ibidi Application Guide

Microscopy With ibidi
The ibidi Imaging Chambers: .  .  .  .  .  .  .  .  .
The Coverslip Bottom .  .  .  .  .  .  .  .  .  .  .  .  .  . 2

Microscopy Techniques:  .  .  .  .  .  .  .  .  .  .  .  .
Principles and Applications  .  .  .  .  .  .  .  . 15

ibidi Polymer Coverslip  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3

Inverted and Upright Microscopy  .  .  .  .  .  .  .  .  .  .  .  .  . 15

ibidi Glass Coverslip  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3

Phase Contrast Microscopy  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 16

Immersion Oil Compatibility of ibidi Labware .  .  .  . 3

Differential Interference Contrast (DIC)  .  .  .  .  .  .  .  . 18

The ibidi Imaging Chambers:
Surfaces and Coatings  .  .  .  .  .  .  .  .  .  .  .  .  . 4ibiTreat (Tissue Culture-Treated) Surface  .  .  .  .  .  .  . 6Hydrophobic, Uncoated Surface .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Bioinert Surface .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Micropatterned Surface  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 7Coatings .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 8Elastically Supported Surface (ESS) .  .  .  .  .  .  .  .  .  .  . 8

The ibidi Imaging Chambers: .  .  .  .  .  .  .  .  .
Chamber Geometry .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 9All-In-One Chambers: All Steps in One Single Slide  . 9Chamber Slides, Removable .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 10
Channel Slides .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 11
Ph+ Slides .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 12

Widefield Fluorescence Microscopy .  .  .  .  .  .  .  .  .  .  . 19
Confocal Microscopy .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 20
Two-Photon and Multiphoton Microscopy  .  .  .  .  .  . 21
Fluorescence Recovery After Photobleaching (FRAP) .  22
Light Sheet Fluorescence Microscopy (LSFM) .  . 22
Förster Resonance Energy Transfer (FRET)  .  .  .  . 23
Fluorescence Lifetime Imaging Microscopy (FLIM) 23
Total Internal Reflection Fluorescence (TIRF)  .  .  .  .  .  . 24
Super-Resolution Microscopy .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 25

ibidi Material Specifications .  .  .  .  .  .  .  . 26
ibidi Bottom Material Specifications .  .  .  .  .  .  .  .  .  . 26
Compatibility With Microscopy Applications  .  .  . 26
Optical Aspects .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 27
Further Aspects .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 27

Microscopy Parameters of Materials .  . 13
Coverslip Thickness . . . . . . . . . . . . . . . . . . . . . . . 13
Numerical Aperture .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 13
Refractive Index  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 14
Material Dispersion / Abbe Number .  .  .  .  .  .  .  .  .  .  . 14
Transmission .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 14
Autofluorescence  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 14.com

The ibidi Imaging Chambers:

The Coverslip Bottom
The outstanding characteristic of the ibidi µ-Slides,µ-Dishes, and µ-Plates is their thin coverslip
bottom, which has excellent features for high-end
microscopy applications. In comparison, the bottomof standard cell culture plastics is about 1 mm
thick—which is more than 5 times the thickness ofthe coverslip and therefore not ideal for imaging.

100 x

ibidi offers labware with the ibidi Polymer Coverslip
Bottom or the ibidi Glass Coverslip Bottom. For
most applications, the ibidi Polymer Coverslip is thematerial of choice, as it offers optimal cell adherence
combined with excellent optical properties. If yourcells require a special coating, or if you are using
a particular microscopy technique such as TIRF or
super-resolution microscopy (STED, STORM), we
recommend using the ibidi Glass Coverslip Bottom.

#1.5 ibidi Polymer Coverslip or#1.5H ibidi Glass Coverslip Bottom

Imaging chambers with the coverslip bottom are
optimized mainly for use with inverted microscopy.
You can find information about the compatibilityof the surface materials with different microscopy
techniques in the chapter “ibidi Material
Specifications”.

POLYMER

GLASS

GLASS

COVERSLIP

COVERSLIP

COVERSLIP

GLASS
SLIDE

#1.5 ibidi Polymer
Coverslip#1.5H ibidi Glass
Coverslip#1.5 glass
coverslip

Standard
glass slide*

Bottom thickness

180 µm(+10/–5 µm)

170 µm(+/–5 µm)

170 µm(+20/–10 µm)

1 mm

Bottom material

Polymer

D 263 M Schott highprecision glass

D 263 M Schott highprecision glass

Glass

Gas permeability

Yes

No

No

No

#1.5

#1.5H

#1.5

* The ibidi 3 Well | 8 Well | 12 Well Chamber Slides, removable are self-adhesive, removable silicone chambers that are mounted
on a standard glass slide. They are suitable for upright and inverted microscopy and enable long-term storage of samples after
immunofluorescence staining.

2

ibidi Polymer Coverslip

POLYMER
COVERSLIP

The ibidi Polymer Coverslip, which is our mostrecommended surface, is a thin plastic coverslip
that forms the bottom of most μ-Slides, μ-Dishes,and μ-Plates. With a standard No. 1.5 coverslip
thickness of 180 µm (+10/­– 5 µm) and immersion
oil compatibility, it meets all optical requirements
for microscopes and is suitable for various imaging
techniques. Due to the gas-permeable materialof the ibidi Polymer Coverslip, carbon dioxide or
oxygen exchange during cell culture is maintained.
The ibidi Polymer Coverslip provides optimal growth
conditions for various cell-based assays and cell
types and is available with different treatments or
coatings. You can find more information about themin the next chapter.

ibidi Glass Coverslip#1.5

Fluorescence microscopy of human endothelial cells (HUVEC)
on the ibidi Polymer Coverslip Bottom. Red: alpha-tubulin, green:
F-actin, blue: nuclei (DAPI). 60x objective lens, oil immersion.

GLASS

COVERSLIP

ibidi offers specialized glass bottom labware with aborosilicate coverglass bottom.

#1.5H

If your cells require a special coating, or if you areusing a particular microscopy technique such as TIRF,
super-resolution microscopy (STED, STORM), or
single molecule microscopy, we recommend using
the ibidi Glass Coverslip Bottom.
The glass coverslip bottom is suitable for all standard
ECM coatings (e.g., collagen, fibronectin, and laminin).
The surface also allows for the application of all common
glass chemistry modifications, such as silanization,
plasma treatments, and chemical activation.
To provide the highest quality necessary for modern,high-performance microscope objectives, only
D 263 M Schott glass coverslips with a thickness
range of 170 μm (+/–5 μm) are used. This thickness
is also known as #1.5H (high performance). Startingwith the µ-Dish 35 mm, high Glass Bottom in 2009,
ibidi was the first company who used the #1.5H
glass as a bottom for slides and dishes.
The ibidi Glass Bottom Dish 35 mm is produced by using
a standard #1.5 glass coverslip with a thickness of
170 µm (+20 µm/–10 µm). This glass bottom material
fulfills the needs of all standard applications where a
cost-effective coverslip is required.

Surface-near F-actin network of a Dictyostelium discoideum
DdLimE-GFP cell. TIRF live cell imaging on the #1.5H ibidi Glass
Coverslip Bottom.

Please note: For most applications, the ibidi Polymer
Coverslip is the material of choice, as it offers
optimal cell adherence combined with excellent
optical properties.

GLASS

θ

COVERSLIP#1.5

Immersion oilor water

Objectiveθ

Immersion Oil Compatibility of ibidi Labware

Objective

Oil immersion is used to increase the resolution of the objective, up to its physical limit. Placing immersion oil
instead of air between the objective lens and the coverslip allows for a significantly higher amount of light to becollected by the lens. This, in turn, increases the resolution and the signal-to-noise ratio in microscopic images.
For high-resolution microscopy, cell culture chambers need to be compatible with immersion oil. The ibidi labware
is compatible with a variety of common immersion oils. For a detailed compatibility list, please visit our website.

3

The ibidi Imaging Chambers:

Surfaces and Coatings
Growth, development, and signaling of cultured
cells strongly depend on the used surface. ibidi's
μ-Slides, μ-Dishes, and μ-Plates have a thincoverslip bottom (ibidi Polymer Coverslip or Glass
Coverslip, both with excellent optical quality).
They are available with several surfaces. The ibidi
μ-Slides, μ-Dishes, and μ-Plates that have an ibidi
Polymer Coverslip bottom can be coated in a similar
process to standard plastic labware, while fully
retaining image quality. ibidi offers Collagen I, IV and
Poly-L-Lysine coatings.

Surfaces and Coatings for theibidi Polymer Coverslip

POLYMER
COVERSLIPGLASSCOVERSLIP

ibiTreat (Tissue Culture-Treated)

Glass Surface

Excellent adhesion of adherent cells,
hydrophilic surface with no need forany additional coating; optimal foreveryday cell culture

Adhesion of adherent cells (coating
might be required), ideal for special
microscopy applications

Hydrophobic, Uncoated Surface
Weak adhesion of adherent cells,suitable for the application of specificcoatings

Bioinert Surface
No adhesion of adherent cells or any biomolecule, stable long-term passivation;ideal for spheroid and organoid cultureµ-Patterned Surface
Spatially defined adhesion of adherent
cells on spots, different spot geometries
available

Elastically Supported Surface(ESS)
Culture of adherent cells on an elastic
surface

Coated Surface
Culture of adherent cells on a Collagen
I, Collagen IV, or Poly-L-Lysine surface;
available for selected µ-Slides

4

Surfaces for theibidi Glass Coverslip

ibiTreat, Uncoated, and Bioinert—A Surface Comparison

ibiTreat
Excellent cell adhesion

Uncoated
Weak cell adhesion

Bioinert
No cell adhesion• Culture of adherent cells• Culture of adherent cells• ECM coatings possible• ECM coatings possible

The hydrophilic ibiTreat surface
provides excellent cell adhesion,even without a defined protein
coating. However, ECM protein
coatings can be done on ibiTreat
without any restrictions. The
ibiTreat surface is ideal for
culture of adherent cells that do
not need any specific coating.

The hydrophobic Uncoatedsurface provides weak celladhesion, if not previously
coated with an ECM protein.
ECM protein coatings can bedone on the Uncoated surface
without any restrictions. The
Uncoated surface is ideal forthe culture of adherent cells that
require a specific coating.

• Culture of suspension cells,
cell aggregates, spheroids,
embryoid bodies (EBs),
organoids• ECM coatings not possible
The hydrophilic Bioinert surface
hinders any protein attachment,
thus inhibiting subsequent cellattachment. Unlike with theibiTreat and Uncoated surfaces,a coating is not possible. The
Bioinert surface is ideal for theculture of suspension cells andcell aggregates.

ibidi Surfaces Show no Autofluorescence
Both the flat, thin bottom material and the excellent optical quality of theibidi Polymer Coverslip enable high-resolution microscopy without any
disruptive autofluorescence.

Fluorescence Intensity
in Thousands [CPS]1800

360 nm (DAPI)

1600

160

480 nm (GFP)

140

1400

120

540 nm (Cy3)

100

120

1200

100

1000

80

800

60

600
400

40

200

20

0

0

80
60
40
20
0

Glass Coverslip D 263M Schott #1.5H
ibidi Polymer Coverslip
Bioinert Surface
Ultra-Low Attachment, Competitor A
Ultra-Low Attachment, Competitor B

5

ibiTreat (Tissue Culture-Treated)
Surface: Excellent Cell Adhesion
ibiTreat is our most recommended surface modification,
because most adherent cells grow well on it without
the need for any additional coating. ibiTreat is the hydrophilic, tissue culture-treated version of the ibidi Polymer
Coverslip. This physical surface modification, which iscomparable to the tissue culture treatment of standard
cell culture vessels, makes the surface hydrophilic andadhesive to virtually all cell types. The ibiTreat surface
appears in more than 20,000 peer-reviewed publications. The adhesion of cells to ibiTreat μ-Slides is evenstrong enough to perform flow experiments that simulate the physiological shear stress of the blood flow.

Hydrophobic, Uncoated Surface:
Weak Cell Adhesion

POLYMER
COVERSLIP

Phase contrast microscopy of human primary keratinocytes on theibiTreat ibidi Polymer Coverslip.

POLYMER
COVERSLIPThe Uncoated surface is a hydrophobic version of theibidi Polymer Coverslip with identical optical properties.
It does not permit direct cell growth without anadditional coating. Therefore, it is not suitable forthe standard cultivation of adherent cells. Due to its
hydrophobicity, it can be used for specific coatings orfor non-adherent suspension cells. However, for cells insuspension we recommend using the Bioinert surface,which completely inhibits cell-surface interactions,even in long-term experiments.
Phase contrast microscopy of MCF7 cells on the Hydrophobic,
Uncoated surface without prior ECM protein coating.

Glass Coverslip Bottom

GLASS

COVERSLIP

ibidi offers specialized glass bottom labware with aborosilicate coverglass bottom. ibidi developed theseglass surfaces specifically for TIRF, super-resolution
microscopy, and single molecule microscopy. The glassbottoms are completely gas-proof and do not allow
for any gas exchange. To provide the highest quality
necessary for modern, high-performance microscope
objectives, only D 263 M Schott glass coverslips witha thickness range of 170 μm (+/–5 μm) are used. This
thickness is also known as #1.5H (high performance).
Please note: In principle, glass bottoms are suitablefor direct cell culture. However, to promote cellattachment, a surface coating might be required
prior to cell seeding.

6

#1.5H

Fluorescence staining of adherent fibroblast cells on the ibidi
Glass Coverslip Bottom. Green: F-actin cytoskeleton (Phalloidin);blue: nuclei (DAPI).

Bioinert Surface:
No Cell Adhesion

POLYMER
COVERSLIP

Culture of suspension cells and spheroids
No Cell Attachment: The Bioinert surface is a thinpolyol hydrogel layer that is covalently bound to
the ibidi Polymer Coverslip. In contrast to standard
ultra-low attachment (ULA) coatings, Bioinert iscompletely non-adherent and allows no bindingof any biomolecule, even in long-term assays.
Therefore, the Bioinert technology provides a stablepassivation for days or even weeks.
No Cell-Substrate Interactions: Bioinert creates
an environment in which cell-cell interactions
dominate over cell-substrate interactions. In fact,
the latter are completely blocked. The stability of
the Bioinert surface allows for long-term assays
on the very same dish—for several days and evenweeks—without the adhesion of any proteins. Even
when using medium with a high fetal calf serum
concentration, Bioinert prevents any cell or proteinfrom adhering to the surface.

Bioinert
surface

Bioinert
surface(0.2 µm)

Culturemediumibidi Polymer Coverslip (180 µm)

No Prior Preparation: Bioinert is ready-to-use. No
pre-hydration step is necessary. The surface will
swell by itself once wetted with buffer or medium.
No Autofluorescence: Both the flat, thin bottom
material and the excellent optical quality of theibidi Polymer Coverslip enable high-resolution
microscopy without any disruptive autofluorescence.

Micropatterned Surface:
Defined Cell Adhesion

POLYMER
COVERSLIP

The ibidi µ-Patterning technology enables spatiallydefined cell adhesion for various 2D and 3D cell
culture applications.
Miniaturized adhesive patterns (e.g., lines, squares,or dots) are irreversibly printed on the non-adhesive
Bioinert surface of the ibidi Polymer Coverslip,
allowing for precisely controlled cell adhesion. Theµ-Patterns are dry-stable, sterile, and ready to use.
The µ-Pattern, the Bioinert surface, and theibidi Polymer Coverslip are all optimized for highresolution imaging and microscopy.

Spheroid formation of NIH-3T3 cells, imaged 14 days after
seeding single cells in the µ-Slide VI 0.4 With Multi-Cell µ-Pattern.
Phase contrast microscopy, 4x objective lens.

µ-Pattern with adherent cells
Passivated Bioinert surface
ibidi Polymer Coverslip

7

Coatings: Collagen I, Collagen IV, and Poly-L-Lysine

POLYMER

Culture of adherent cells that require a specific extracellular matrix (ECM) on the surface

COVERSLIP

For selected µ-Slides, ibidi offers collagen I, IV,and poly-L-lysine precoated surfaces on the ibidi
Polymer Coverslip.

Collagen I
Collagen type I is the most common type of collagenin the human body. Building collagen fibers, it ispresent in many parts of the body, such as skin andbone. To promote better cell adherence, culture
vessel surfaces, such as the ibidi Polymer Coverslip,
can be coated with collagen I. In addition, collagen I
3D gels are frequently used for cell cultivation
to stimulate an in vivo-like 3D environment. ibidi
provides ready-to-use µ-Slides that are coated withcollagen I. For researchers who want to do their own
coating, ibidi offers a high-quality Collagen Type I,
Rat Tail that is made from rat tail tendon.

RAT1 cells on the ibidi Polymer Coverslip coated with collagen IV.

Collagen IV

Poly-L-Lysine (PLL)

Collagen type IV is one of the major constituents ofthe basement membrane, which is a thin layer of theextracellular matrix (ECM). In cell culture, collagenIV is used as a substrate to promote the adherence
and proliferation of cells. Collagen IV coatings havebeen tested for a variety of standard cell lines, suchas epithelial, endothelial, neural, and muscle cells.
ibidi uses mouse collagen IV (Corning, No. 356233)
for the coating.

Poly-L-lysine (PLL) is a polymer of the essential
amino acid L-lysine. This polymer is one of the mostcommonly used adhesion substrates for cell culture.
It is suitable for a large variety of cell types, especiallyfor neuronal cultures. Adhesion is mediated by an
integrin-independent mechanism when PLL is used.
ibidi uses a commercially available PLL solution(Sigma-Aldrich, No. P4832) for the coating.

Elastically Supported Surface (ESS)As the uncoated ESS surface is hydrophobic
and does not allow for any direct cell growth, we
recommend extracellular matrix coatings.

8

12

Stiff

9

10
8
10
10

7

Soft

6

10
5
10
4

10
3
10

ss
la
G

es
id
Co i Pove lym
rs e
lip r

tis

di
sh

ib

Pe
tri

su
d
ar
H

iu

m

tis

is
s
ft
t

ed

su
e

e

2

ue

10

M

MPa

GPa

11

10
10
10

So

The elasticity of the ibidi elastically supported
surface (ESS) is comparable to that in cells andtissue. The µ-Dish 35 mm, high ESS is available in threedifferent elasticities, which cover the whole rangeof elasticities found in living tissue.

10

kPa

In living tissue, cell growing conditions are different
from those in in vitro cultures. The surface stiffness/elasticity affects cell proliferation, differentiation,and overall cellular function. The elasticity(Young’s modulus) of most cell culture plastics is~1 gigapascal (GPa). Young’s Modulus of glassis ~70 GPa. In contrast, the Young’s Modulus of
mammalian cells is <100 kPa. Therefore, the natural
cell environment is at least 100,000 times more elastic
than the standardly applied cell culture surfaces.

Surface elasticity [Pa]

Culture of adherent cells on an elastic surface

The ibidi Imaging Chambers:

Chamber Geometry
Chamber GeometryOpen-Well Format

Channel Format

Specialized Geometry• Common formats• Excellent imaging• Easy handling• Homogeneous cell distribution• Designed for specific uses
(e.g., gradients or gel matrices)• Large or low volume• Low volumeYour cell-based assays are as versatile as our chambers: We provide imaging chambers with various geometries
that are specifically tailored to your assay. In order to guarantee the highest imaging quality at optimized
experimental conditions, we have developed our imaging chambers using a combination of geometrical patterns
and physical features. The all-in-one chambers reduce the time and number of experimental steps needed incell-based assays. In addition, ibidi provides labware with specialized geometrical designs for functional cellbased assays, such as angiogenesis, chemotaxis, wound healing, or cell migration assays.

All-In-One Chambers: All Steps in One Single Slide
All-in-one chambers have been developed to optimize
the daily lab routine by providing an all-in-one solution
for cell cultivation, treatment, and live cell imaging.
The design of the ibidi μ-Slides, μ-Dishes, andμ-Plates allows for all of the experimental steps—
from cell cultivation to microscopic imaging—to be
conducted in one single chamber.
The open-well format of the µ-Slide 2 Well | µ-Slide
4 Well | µ-Slide 8 Well high, and the μ-Dish 35 mm, highallows for the use of standard immunofluorescence
protocols—with all procedures done in one single
slide. After fixation and staining, the sample canbe observed through the coverslip bottom using
high-resolution microscopy. There is no need for an
additional glass coverslip.

Cultivation

Fixation

Staining

Imaging

40 x

The ibidi all-in-one chambers reduce the number of experimental
steps needed in immunofluorescence assays.

Channel formats like the μ-Slide VI 0.4 are ideal foran exact exchange of small amounts of reagents.
These μ-Slides reduce the number of experimental
steps needed for immunofluorescence assays. Dueto the low volume, the channel slides help saving a
lot of money for antibodies.

9Chamber Slides, Removable:
Long-Term Storage of
Immunostained Samples
With the 3 Well | 8 Well | 12 Well Chamber Slides,
removable, ibidi provides self-adhesive, removable silicone chambers that are mounted on a standard glass
slide (1 mm thickness). They are suitable for uprightand inverted microscopy and enable long-term storage of samples after immunofluorescence staining.
All steps in the immunofluorescence assay can bedone in one single slide, which simplifies the overall
procedure and requires only a small number of cells
and a low amount of antibody and medium.
Please note: In principle, glass bottoms are suitable fordirect cell culture. However, to promote cell attachment,a surface coating might be required prior to cell seeding.

Immunofluorescence using the 12 Well Chamber, removable
60 x

Seeding

Cultivation

Chamber removal

Fixation & Staining

Mounting & Imaging

Storage

Cultivation
Lid
Removable silicone
gasket (wells)
Standard glass slide

Microscopy
40 x
Glass coverslip
Mounting medium
Standard glass slide

The ibidi Chamber Slides, removable, allow for cell cultivation(top) and upright microscopy of cells (bottom) after immunofluorescence staining.

10

Triple immunofluorescence of Madin-Darby canine kidney(MDCK) cells.
Red: mitochondria, stained with MitoTracker™ Red CMXRos;Green: F-actin, stained with Alexa Fluor™ 488 Phalloidin;
Blue: nuclei, stained with DAPI.

Channel Slides: Homogenous
Cell Growth and Optimized
Phase Contrast Microscopy
When using the ibidi channel µ-Slides, the cells arecultured within a channel that has one Luer port ateach side. Channel slides provide several advantages
over open well slides, such as homogeneous
cell distribution, no meniscus formation, and nocondensation.

Homogeneous Cell Distribution

Condensation water
20°C

The ibidi channel slides provide the ideal geometry
for homogeneous cell distribution.
Cell densities in open wells are very de­pendent on
handling during cell seeding. Further, cell distribution
often varies depending on the well region. Due tothe closed channel geometry, cell distribution in theibidi channel slides does not vary depending on theirpositions inside the slide, or with their handling andtreatment during and after cell seeding.

37°C

Light

20°C

37°C

The cell distribution can vary when using an open well (left).
Using a channel µ-Slide, the cells are distributed homogeneously
across the whole channel (right).

No Lid—No Condensation
Condensation, which occurs on the lid of the culture
vessel after being removed from the incubator,
disrupts phase contrast microscopy. When using
the ibidi channel slides, condensation inside theoptical pathway is intrinsically impossible.
Condensation can reduce the quality of phase contrast
microscopy in an open well (left). Excellent phase contrast images
are achieved using a channel µ-Slide (right), where condensation
is intrinsically impossible.

Optimized Phase Contrast Microscopy
The meniscus formation in open well chambers at the
air-water-interphase is a natural and unavoidable effectthat can disturb phase contrast microscopy. In channel
slides (e.g., the ibidi µ-Slide VI 0.4), meniscus formationis geometrically impossible. Therefore, phase contrast
in channel slides is superior to small open wells.
Find more detailed information about the principleof phase contrast microscopy and ibidi labware
solutions in the chapter “Phase Contrast”.

When using an open well (left), the meniscus causes poor
contrast in microscopy. Only the small center part exhibits
satisfying phase contrast. When using a channel µ-Slide (right),
no meniscus forms, resulting in excellent phase contrast images
across the whole channel.

11Ph+ Slides: Excellent MeniscusFree Phase Contrast Microscopy
The µ-Slides and µ-Dishes with Ph+ feature (Phase
Contrast +) are designed for excellent phase contrast
microscopy without meniscus, which naturally
disturbs the phase contrast effect in standard open
wells. The Ph+ version provides a special intermediate
plate in the center of the well. This plate flattens themeniscus and creates a parallel beam throughout theplate, thereby increasing the area of well-contrasted
cells. Openings near the corners provide access to thewells for the easy filling and aspiration of liquids. This
innovative technique supports meniscus-free phase
contrast microscopy in a very convenient manner.
Depending on your application, you can choose
between the following labware formats:• µ-Slide 2 Well Ph+• µ-Slide 2 Well Ph+ Glass Bottom• µ-Slide 4 Well Ph+• µ-Slide 4 Well Ph+ Glass Bottom• µ-Dish 35 mm Quad

Phase contrast microscopy of MCF7 cells.

Working with the Ph+ Slides Diminishes the Meniscus Effect12µ-Slide 2 Wellµ-Slide 2 Well Ph+• Poor phase contrast• Excellent phase contrast• Excellent fluorescence microscopy• Excellent fluorescence microscopy

Illustration of the perturbing effect of a meniscus. Light is refracted
on the air-water-interface, leading to poor contrast in microscopy.
Only the small center part exhibits satisfying phase contrast.

Working with the Ph+ slides diminishes the meniscus andincreases the area of well-contrasted cells.

Microscopy Parameters of Materials

Coverslip Thickness
The thickness of the coverslip is a crucial parameterthat defines imaging quality. Most of the objective
lenses used for microscopy are corrected to thestandard coverslip thickness of 0.17 mm (170 µm+20/–10 µm, #1.5). Thinner or thicker coverslips
require the use of the correction collar on the objective
lense, which then prevents the formation of blurred
images by spherical and chromatic aberrations.

Focus above thecoverslip

Focus inside thesubstrate~170 µm

1 mm
x100

x100

Light

Light
CCD-camera

CCD-camera

The ibidi Polymer Coverslip is the standard bottomof all ibidi μ-Slides, μ-Dishes, and μ-Plates. With athickness of 180 µm (+10/–5 µm), the ibidi Polymer
Coverslip provides ideal prerequisites for brilliant
inverse high-resolution microscopy.
For special microscopy applications, many ibidi
labware products, such as the μ-Dishes 35 mm, high,or the µ-Slide 2 Well | 4 Well | 8 Well high are alsoavailable with a 170 µm (+/–5 µm) coverslip glassbottom #1.5H.

Numerical Aperture
The numerical aperture (NA) is an important value
for microscope objectives, which defines theirresolution and luminous intensity. It measures theability of the objective to gather light and resolve
fine specimen detail at a fixed object distance. The
higher the NA, the greater the ability of an objective
is to resolve details of a specimen. The NA is
imprinted on every objective.
The numerical aperture is defined by: NA = n sin θ.
n = refractive index of the medium in which thelens works (e.g., 1.52 for immersion oil)

NA = 0.50

θ = 30.0°

Example of a 20x objective
lens with a numerical apertureof 0.5 (20x/0.5).

θ = half-angle of the maximum cone of light thatcan enter into the lens
For dry objectives, the maximal NA is ~0.95. For
immersion objectives, the maximal NA is ~1.4.

Find more information about microscopy parameters of the ibidi surfaces and their compatibilities
with different microscopic techniques in thechapter “ibidi Material Specifications”

13

Refractive Index

Material

Refractive Index nD

The refractive index nD measures the speed oflight inside a specific material, as compared tothe absolute vacuum. It is an important value forcalculating the numerical aperture (NA).

Vacuum1 (by definition)

Air

1.0003

Water

1.33

The refractive index is defined by: nD = c/η

Glycerol

1.47

Immersion oil

1.52

Glass coverslip

1.52

ibidi Polymer Coverslip

1.52

c = velocity of light in free spaceη = light velocity in a particular medium
The refractive index is wavelength-dependent. As astandard, nD is used to characterize the refractive
index of optical materials, which represents 589 nm.
The refractive index is often referred to as “opticaldensity”. Most objectives are designed for use withcoverslips that have a standard refractive index of
1.52, including glass and the ibidi Polymer Coverslip.

includes ibiTreat, Uncoatedand Bioinert

Immersion oil, which can be seen as an extended
front lens of immersion objectives, also has arefractive index of 1.52.

Material Dispersion / Abbe Number
equal in that material, leading to reduced separationof different wavelengths. Therefore, materials witha higher Abbe number give less color dispersion andprovide a better optical quality for microscopy.

Dispersion in an optical material is quantified using
the Abbe number. It is calculated from the refractive
indices of three different wavelengths. Using amaterial with a high Abbe number means that therefraction index of different wavelengths is nearly

A material with an Abbe number equal to or larger
than 55 is considered to be well-suited for highresolution microscopy. For example, the ibidi Polymer
Coverslip has an Abbe number of 56 and the D 263 M
Schott borosilicate glass has an Abbe number of 55.

Transmission
The transmission of bottom material describes
its ability to permit the passage of light throughit at specific wavelengths. The more that light isabsorbed, the less it can contribute to fluorescence
excitation and image acquisition. To be suitable fordifferent microscopy applications, a material shouldhave a high transmission ability across various
wavelengths, such as the ibidi Polymer Coverslip.

Autofluorescence
Autofluorescence describes the intrinsic fluorescenceintensity of the pure material (e.g., the ibidi Polymer
Coverslip or a glass coverslip) without any fluorescentsample. The autofluorescence is emitted by thematerial and can emerge as noise or background
during the imaging process. Autofluorescence canbe disruptive, especially when trying to image faintfluorescent signals. All materials and culture media
show some degree of autofluorescence, which varies
with the excitation/emission wavelength and strongly
depends on the material type.

14

Transmission [%]Material dispersion is defined as a variation in therefractive index that depends on the wavelength.
In other words, dispersion is a measurement for
chromatic aberrations.

100
80

ibidi Polymer Coverslip
Glass Coverslip D 263M
Polystyrene (PS)

60
40
20
0
200

300

400

500

600

visible light

UV C UV B UV A

700

800
IR

Wavelength [nm]Autofluorescence Influences the
Signal-to-Noise Ratio

Low autofluorescence using
the ibidi Polymer Coverslip.

High autofluorescence using
a standard culture flask.

Microscopy Techniques:

Principles and Applications
Inverted Microscopy

Upright Microscopy

100x

100x

Schematic of an inverted microscope. Note the thin vessel
bottom, through which the cells are observed.

Schematic of an upright microscope. Note that the cells are
squeezed between the slide and a coverslip.

Applications

Applications

Inverted microscopy is a very popular techniquefor live cell imaging. Here, living cells are observed
through the vessel bottom. This technique has
several advantages over upright microscopy.

In cell biology, upright microscopes are used forphase contrast or widefield fluorescence microscopyof living cells or fixed samples that are squeezed
between a slide and coverslip.

Most adherent cells naturally sink to the vessel bottom
and adhere to the surface, meaning that they are spread
across one focal plane. When growing in vesselsthat are suitable for inverted microscopy, cells haveaccess to larger amounts of medium than if they weresqueezed between a coverslip and a slide. In addition,
medium exchange and sample access from the top are
possible. Another very important advantage is sterility:
as there is no contact between the objective and thesample, sterile working conditions are guaranteed.

In principle, upright microscopes can be used for live
cell imaging. However, they have several disadvantages
when compared to inverted microscopes. The working
distances in upright microscopes are longer, leading tolower resolution and weaker fluorescence signals. In
contrast to inverted microscopes, upright microscopes
do not allow for the use of objectives with highnumerical aperture directly on the coverslip bottom, on
which the cells adhere. Therefore, ibidi recommends
inverted microscopes for live cell imaging.

Principle

Principle

In an inverted microscope, the source for transmitted
light and the condenser are placed on the top of thestage, pointing down. The objectives are locatedbelow the stage, pointing up. The cells are observed
through the bottom of the vessel. For successful
imaging, the vessel bottom must have the highest
optical features, which are given for the ibidi Polymer
Coverslip and the ibidi Glass Coverslip.

In an upright microscope, the source of transmitted
light and the condenser are located below the stage,
pointing up. The objectives are placed on top of thestage, pointing down. The specimen is observed fromthe top through the lid of a petri dish or a coverslip. In
some systems, the objective lens is dipped into theculture medium without any coverslip.

ibidi Solutions

ibidi Solutions• All ibidi μ-Slides, μ-Dishes, and μ-Plates
are designed for use with different types of
inverted microscopes.

• The 3 | 8 | 12 Well Chamber, removableand the μ-Slide VI - Flat are compatible withupright or inverted microscopes.

• The ibidi Stage Top Incubation System isideal for establishing in vivo-like conditions
on every inverted microscope that has amultiwell plate holder or a K-frame.

• The ibidi µ-Dish 50 mm, low can be used withupright microscopes when a dipping lens isapplied.

15

Phase Contrast
Applications
Phase contrast is by far the most frequently usedmethod in biological light microscopy. It is anestablished microscopy technique in cell culture
and live cell imaging. When using this inexpensive
technique, living cells can be observed in their naturalstate without previous fixation or labeling.

Principle
Unstained living cells absorb practically no light.
Poor light absorption results in extremely small
differences in the intensity distribution in the image.
This makes the cells barely, or not at all, visible in abrightfield microscope. When light passes throughcells, small phase shifts occur, which are invisible
to the human eye. In a phase contrast microscope,these phase shifts are converted into changes inamplitude, which can be observed as differences inimage contrast. However, this label-free technique
is strongly dependent on the correct alignment ofcomponents in the optical pathway. This alignment
can be disturbed by the naturally occurring meniscus
effect, causing weak phase contrast.

Phase contrast microscopy of Rat1 cells.

An important issue to be considered in phase
contrast microscopy is the meniscus, which isnaturally formed at the air-liquid interface. Thisphenomenon can significantly reduce the image
quality, especially in small culture wells like thestandard 96 well plates. The diffraction, due to themeniscus, disarranges the correct alignment of
the phase ring and phase plate inside the opticalpathway.

Light from bulb
Annular ring
(Phase ring)
Condenser lenssystemSpecimenwith / without
meniscus
Objective lenssystemPhase plate

Back
focal plane

ibidi Solutions
ibidi has developed several solutions to overcome
this problem—and guarantee excellent phase
contrast images:• µ-Slide Angiogenesis and µ-Plate
Angiogenesis

Beam path with meniscus

Beam path without meniscus

No alignment, bad phase
contrast.

Correct alignment, goodphase contrast.

• Channel µ-Slides• µ-Slides Ph+
For detailed information, please refer to “Phase
Contrast in Channel Slides” or read ourApplication Note 03 (PDF).

16

F. Zernike. „Phase contrast, a new method for the microscopic
observation of transparent objects“.
Physica, 1942, part I: 10.1016/S0031-8914(42)80035-X,
part II: 10.1016/S0031-8914(42)80079-8.
E. Horn, R Zantl. Phase-Contrast Light Microscopy of Living Cells
Cultured in Small Volumes. Microsc Anal, 2006, 20(3):5–7

ibidi Solutions for Phase Contrast
ibidi µ-Slide Angiogenesis andµ-Plate Angiogenesis
Theµ-SlideAngiogenesis
andµ-PlateAngiogenesis 96 Well are not only designed forangiogenesis and tube formation assays, they also
provide the ideal cell culture vessel for brilliant phase
contrast images. A geometrical trick, the „well in awell“ technology, inhibits the meniscus formation
and results in good phase contrast over the entire
observation area.

1)

1)

2)

2)

10x

10x

Standard wellµ-Slide / Plate Angiogenesis

1) Meniscus on air-liquid interface: poor phase contrast in
most of the observation area.

1) Planar air-liquid interface:good phase contrast all over
the observation area.

2) Meniscus on the gel surface:
not possible to focus on all cells
simultaneously.

2) Planar gel surface: all cells
are in one optical plane.

96 well plate / small open well

Channel or parallel plates

Strong meniscus, low contrast
near the edges.

No meniscus, good phase
contrast over the entire area.

Standard well

Ph+ wellStrong meniscus effect.

No meniscus effect.

ibidi Channel µ-Slides
The ibidi channel μ-Slides provide ideal opticalconditions for phase contrast microscopy. When
culturing cells, the channel is filled with medium
from bottom to top. This geometrically inhibits
meniscus formation and allows for excellent phase
contrast across the whole channel.

ibidi µ-Slides Ph+
The ibidi µ-Slides Ph+ are specifically designed forphase contrast microscopy. A special intermediate
plate in each well avoids meniscus formation andguarantees brilliant phase contrast—no matter
which part of the well is being imaged.

17Differential Interference Contrast(DIC)
Applications
DIC is a more expensive, label-free microscopy
technique with a high sensitivity to thin cellularmaterial, even when it is located within thick tissue.
It is useful for rendering contrast in transparent
samples and gives brilliant pseudo-3D relief shading
images. Although DIC images look very appealing,
the pseudo-3D effect might be misleading in somecases, making it seem that the cells have structuresthat they do not have. As an example, areas inside
a living cell with a different refractive index, likevacuoles and chromatin, appear as bumps, which isactually an optical impression.
DIC microscopy of Rat1 cells. Scale bar 20 µm.

Principle

DIC Compatibility of Different Materials

Similar to phase contrast, DIC microscopy is acontrast-enhancing technique. DIC uses polarized
light to convert phase delays into intensity changes(contrast). The effect is called differential, because
contrast is created only in neighboring areas. Unlike
in phase contrast, the DIC image is not built globallyover the entire image. Instead, adjacent structureswith different refractive indices are contrasted whenin close contact with each other.

Compatible

DIC is less sensitive to meniscus formation than phase
contrast. However, DIC needs low birefringence of the
microscopy chamber and the lid, making it incompatible
with standard polystyrene cultureware. Since manyfactors can influence the recovery kinetics, the FRAP
experiments have to be planned well and the resulting
data must be analyzed carefully. It is then possible toobtain the diffusion coefficient and molecule mobility
parameters using mathematical modelling.

• DIC Lid for µ-Slides | DIC Lid for µ-Dishes• Open µ-Slides | µ-Dishes | µ-Plates without lid• Glass coverslips, glass slides• ibidi Polymer Coverslip• ibidi Glass Coverslip• All ibidi Heating Systems• Water, cell culture medium• Cell culture flasks, Petri dishes
Not compatible• Lids of all µ-Slides, µ-Dishes and µ-Plates• Channel µ-Slides and Ph+ µ-Slides

ibidi Solutions• Unlike other polymers on the market, the ibidi
Polymer Coverslip is DIC compatible.
• Glass coverslip bottoms are 100% compatible
with DIC microscopy.
• ibidi offers a special DIC Lid for µ-Dishes
and a DIC Lid for µ-Slides.

18

Widefield Fluorescence
Microscopy
Applications
Fluorescence microscopy is applied for the detectionof specific structures, molecules, or proteins withina cell. When using this technique, almost any
component of living and fixed cells or tissues can be“stained” and thereby specifically imaged—up to thehighest magnifications. Immunofluorescence assays
are widely analyzed using widefield fluorescencemicroscopy. It is also possible to visualize several
structures in parallel, which then appear as different
pseudocolors in the obtained image.

Principle
Widefield fluorescence microscopy is a variationof light microscopy and the easiest fluorescenceimaging mode. The underlying key principle is the useof fluorescent molecules—so-called fluorophores—
for the labeling of defined cellular structures. Thesemolecules, such as green fluorescent protein (GFP),
absorb light at a specific wavelength (excitation) andemit it at a specific higher wavelength (emission).
To visualize the molecule of interest, fluorophorecoupled specific antibodies or proteins, for example,
are transferred into the cell. The specimen isthen illuminated at the excitation wavelengthand viewed through a filter that allows only theemitted wavelength to pass through. Whereas the
background is dark, the structures with a boundfluorophore emit light, indicating the presence ofthe structure of interest.
In contrast to confocal microscopy, the whole
specimen is exposed to light in widefield
fluorescence microscopy. Fluorescence signals
from all focal planes are detected, which leads tolower contrast in thick samples like spheroids andtissue. Therefore, widefield microscopy is best
applied with thin specimens with low backgroundautofluorescence, like adherent cells.

Fibroblasts were cultivated on the ibidi Polymer Coverslip for 24hours following paraformaldehyde fixation. F-actin and nuclear DNA
were stained with Phalloidin (green) and DAPI (blue), respectively, before mounting with ibidi Mounting Medium. Widefield
fluorescence microscopy, Carl Zeiss Axiovert 100, objective lensPlan-Neolfluar 100x/1.3 oil, scale bar 10 µm.

Detector

Excitation
filterEmission
filterDichroic
mirrorLight pathways in a widefield microscope.

ibidi Solutions
Widefield fluorescence is possible without
restrictions when using any of the ibidi labwarethat contains the ibidi Polymer Coverslip or theibidi Glass Coverslip Bottom.

19

Confocal Microscopy
Applications
Confocal microscopy is broadly used to resolve
the detailed structure of specific objects within thecell. Similar to widefield fluorescence microscopy,
various components of living and fixed cells or
tissue sections can be specifically labeled using
immunofluorescence, for example, and thenvisualized in high resolution.
As a distinctive feature, confocal microscopy enables
the creation of sharp images of the exact plane of
focus, without any disturbing fluorescent light fromthe background or other regions of the specimen.
Therefore, structures within thicker objects can beconveniently visualized using confocal microscopy.
Furthermore, by stacking several images fromdifferent optical planes, 3D structures can beanalyzed. The sample penetration depth is limited,
however, when using confocal microscopy. Thicker
objects, like large spheroids, organoids, tissue, andsmall animals, should instead be optimally imaged
using two-photon microscopy or LSFM.

Z-stack of an FDA/PI-stained MCF-7 spheroid, acquisition by
confocal microscopy. Green: FDA-stained living cells. Red: PIstained dead cells in the necrotic center of the spheroid.

Principle
Similar to the widefield microscope, the confocal
microscope uses fluorescence optics. Instead ofilluminating the whole sample at once, laser light isfocused onto a defined spot at a specific depth within
the sample. This leads to the emission of fluorescentlight at exactly this point. A pinhole inside the opticalpathway cuts off signals that are out of focus, thus
allowing only the fluorescence signals from theilluminated spot to enter the light detector.
By scanning the specimen in a raster pattern, imagesof one single optical plane are created. 3D objects
can be visualized by scanning several optical planes
and stacking them using a suitable microscopy
deconvolution software (z-stack). It is also possible
to analyze multicolor immunofluorescence stainings
using state-of-the-art confocal microscopes that
include several lasers and emission/excitation filters.

Excitation

Emission

Detector
ConfocalpinholesDichroic
mirrorLaser

Objective
3D sample
Focal point

Excitation and emission light pathways in a basic confocal microscope configuration.

ibidi Solutions
Confocal microscopy is possible without
restrictions when using any of the ibidi labwarethat contains the ibidi Polymer Coverslip or theibidi Glass Coverslip Bottom.

20

Two-Photon and Multiphoton Microscopy
Applications
Two-photon microscopy (also called multiphoton
microscopy) can be used for live cell imagingof thick biological specimens, as it has several
advantages over confocal microscopy. Molecules
can be visualized deeply within the specimen witha maximal penetration depth of about 1 mm. This
enables 3D imaging of tissue slices, organoids,
whole organs, embryos, or even whole animals.
In contrast to confocal microscopy, two-photon microscopy works with higher wavelengths leading toless photobleaching or photodamage, which is especially important when working with living samples.

Principle
Just like widefield or confocal fluorescencemicroscopy, two-photon microscopy is based on
fluorophore excitation, which results in the emissionof light. In classic fluorescence microscopy, afluorophore is excited by absorbing one single
photon of a certain wavelength. When using twophoton microscopy, two or three photons of a higher
wavelength do the work of one: When they hit thefluorophore at the very same time (typically withinseveral femtoseconds), they are absorbed, resulting
in fluorophore excitation and emission of light.

Rotating 3D rendering of a confocal image stack showing the dorsal germ ring of a zebrafish embryo at the onset of gastrulation(6 hours post fertilization). GFP (white) is expressed in internalizing prechordal plate progenitors and lyn-TagBFP (red) marks themembrane in all cells. The embryo was injected with dextranrhodamine to label the interstitial fluid (blue). The image wasrecorded at the Bioimaging Facility of the Institute of Science andTechnology Austria (IST), using a multiphoton LaVision BioTec
TriM Scope microscope.

In this process, photons combine their energy,
which allows low-energy infrared photons to
excite standard fluorophores, such as GFP. The
infrared light penetrates tissue more deeply than
the standard excitation light used in fluorescencemicroscopy. Due to its low energy level, infrared
light is less damaging, and therefore especially
useful when working with living samples.
In order to increase the likelihood that two photons hit
the fluorophore simultaneously, lasers with very highintensity are needed. Their infrared light only leads
to excitation in the focus of the objective, because
only in this area, the critical number of photons pertime and space is reached. Therefore, all emitted lightcomes from one focal point in the specimen, strongly
reducing background noise. The image is created justas in confocal microscopy: The laser scans across the
sample, recording the image intensity point by point.
R.K.P. Benninger, D.W. Piston. Two-photon excitation microscopy
for the study of living cells and tissues. Curr Protoc Cell Biol,
2013, 10.1002/0471143030.cb0411s59

ibidi Solutions
Two-photon microscopy is possible without
restrictions when using any of the ibidi labware
containing the ibidi Glass Coverslip Bottom.
Optically, the ibidi Polymer Coverslip is 100%
compatible, but be aware that the polymer isonly temperature-stable up to a maximum of
80°C/175°F. Depending on the microscopy setup
being used (e.g., wavelength, laser intensity,
objective lens, heat transfer), it is possible toexceed this temperature limit, which will damage
the ibidi Polymer Coverslip. We recommend
trying a free sample of your chosen product totest its compatibility with your specific setup.

21

Fluorescence Recovery After
Photobleaching (FRAP)Light Sheet Fluorescence
Microscopy (LSFM)

Applications

Applications

FRAP is a fluorescence microscopy method forstudying the mobility of fluorescently-labeled molecules in living cells. It is applied for the analysis ofmolecule diffusion within the cell, fluidity of bio
membranes, and protein binding.

LSFM is applied for live cell 3D imaging of thickbiological samples as a whole, such as embryos,
spheroids, organoids, and whole animals. The
strongly reduced background signal, minimized
phototoxicity, and increased scanning speed make
LSFM superior to epifluorescent microscopy for the
analysis of thick living specimens.

Principle

FRAP-based visualization of the F-actin diffusion in a primarydendritic cell using LifeAct.

Principle
FRAP can be conducted using a modern confocal
microscope. It needs fluorescent labeling of the molecule of interest. A typical FRAP experiment involves
three distinct phases. First, the initial fluorescence ismeasured in the region of interest. Next, the fluorescent molecules are photobleached within a selected
area. This is done by focusing the laser beam onto
the respective area, leading to intense illuminationand extinction of the fluorophores. As a result, adark area in the otherwise fluorescent sample canbe observed. Finally, fluorescent molecules from thesurroundings can diffuse through the sample andexchange the photobleached molecules with intact
ones. This fluorescence recovery is measured overtime, making it possible to obtain the diffusion coefficient and molecule mobility parameters.

Researchers can choose between several
LSFM/SPIM (Single/Selective Plane Illumination
Microscopy) systems that differ in certain aspects
but all have the following main features: in contrast
to confocal or widefield fluorescent microscopy
only a thin section of the specimen is illuminatedby a light sheet. Another important difference is theseparation of the optical pathways of illuminationand detection. To avoid the measurement of out-offocus fluorescence, emitted light is detected on adifferent axis to the illumination (e.g., orthogonal). By
doing this, unnecessary out-of-focus fluorescenceis not excited, which prevents photobleaching andphotodamage in regions that are currently not being
scanned. Finally, with LSFM, the image is created byscanning a plane of light (optical sectioning), insteadof point by point, which markedly increases scanningspeed when compared to confocal microscopy.
These advantages—reduced cellular stress, background fluorescence, and time consumption—areespecially important when doing 3D live cell imaging of sensitive biological samples.

Detector

The principle setupof LSFM.

ibidi Solutions
FRAP and LSFM are possible without restrictions when using any of the ibidi labware that contains the ibidi
Polymer Coverslip or the ibidi Glass Coverslip Bottom.
Please note: The suitability of the ibidi labware for LSFM depends on the well/channel geometry, the used
LSFM system and its optical setup. It has to be checked individually before each experiment.

22

Förster Resonance Energy
Transfer (FRET)

Fluorescence Lifetime Imaging
Microscopy (FLIM)

Applications

Applications

FRET determines the precise location and spatial
proximity of fluorescently labeled molecules andtheir interactions in living cells. Using this technique,protein-protein interactions or conformation changes,
for example, can be analyzed using a standard
widefield or confocal fluorescence microscope.
Using specific calcium-sensitive biosensors, FRET
can also be applied for the visualization of changes
in cellular calcium concentration.

FLIM is used for analyzing the distribution of specificcellular components, such as proteins or nucleic acids.
It can be used to gain information about the molecules
and to visualize the state of the environment that surrounds the respective molecule in living cells. FLIM
helps to measure several factors, such as calcium ion
amount, pH, oxygen concentration, molecular interaction, and molecular binding. Under optimal conditions,
it is even possible to detect and identify single molecules. The penetration depth using FLIM is higher
compared to standard fluorescence microscopy,
which enables the analysis of thicker samples.

Principle

FRET-based visualization of cytoplasmatic calcium concentration.
HEK293 cells expressing NK-I on the outer cell membrane and thecalcium biosensor Yellow Cameleon 3.6 (YC3.6) in the cytoplasm.
Addition of the fluorescently labelled NK-I ligand (SP-TAMRA)
results in a red glow of the cell membrane. Upon receptor activation, calcium release induces a change in the YC3.6 fluorescence
properties: CFP excitation yields simultaneous CFP and YFP emissions by a FRET phenomenon, appearing as a green glow in thecytoplasm. Provided by M. Roelse, Wageningen, The Netherlands.

Principle
A donor fluorophore in its excited state can transfer
its excitation energy to an acceptor fluorophore in a
non-radiative fashion. Typically, this happens throughdipole-dipole coupling in a distance of less than 10
nm. Beyond that distance (Förster radius), the two
fluorophores show normal fluorescence behavior.
Single fluorescent protein

433 nm

CFP

475 nm475 nm

A

FRET

YFP

In contrast to standard fluorescence microscopy
where the intensity is used to create an image ofthe specimen, FLIM uses the lifetime of the signal
by analyzing the fluorophore’s exponential decay rate.
The fluorescence lifetime is specified as the averagetime that a fluorophore stays in the excited state
before emitting a photon and returning to the groundstate. In the excited state, each type of fluorophore
has its own lifetime. By detecting differences inlifetime, it is possible to distinguish fluorophores thathave the same excitation and emission spectrum.
The fluorescence lifetime depends on the local environment. While it is affected by factors such as molecularinteraction or ion concentration, it is not influenced byfluorophore concentration, photobleaching, or excitation light intensity. The resulting FLIM image contrasts
the lifetime of the individual fluorophores, which canthen be used to define and interpret the environmental
factors of the molecules of interest. The lifetime alsochanges when an energy acceptor molecule is in close
proximity. This combination of FLIM and FRET gives
detailed insight into sub-molecular binding processes.
H.C. Ishikawa-Ankerhold, R. Ankerhold, G.P.C. Drummen. Advanced
Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET
and FLIM. Molecules, 2012, 10.3390/molecules17044047

528 nmB
 FRET 

433 nmCFP
A

YFP

528 nmB

FRET protein interaction assay. A membrane receptor A and its
ligand B are tagged with CFP and YFP, respectively. When theligand binds to the receptor, the YFP is excited by FRET.

ibidi Solutions
FRET and FLIM microscopy are possible without
restrictions when using any of the ibidi labwarethat contains the ibidi Polymer Coverslip or theibidi Glass Coverslip Bottom.

23

Total Internal Reflection
Fluorescence (TIRF)
Applications
TIRF is a fluorescence-based technique used toimage the processes that occur in and near themembrane of living cells. Although TIRF cannot be
used to visualize structures located deep within a
specimen, it allows for near-membrane imaging
close to the interface with a high signal-to-noise ratio.
TIRF is very useful visualizing membrane processes,
such as receptor-ligand interactions, endocytosis,
viral infection, or cell adhesion to surfaces.

Principle
For TIRF, two optical media with different refractive
indices, such as water (nD=1.33) and glass (nD=1.52),
are needed. If the total internal reflection of incident
light occurs at the interface of these media, anevanescent field is created. This evanescent field is
an area in which the totally reflected light is still able
to excite fluorophores. It extends about 100–200 nm
deep into the specimen, leading to the excitation offluorophores. Only the fluorescent events occurringat the section of the specimen close to the glass/sample interface are then visualized.

Surface-near F-actin network of a Dictyostelium discoideum
DdLimE-GFP cell. TIRF Live cell imaging on a Glass Coverslip#1.5H.

Water
Cell
Evanescent field
Coverslip

βG

Laser

The principle of TIRF microscopy.

ibidi Solutions
The ibidi Glass Coverslip Bottom provides ideal
optical conditions for TIRF. It is possible to use
any of the ibidi labware, without restrictions,containing the ibidi Glass Coverslip Bottom.
Please note: TIRF is generally possible with theibidi Polymer Coverslip, but we recommend using
the ibidi Glass Coverslip.

24Super-Resolution Microscopy(STED, SIM, (F)PALM, (d)STORM)
Applications
Super-resolution
microscopy
enablesthevisualization of the smallest structures in living cells
that cannot be resolved using standard widefield or
confocal fluorescence microscopy. This techniqueprovides a spatial 3D-resolution that is well below
the diffraction limit. It creates new views on the
structural organization of cells and the dynamics of
biomolecular assemblies, that are close to a nearmolecular resolution.

Principle
Resolution is described as a function to discriminate
two dots from each other. It is dependent on the
wavelength and the numerical aperture and isphysically limited by Abbe’s law.
When using widefield and confocal fluorescencemicroscopy, the diffraction barrier limits the
maximal resolution to about 200 nm. Superresolution microscopy breaks the diffraction barrier,enabling “nanoscopy” with substantially improved
optical resolution of down to 5–20nm. This method
uses the physical or chemical properties of adjacentfluorophores to resolve them from each other. For
example, while one fluorophore’s state is “on”,
the neighboring fluorophore’s state is “off”, whichenables their differentiation.

dSTORM image of plasma membrane glycans on the ibidi
Polymer Coverslip. Membrane glycans of SK-N-MC neuroblastoma cells were stained through the metabolic incorporationof azido-sugar analogues followed by copper-catalyzed azidealkyne cycloaddition (CuAAC). Inlet: comparison to widefieldmicroscopy. Provided by Markus Sauer, Würzburg.

Several super-resolution microscopy techniques
were developed, each with its own advantages anddisadvantages. There are deterministic and stochastic
functional techniques. Common examples are:• Stimulated emission depletion (STED)• Saturated structured illumination microscopy (SSIM)• REversible Saturable Optical Linear
Fluorescence Transitions (RESOLFT)• Photoactivated localization microscopy (PALM)• Fluorescence photoactivation localization
microscopy (FPALM)• Stochastic optical reconstruction microscopy(d)STORM

S.W. Hell. Far-field optical nanoscopy. Science, 2007, 10.1126/
science.1137395
S.W. Hell. Microscopy and its focal switch. Nat Methods, 2009,10.1038/nmeth.1291
B. Huang, H. Babcock, X. Zhuang. Breaking the diffraction
barrier: super-resolution imaging of cells. Cell, 2010, 10.1016/j.
cell.2010.12.002
S.J. Sahl, S.W. Hell, S. Jakobs. Fluorescence nanoscopy in cellbiology. Nat Rev Mol Cell Biol, 2017, 10.1038/nrm.2017.71

ibidi Solutions
In general, we can recommend the ibidi Glass
Coverslip Bottom for super-resolution microscopy
techniques. The ibidi Polymer Coverslip, however,cannot yet be guaranteed to work with every
microscopic technique due to the large numberof techniques available.
Please note: dSTORM is possible with the ibidi
Polymer Coverslip. If you would like to takeadvantage of the ibidi Polymer Coverslip Bottomfeatures and test them with your own superresolution microscopy experiments, please checkout our free sample! Then, give us feedback on
how the ibidi Polymer Coverslip worked with yourtechnique of choice.

25

ibidi Material Specifications

ibidi Bottom Material Specifications
POLYMER
COVERSLIP

#1.5

GLASS

COVERSLIP#1.5H#1.5 ibidi#1.5H ibidi
Polymer Coverslip Glass Coverslip
Bottom thickness
Bottom material
Surfacemodifications

180 µm(+10/–5 µm)
Polymer
ibiTreat (tissue culturetreated), Hydrophobic,
Uncoated, Bioinert,µ-Patterning, ElasticallySupported Surface(ESS), Collagen I, IV,
Poly-L-Lysine coating

170 µm(+/–5 µm)

GLASS

COVERSLIP

GLASS
SLIDE

#1.5 glass
coverslip

Standard
glass slide*

170 µm(+20/–10 µm)

1000 µm (+/–50
µm)

#1.5

D 263 M Schott
D 263 M SchottSoda-lime glass
high precision glass high precision glass

Uncoatedglass

Uncoatedglass

Uncoatedglass

Standard
polystyrene
plates & dishes
500–1000 µm
Polymer

Tissueculture-treated,
Untreated

Compatibility With Microscopy Applications
POLYMER
COVERSLIP

#1.5

GLASS

COVERSLIP#1.5H#1.5 ibidi#1.5H ibidi
Polymer Coverslip Glass Coverslip

26

GLASS

COVERSLIP

GLASS
SLIDE

#1.5 glass
coverslip

Standard
glass slide*

Standard
polystyrene
plates & dishes#1.5

Brightfield
Microscopy

++

++

++

++

++

Phase Contrast

++

++

++

++

++

Differential Interference Contrast (DIC)

++

++

++

++

-

Widefield
Fluorescence

++

++

++

++

-

Confocal Microscopy

++

++

++

++

-

Two-Photon and Multiphoton Microscopy

++

++

++

++

-

Fluorescence
Recovery After
Photobleaching (FRAP)

++

++

++

++

-

Förster Resonance
Energy Transfer (FRET)

++

++

++

++

-

Fluorescence
Lifetime Imaging
Microscopy (FLIM)

++

++

++

++

-

Lightsheet Fluorescence Microscopy(LSFM, SPIM)

++

++

++

++

-

Total Internal Reflection
Fluorescence (TIRF)

+

++

+

+

-

Super-Resolution
Microscopy

+

++

+

+

-

Optical Aspects
POLYMER
COVERSLIP

#1.5

GLASS

COVERSLIP#1.5H#1.5 ibidi#1.5H ibidi
Polymer Coverslip Glass Coverslip
Refractive index(nD 589 nm)

1.52

1.52

GLASS

COVERSLIP

GLASS
SLIDE

#1.5 glass
coverslip

Standard
glass slide*

Standard
polystyrene
plates & dishes

1.52

1.52

1.58

#1.5

Abbe number

56

55

55

64

31

Autofluorescence

Low

Low

Low

Low

Very highVery high(even ultraviolet)High(ultraviolet
restrictions)High(ultraviolet
restrictions)High(ultraviolet
restrictions)High(ultraviolet
restrictions)

COVERSLIP

GLASS

GLASS
SLIDE

#1.5 glass
coverslip

Standard
glass slide*

Standard
polystyrene
plates & dishes

Transmission

Further Aspects
POLYMER
COVERSLIP

#1.5

GLASS

COVERSLIP#1.5H#1.5 ibidi#1.5H ibidi
Polymer Coverslip Glass Coverslip#1.5

Gas permeability

Yes

No

No

No

Yes

Material flexibility

High

Low

Low

Very lowHigh

Breakable

No

Yes

Yes

Yes

No

Compatibility withprotein coatings

Yes

Yes

Yes

Yes

Yes

Low
(DIC-compatible)

Low(DIC-compatible)

Low(DIC-compatible)

Low(DIC-compatible)

Very high

Yes

Yes

Yes

Yes

No

Yes

No
(Yes, if mountedwith a standard
glass coverslip)

No

Birefringence (DIC)
Immersion oil
compatibility
Correct thickness
for high resolution
microscopy
objective lenses

Yes

Yes

* With the 3 Well | 8 Well | 12 Well Chamber Slide, removable, ibidi provides self-adhesive, removable silicone
chambers that are mounted on a standard glass slide. They are suitable for upright and inverted microscopy andenable long-term storage of samples after immunofluorescence staining.

27

Manufacturer / Supplier

North American Headquarters

ibidi GmbH
Lochhamer Schlag 1182166 Gräfelfing
Germany

ibidi USA, Inc.
2920 Marketplace Drive,
Suite 102
Fitchburg, WI 53719
USA

Toll free within Germany:
Phone: 0800 / 00 11 11 28
Fax:
0800 / 00 11 11 29

28

Toll free within the US:
Phone: +1 844 276 6363

International calls:
Phone: +49 89 / 520 46 17 - 0
Fax:+49 89 / 520 46 17 - 59

International calls:
Phone: +1 608 441 8181
Fax:
+1 608 441 8383

All ibidi products are for research use
only! Errors and omissions excepted.

E-Mail: info@ibidi.com
ibidi.com

E-Mail: ibidiusa@ibidi.com
ibidi.com© ibidi GmbH
FL_AG_031, V 2.0 2021 /11

For free samples, application notes, and handling movies,
please visit us at:.com