different materials

Figure 4: CELLview Dish (Item No. 6278XX and 6279XX)

POLYSTYRENE AND CYCLOOLEFIN YIELD HIGH RESOLUTION SIMILAR TO GLASS
Fluorescence Microscopy of HELA cells,
63x (LSM 710 Zeiss)

Polystyrene(µClear® bottom)
190 µmCELLview(glass bottom)
175 µmcytoplasm

nucleuscytoskeleton

SCREENSTAR(cycloolefin bottom)
190 µmFigure 3: Fluorescence microscopy with an inverted microcscope (LSM 710, Carl Zeiss AG, Germany, 63x with oil).
Comparison of different base materials of ultra-thin bottoms: polystyrene (µClear® film), glass (CELLview) and cycloolefin(SCREENSTAR). All materials gave a very high resolution.

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N° 18

4/ SUBSTRATES IN MICROSCOPY

susceptible to scratching and dust contamination. Glassbottom microplates such as the 96 well CELLview featureexcellent intra- and inter-well flatness due to the rigidityof the glass bottom. As microscope objectives are adapted to the bottom thickness and refractive index of coverglass, generally the best optical quality with highest resolution can be achieved using glass substrates (Fig. 7).

4.1/ GLASS
4.1/ GLASSThe typical substrate for biological samples in microThe
substrate
for biologicalin microscopytypicalis glass.
Glass displays
excellentsamples
optical properties
scopy
isglass.
Glass
displays
excellent
opticalproperties(Fig. 3, 5, 6, 7) as it is highly transparent, clean, and less(Fig. 4, 6, 7, 8) as it is highly transparent, clean, and less

OUT-OF-FOCUS BLUR OF DIFFERENT MATERIALS
1 µm
2 µm

2 µm

2 µm

1 µm

1 µm

2 µm
2 µm

2 µm
1 µm

2 µm

2 µm

1 µm

1 µm

Polystyrene 190 μm

2 µm

1 µm

1 µm

Glass 175 μm

1 µm

Cycloolefin 190 μm

Figure 5: Confocal imaging(LSM 710, 63x, oil immersion, Zeiss AG, Göttingen, Germany) of fluorescent beads in microplates with different film bottoms. Images
display the out-of-focus blur obtained with different materials (polystyrene, glass and cycloolefin). Cycloolefin shows approximately
the same low level of out-of-focus blur as glass.

AUTOFLUORESCENCE OF DIFFERENT MATERIALS

7000

800

6000

700

5000

600

4000

500

3000

400

2000

300

1000

C) Fluorescence Spectrum
Cycloolefin
7000

800

6000

700

5000

600

4000

500

3000

400

2000

300

1000

0

Emission / nm

B) Fluorescence Spectrum
Glass
Emission / nm

Emission / nm

A) Fluorescence Spectrum
Polystyrene

7000

800

6000

700

5000

600

4000

500

3000

400

2000

300

1000

0

0

300 400 500 600 700 800

300 400 500 600 700 800

300 400 500 600 700 800

Excitation / nm

Excitation / nm

Excitation / nm

Figure 6: Autofluorescence of polystyrene (A), borosilicate glass (B) and cycloolefin (C) in the UV
The graphs display autofluorescence in a 2D diagonal plot, where the diagonal peak represents equal excitation and emission
wavelengths. Due to energetic loss, emitted light generally exhibits a higher wavelength than that of excitation, therefore
autofluorescence only occurs when the emission wavelength is above the excitation. The lowest autofluorescence can be detectedin the cycloolefin microplates (C), followed by the glass bottom microplate (B).

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N° 18Control: Cell division with correct
chromosome alignment (a, b)

siRNA-1: Cell division with chromosome alignment problems(c) multipolar spindle(d) cell death

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siRNA-2: Cell division with(e) chromosome segregation problem(f) polylobed nucleus

Figure 7: Phenotypic profiling of human genes by time-lapse microscopy in glass bottom microplates(96 well CELLview; Item No. 655891). Maximum projection of confocal still images of HeLa cells stably expressing GFP–tubulin (green)and H2B–mCherry (red) after RNAi knockdown show phenotypes during cell division and mitotic consequences such as multipolar
spindle, polylobed nucleus and cell death. Images are courtesy of Dr. Beate Neumann, Advanced Light Microscopy Facility, European
Molecular Biology Laboratory, Heidelberg, Germany.

INFLUENCE OF BOTTOM THICKNESS ON RESOLUTION AND IMAGE QUALITY
Tolerance +/- 20 µm
175 µmCoverslip Glass &CELLview
In Focus

190 µmμClear® &SCREENSTAR

250 µmCompetitor

Out of Focus

Adipogenesis of hMSC(# R7788-115, StemPro® human ADSC,
Invitrogen, Carlsbad, USA)LSM 710, Zeiss, 20x
Lipid Vesicles
Nucleus
Cytoskeleton

190 µm

250 µmFigure 8: Influence of bottom thickness on resolution and image quality
The adipogenesis of human mesenchymal stem cells (hMSC) was analysed in microplates with 190 μm film bottom and competitive
microplates with 250 μm film bottom. The 190 μm film bottom demonstrates exceptional performance, giving high image quality
with excellent resolution due to the bottom thickness being within the tolerance range of the microscopic system (Operetta® HighContent Imaging System, PerkinElmer, Waltham, Massachusetts, USA). The 250 μm film bottom of the competitive microplate yieldblurry images, incapable of sharp focus and magnifications above 20x.

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4.2/ POLYSTYRENE
Polystyrene is the most widely used base material for microplates. Polystyrene film bottom plates are an optimal
solution for routine high-throughput and imaging applications. In general, polystyrene microplates are reasonably
priced due to highly automated production processes andrelatively low material costs. The 190 μm polystyrene
film bottom of the Greiner Bio-One black and white μClear®
microplates is perfectly suited for basic microscopic
applications as it fits into the tolerance window of mostmicroscopic objectives (Fig. 8) and does not require any
special adaptations or corrective lenses. Although polystyrene substrates are well suited for many cell-based
assays, they are not optimized for high magnifications
i. e, use of high numerical aperture (NA) objectives withshort working distances. This does not only refer to thestandard plate geometry but also to the optical propertiesof the polystyrene polymer. Major advantages are the highvariety of cell culture surfaces that facilitate optimal cell
culture condition for various applications. CELLSTAR®
surfaces for adherent cells are tissue culture treated toprovide a perfect support for adherence and growth ofmany cultures. Moreover, polystyrene can easily undergodifferent modifications to generate specialised surfaces for sensitive or stressed cultures such as the Greiner
Bio-One Advanced TC surface or CELLCOAT® protein
coatings (Poly-D-Lysine, Collagen I, etc.), as well as modifications to inhibit cell attachment for suspension or
spheroidal cell cultures such as with the Greiner Bio-One
CELLSTAR® cell-repellent surface. The result of this flexibility is a large range of specialised surfaces within thepolystyrene microplate product range for numerous andvaried applications.

during the harsh immunochemical staining procedures
typically required in fluorescence microscopy. Additionally, the surface modification required to improve cellattachment to glass is time-consuming and expensive.
To overcome the drawbacks for use of both glass andpolystyrene as cell culture substrates, cycloolefins havebecome increasingly popular as a base material for microplate manufactures.
Cyclic olefins have become increasingly popular for use asa base material for microplates. The material possesses
excellent optical features with a high spectral transmission
and a low background in the lower UV range. With a refractive index which is very close to that of glass, cyclic olefinbottom plates combine excellent optical properties withimproved cell growth and cell attachment due to their organo-polymeric nature. Although cyclic olefin bottom
plates typically do not achieve the planarity of glass substrates, they show a very good inter- and intra-well flatness.

RECESSED MICROPLATE BOTTOMOF SCREENSTAR ANDCELLVIEW MICROPLATES

4.3/ CYCLOOLEFINS
Although polystyrene substrates are well suited for manycell-based assay and imaging applications, with morechallenging microscopic analyses, polystyrene cannot
achieve the image quality of glass. Glass substrates canalso show limitations for use in demanding cell culture
applications, e.g. with cultivation of primary cells on glass,there is an imminent risk for cells to be washed away

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Figure 9: 40x water immersion objective for microscopic
detection in a 96 well microplate.
Within the standard 96 well microplate design, the distance
between well undersides and microplate skirt bottom causesinterference of oil or water immersion objectives with themicroplate skirt.
This is not the case with the SCREENSTAR or the CELLview
microplates, as the recessed well bottoms enable a close
proximity for the objectives to fully access all microplate wells.

N° 185/ SCREENSTAR AND CELLVIEW:MICROPLATES FOR HIGHRESOLUTION MICROSCOPY
The increasing significance of microscopic methods indrug discovery, with a trend of focusing on subcellularlevels with higher resolution, leads to a demand for advanced microscopic technologies. Improved microscopic

A

FORUM

devices and sophisticated software programs for data
analysis in the subcellular level has been launched in the
recent decade. However, the exploitation of the technicalbenefits in high-content screening has been somewhat
limited due to restrictions in the design and performanceof the available specimen supports.
The abundantly available microscope slide is not suitablefor high-content screening applications where multiple
samples must be analysed in parallel. Rather, microplates
have become the standard substrate of choice in highcontent screening, as they facilitate an easy automation
and analysis.

RECESSED MICROPLATE BOTTOM

B

C

2

8

7,3

1,7

One of the major drawbacks of a classic polystyrene microplate is the distance from the microplate well undersides
to the plate skirt bottom. In microscopy, a distance ofgreater than 1 mm can prohibit access to peripheral wells,
especially when high-magnification objectives that almost
touch the well undersides, separated just by a thin oil orwater layer, are used. For such demanding applications,
it is critical that the microplate well undersides are in very
close proximity to the bottom of the microplate skirt toallow full access of the objective to all peripheral microplate
wells. The CELLview and SCREENSTAR microplates provide a recessed, optically transparent bottom to allow fulluse of high-magnification oil or water immersion objectives
with access to all microplate wells, including perimeter andcorner positions (Fig. 9).

1,45

5.1/ 1536 WELL SCREENSTAR MICROPLATE
Figure 10: 1536 well SCREENSTAR MicroplateA) Black microplate with clear cycloolefin film bottom (190 μm)
for high-content screening
Smooth microplate top absent of alphanumeric coding
facilitates flush lid mounting for use within automated systems
and improves heat sealing
B + C) Well design of a 1536 well SCREENSTAR microplate
Plate height:
8 mm
Well depth:
7.3 mmWell diameter top:
1.7 mmWell diameter bottom:
1.45 mm
Distance microplate rim to internal well bottom:
0.7 mmDistance microplate rim to external well bottom:
0.51 mm
Film bottom thickness:
190 μmAs classic polystyrene microplates are not always preferred for advanced microscopic applications, Greiner Bio-One
has developed cycloolefin-based microplates especiallydesigned for microscopy. The initial microplate launchedwithin this portfolio was the 1536 well SCREENSTAR, ablack 190 µm film bottom microplate for high-throughput
and high-content screening applications (Fig. 10). This
plate is followed by two microplates in 384 well and 96
well format.

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5.2/ 96 WELL SCREENSTAR AND 96 WELLCELLVIEW MICROPLATE

can deal with an overall flatness (inter-well flatness) below 0.2 mm, which is specified for the SCREENSTAR andCELLview microplates.

5.3.1/ PLATE DIMENSIONS AND TOLERANCESThe 96 well SCREENSTAR and CELLview microplates(Fig. 11) meet the ANSI 1-2004 through ANSI 4-2004 standards, which define footprint, height dimensions, outerflange, and well position dimensions. The intra-well andinter-well flatness are described by the ANSI 6-2012:„Microplates - Well Bottom Elevation“. Most optical devices

Demanding cell biology applications, such as detectionof subcellular reactions, require prolonged cell culture,Ø 6,96

B

0,19

14,4

10,9

A

5.3.2/ IMPROVED CELL CULTURE
CONDITIONS (ENLARGED WORKINGVOLUME AND PERIMETER DITCH)

D

14,4

0,190

Ø 6,9613,7

C

Ø 6,58

Ø 6,49Figure 11:A) Well bottom distance in a 96 well microplate(Item No. 655090)
C) Well bottom distance in a 96 well SCREENSTAR microplate(Item No. 655866 and 655891)
A basic requirement for the 96 well SCREENSTAR and CELLview
microplate was the compatibility of high-magnification objectives
such as oil and water immersion lenses. These objectives almost
touch the microplate well undersides, separated just by a thin oilor water layer. For this technology, the microplate well undersides
must be close to the microplate skirt bottom (Fig. 9) to easily allow
interchange of microscopic objectives and complete accessof peripheral wells.

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B) Well design of a 96 well μClear® PS microplate(Item No. 655090)
Plate height:
Well depth:
Well diameter top:
Well diameter bottom:
Distance microplate rim to internal well bottom:
Distance mictoplate rim to external well bottom:
Film bottom thickness:

14.4 mm10.9 mm
6.96 mm
6.58 mm
3.5 mm3.31 mm
190 μmD) Well design of a 96 well SCREENSTAR microplate(Item No. 655866)
Plate height:
14.4 mm
Well depth:
13.7 mm
Well diameter top:
6.96 mm
Well diameter bottom:
6.49 mm
Distance microplate rim to internalwell bottom: 0.7 mm
Distance microplate rim to external well bottom: 0.51 mm
Film bottom thickness:
190 μmN° 18FORUM

to include critical media changes. Low working volumes
and high evaporation during such long-term cell culture
could adversely affect the consistency and performanceof a cell-based assay.
To reduce evaporation, the 96 well SCREENSTAR andCELLview microplates offer a ditch around the perimeter
which can be filled with either sterile media or water(Fig. 12). This can help create a saturated water vapour
barrier to significantly reduce evaporation.
With a working volume of 440 µl/well in SCREENSTAR andCELLview, the volume is increased nearly 100 µl in comparison to a standard 96 well chimney well design (Table 3).

Figure 12: Detail of the 96 well SCREENSTAR microplate
demonstrating the perimeter ditch which can be filled withsterile water or media
Well depth:
13.7 mm
Working volume:
25 - 440 μl
Growth area:
33 mm²
Total volume:
483 μlWell diameter (bottom): 6.49 mm

The larger working volume enables use of more media topotentially enlarge time periods between media changes.
The nutrition of cells can additionally be improved due tothe application of a higher volume of media.

Description

SCREENSTAR / CELLview

Standard Chimney Well

127.76 x 85.48 mm

127.76 x 85.48 mm

14.4 mm

14.4 mm11.01 x 7.87 mm

11.01 x 7.87 mm

Top

6.96 mm

6.96 mm

Bottom6.49 mm

6.58 mmSkirt

2.0 mm

2.0 mm

Pitch

9 mm

9 mm

Well depth

13.7 mm10.9 mm

Total volume

483 μl392 μlLength / Width
HeightA1 position

Working volume

25 - 440 μl

25 - 340 μl

Growth area

33 mm

34 mm2

2

Table 3: Comparison of the major microplate and well dimensions
between SCREENSTAR / CELLview and a standard 96 well chimney well microplate

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N° 18

FLUORESCENCE MICROSCOPY OF HELA CELLSCULTIVATED ON DIFFERENT MICROPLATE SURFACES
40 x Leica DM IL Fluo (Leica Microsystems GmbH, 35578 Wetzlar, Germany)

μClear® PS 190 μm

Competitor PS 250 μm

Phalloidin

DAPI

SCREENSTAR 190 μm Cycloolefin

excellent resolution & best image quality

63 x Oil Immersions (Leica Microsystems GmbH, 35578 Wetzlar, Germany)

Phalloidin

μClear® PS 190 μm

DAPI

SCREENSTAR 190 μm Cycloolefin
excellent resolution & best image quality

Figure 13: Optical quality of different clear bottom microplates.
SCREENSTAR microplates show excellent image quality with highest resolution.

6/ ORDERING INFORMATIONSCREENSTAR Microplates – 96 / 384 / 1536 Well
Bottom: Cycloolefin film, Raw material: COP, Surface treatment: TC
Item no.

Well format

Growth area / unit

Well profile

65586696

33 mm²

F-bottom /Chimney Well

781866384

8.1 mm²

7898661536

2.1 mm²

Product colour

Working volume (well)

Lid

SterileQty. inner / outerblack

25 μl - 440 μl

yes+

1 / 16

F-bottomblack

10 μl - 110 μl

yes+

8 / 32

F-bottomblack

3 μl - 15 μlno+17 / 68

Well format: 96, Growth area / unit: 33 mm², Well profile: F-bottom / Chimney Well, Bottom: glass,
Raw material: COP, Working volume (well): 25 μl - 440 μl, Lid: yes
Item no.

Surface treatment

Product colour

SterileQty. inner / outer655891

TC

black

+

1 / 16655981

Advanced TC

black

+

1 / 16

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Bio-One GmbH is expressly prohibited. Media owner: Greiner Bio-One GmbH / Represented by Managing Directors Jakob Breuer and Heinz Schmid.
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PHONE +49 7022 948-0 / FAX +49 7022 948-514 / E-MAIL info@de.gbo.com
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Find the contact details of your local partner on our website.

F073 787 [rev. 07.2022]

CELLview Plates